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Deciphering Mode of Action of Functionally Important Regions

Larry H. Bernstein, MD, FCAP, Curator

LPBI

 

Deciphering Mode of Action of Functionally Important Regions in the Intrinsically Disordered Paxillin (Residues 1-313) Using Its Interaction with FAT (Focal Adhesion Targeting Domain of Focal Adhesion Kinase)

Intrinsically disordered proteins (IDPs) play a major role in various cellular functions ranging from transcription to cell migration. Mutations/modifications in such IDPs are shown to be associated with various diseases. Current strategies to study the mode of action and regulatory mechanisms of disordered proteins at the structural level are time consuming and challenging. Therefore, using simple and swift strategies for identifying functionally important regions in unstructured segments and understanding their underlying mechanisms is critical for many applications. Here we propose a simple strategy that employs dissection of human paxillin (residues 1–313) that comprises intrinsically disordered regions, followed by its interaction study using FAT (Focal adhesion targeting domain of focal adhesion kinase) as its binding partner to retrace structural behavior. Our findings show that the paxillin interaction with FAT exhibits a masking and unmasking effect by a putative intra-molecular regulatory region. This phenomenon suggests how cancer associated mutations in paxillin affect its interactions with Focal Adhesion Kinase (FAK). The strategy could be used to decipher the mode of regulations and identify functionally relevant constructs for other studies.
Neerathilingam M, Bairy SG, Mysore S (2016) Deciphering Mode of Action of Functionally Important Regions in the Intrinsically Disordered Paxillin (Residues 1-313) Using Its Interaction with FAT (Focal Adhesion Targeting Domain of Focal Adhesion Kinase). PLoS ONE 11(2): e0150153. doi:10.1371/journal.pone.0150153

Genomic data suggests that a large proportion of eukaryotic proteins appear to adopt disordered structures in physiological conditions [1, 2]. Mutations/modifications in such IDPs are shown to be associated with various diseases (like cancer) [3]; therefore, understanding their structural behavior is critical for various applications like drug-targeting, mapping protein interactions, deciphering mode of action and finding functional relevance. However, deciphering mode of action in IDPs has been challenging given that unstructured segments render poor chemical shift dispersions and electron density in major techniques like NMR and X-ray, respectively [4]. For example, it took almost 10 years to decipher the mode of action of Sic1, a disordered protein involved in inhibition of a cyclin-dependent kinase [5]. One way to map and study the functional regions is to make truncated constructs by dissecting the whole construct rationally. A limited number of dissection constructs are usually generated; this is due to the time-consuming and challenging process of generating soluble and functionally relevant constructs when studies are performed in-vivo and constructs are prepared and tested sequentially. Here we present a simple high throughput (HTP) screening strategy (Fig 1a), which focuses on finding functionally relevant regions in IDPs based upon its interaction with a binding partner. Close to thirty dissection constructs of the IDP were generated and studied in parallel to understand the importance and functionality of the various regions of the protein. We perform cell-free expression followed by solubility check and GST pull-down interaction study in HTP format. Though both cell-free expression and GST pull-down assay have been individually performed in HTP format [6, 7], we did not find previous studies that combine the two methods in HTP format. Although the nature of interaction of IDPs with respective binding partners may vary, our strategy may be used to derive crucial insights into “structural behavior” of the unstructured segments in modulating the interaction. The strategy can also be used to identify functionally important regions in the IDP that would be suitable for further structural studies.

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Fig 1. Dissection of paxillin constructs (residues 1–313) followed by expression and interaction studies.

(a) Timeline for overall-strategy. (b) Illustration of solubility and activity level of linear dissected human paxillin (residues 1–313). (c) Phosphor screen image of filter assay for optimization of temperature for paxillin constructs (left). Tabular representation of paxillin constructs, negative and positive controls corresponding to each well in filter assay [1].(d) Phosphor screen image of 10% SDS PAGE of 35S labeled cell-free expressed samples after GST pull-down assay of the paxillin constructs A1–E1; The right panel shows fraction of interaction of each construct with respect to B2 (since B2 showed maximum level of interaction) (e) Illustration of solubility and activity of dissected C3 constructs. All experiments were performed in triplicates and averaged. To rule out non-specific interactions that might occur with GST tagged FAT, GFP that was expressed in cell-free system and a reaction without DNA template were used as negative controls.    http://dx.doi.org:/10.1371/journal.pone.0150153.g001

Disorder/Intrinsic disorder seems to be a common feature of hub proteins in eukaryotes [2], thus highlighting the need for studying the mode of action of unstructured segments in such proteins. Here we used paxillin (residues 1–313), an intrinsically disordered construct, for demonstrating this approach. Paxillin (residues 1–313) consists of multiple protein interaction sites that are connected by flexible disordered sequences [8]. The disordered regions in paxillin have been detrimental in efforts to study the complete structure of the protein due to the demerits mentioned previously. This explains the lack of structural details of regulation of paxillin binding. Residues 1–313 of paxillin consist of five leucine-rich sequences LD1-LD5 (with consensus sequence: LDXLLXXL), termed LD motifs, which are highly conserved between species and other family members such as Hic-5, leupaxin and PAXB [8]. Paxillin interacts with multiple proteins involved in cell migration, actin rearrangements and cell proliferation [9]. Mutations in paxillin are shown to be associated with lung cancer [3, 10]; and the differential expression of paxillin is associated with various forms of cancer and other diseases such as Alzheimer’s and inflammation [1113]. This implies the importance of studying the structural and functional characteristics of paxillin. Most paxillin studies focus on interactions of LD motifs with proteins such as focal adhesion kinase (FAK), vinculin and v-crk, providing clues towards their importance in deciphering the functionality of paxillin [8, 14, 15]. Though regions of paxillin that bind to various partners were deciphered through previous studies, the basis of effect of mutations in paxillin on binding its partners was not explained. Mutations in paxillin, some that were observed to be associated with cancer were positioned in the intrinsically disordered regions between the LD motifs and not on the motifs themselves [3, 10]. For example, P30S, G105A and A127T mutations lie between LD1 and LD2 motif; P233L and T255I mutations lie between LD3 and LD4 motifs. This shows that the LD motifs alone do not govern the functionality, but unstructured regions linking the LD motifs could play a major role. In normal conditions, FAT (Focal adhesion targeting domain of FAK) binds hydrophobically through its HP1 (Hydrophobic patch 1) and HP2 (Hydrophobic patch 2) sites to paxillin LD motifs—LD2 and LD4 [16, 17], which lead to activation of binding sites for other proteins on paxillin. LD2 preferentially binds to the HP2 site, whereas LD4 preferentially binds to the HP1 site [18]. In a state of cancer caused by mutations in paxillin, the LD interactions could be hindered, as mutations in the unstructured segments result in abnormal binding of FAK to either of the LD motifs [9]. Here we wanted to locate the region involved in the structural modulation of paxillin-FAT interaction by adopting a simple approach (Fig 1) that involves dissected proteins generated using cell-free protein expression coupled with protein-protein interaction study. We map the disordered proteins’ structural importance to understand the function and modulation of paxillin-FAT interaction in days rather than months (Fig 1a).

Dissection and identification of fragments of paxillin (residues 1–313) with functional relevance

We dissected paxillin (residues 1–313) (Fig 1b) into nested sets using PCR such that each of the constructs had either or both LD2 and LD4 motifs (S1 Fig and S1 Table). Further, these constructs were expressed in soluble form using small-scale cell-free expression system in a 96 well format (Fig 1c). However, all constructs except A6, B6, C4 and C5 expressed detectable amounts of protein (S2a Fig and S2 Table). The failure in expression of the above constructs could be due to the instability of the smaller peptide fragments that might be susceptible to proteolytic cleavage [19]. Soluble protein from small-scale expression of the dissected constructs namely A1, A2, A3, A4, A5, B1, B2, B3, B4, B5, C1, C2, C3, D1, D2, and E1 were pulled down and analysed (Fig 1d). Although constructs A1–A5, B1–B5, C1, C2, D1 and E1 interacted successfully, C3 (containing LD2) and D2 (containing LD4) failed to interact (Fig 2c) despite containing LD motifs. However, based on previous reports [8, 16, 17], we expected all constructs containing either LD2 and/or LD4 to interact with the FAT domain. Therefore, this led us to suspect that intra-molecular auto-inhibition in unstructured segments modulated binding of FAT to LD motifs in paxillin.

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Fig 2. Regulatory and masking regions around paxillin’s LD2 and LD4 and their circular dichroism spectra.

(a) CD spectra of paxillin LD peptides (LD1-LD5) and constructs: B2, C3, C35 and D2. CD spectra of LD2, LD4, C35 and D2 constructs showed negative bands at 222nm and 206nm and a positive band at 192nm that confirms the presence of alpha helical content thus may behave as folded effector binding sites. However, LD1, LD3, LD5, B2 and C3 do not show the characteristic peaks of secondary structures, thus may behave as unfolded effector binding sites. (b) LD2 regulatory region (54–130) and masking region (167–224) evidenced by constructs B3, B4 and B5. (c) LD4 regulatory region (216–257) and masking region (280–313) evidenced by constructs D1, D2 and E1.
http://dx.doi.org:/10.1371/journal.pone.0150153.g002

Identification of regulatory regions and their mechanisms

To investigate the non-interaction of C3, a series of C3 deleted constructs (C31 –C310) (Fig 1e,S1 Table) were generated to determine the internal region that influenced the non-functioning of C3. C36 linear template could not be amplified for expression. As solubility of C3 could play a critical role in determining interaction, the homogeneity of the sample was confirmed by capillary electrophoresis under non-reducing conditions [20] (See S3 Fig). The linear templates—C31, C32, C33, C34 and C35 were successfully expressed in soluble form, The other C3 deleted constructs did not express due to issues related to small size as described earlier. Surprisingly, none of the C3 deleted constructs interacted with FAT despite the presence of the LD2 motif, although constructs such as B3, B4 and B5 that contain regions overlapping with C3 showed interaction (S2b and S2c Fig, Fig 1d and 1e). Here B3 that included the whole of C3 and unstructured segment 54–130 showed interaction (Fig 1b). Constructs B4 and B5 also containing residues 54–130 showed interaction despite differing from B3 by lacking regions 167–224 and 155–224, respectively. Interestingly, the non-interacting constructs C3 and C35 do not contain 54–130 residues, but include the regions 167–224 and 167–189, respectively (Fig 1b). Here constructs containing region 167–189 but lacking 54–130 did not interact with FAT despite LD motif alone showing interaction (switch off) (Fig 3a). Whereas, if 54–130 was included, interaction was reinstated (switch on) (Fig 3a). This clearly shows that interaction of LD2 in construct C35 is masked by residues 167–189 (masking region) (Fig 2b). The constructs B3 and B4 binding to FAT despite the presence of the masking region led us to conclude that the region 54–130 (regulatory region) acts to remove the masking effect (Fig 2b).
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Fig 3. Binding studies of paxillin constructs using Bio-layer Interferometry on OctetRed96.

(a) Switch off in C3 and D2 on LD2 and LD4 respectively; Hypothesis of partial switch on when regulatory region of LD2 is absent, as evidenced in C2. (b) Concentration calibration curves depicting binding of constructs B2, C35, ‘54–189’, ‘79–189’, ‘105–189’ with GST-FAT. The data is representative of a single experiment. Each experiment was performed at-least thrice. (c) Illustrations of C35, C35_1, C35_2 and C35_3.  http://dx.doi.org:/10.1371/journal.pone.0150153.g003

Similar to LD2, LD4 in construct D2 containing 216–257 (masking region) requires additional residues of paxillin 280–313 (regulatory region) for FAT binding (Fig 2c), which was demonstrated by showing the interaction with constructs D1 (spanning region 216–313) (Fig 1b) and E1 (spanning region 258–313). To visualize the non-binding of FAT to C35, in-silico methods were employed to model the C35 construct and docked with the crystal structure of FAT (1K05, residues 916–1050 [21]) (Fig 4). The docking results showed a clear masking effect in the C35 construct by the 167–189 (masking region) residues. The constructs B2, C3 and C35 were also structurally characterized using CD analysis (Large scale cell-free expression was performed for this purpose, see S3 Fig). The percentage of alpha helical content was found to be much higher in C35 (95.32%) as compared to B2 (12.43%) (Fig 2a, S3 Table). Therefore, the dissection(s) of B2 to C35 allowed the identification of structured regions (C35) as compared to the disordered B2. Further, it showed that the LD2 peptide and C35 have significant alpha-helical structures that do not translate into functional similarity as evidenced by the inability of C3, C35 and D2 to bind to FAT. Moreover, LD2 peptide binds to FAT while C35 does not (Fig 1e and S2b and S2c Fig). A similar observation was made when comparing the ability of LD4 peptide and the inability of D2 to bind to FAT despite both having detectable α-helical content (Fig 1b). Thus, these results confirm the existence of masking and regulatory regions (Fig 2b and 2c) that determine switch on and off and in turn, intra-molecular auto-inhibition. C2 showed activity despite missing regulatory regions for both LD2 and LD4 (similar activity observed in C1). This could be because the unfolded nature of LD3 effector binding site that is located between LD2 and LD4 is flexible to mask only a single LD motif but not both (Partial switch on, Fig 3a).

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Fig 4. In-silico analysis of non-binding of C35.

(a) LD2 crystal structure from PDB id: 1K05 (left) being compared with the LD2 structure in the side view and top view of C35 structure showing the masking of the hydrophobic binding region predicted through HMM based SAM-T08 software. The LD2 binding region and the masking regions are depicted by the bracketed region. (b) Docking control showing FAT (co-ordinates from PDB id: 1K05) and LD2 (co-ordinates from PDB id: 2L6F, NMR model # 1) interaction using Hex 6.3 software. (c) Docking of C35 with FAT showing non-interaction due to masking effect. The sidechains of the active residues are shown as red sticks. The hydrophobic patch—HP2 in FAT molecule, which preferentially binds to LD2 is shown as a space filling model in orange (part of helix 1 of FAT) and grey (part of helix 4 of FAT) colors.   http://dx.doi.org:/10.1371/journal.pone.0150153.g004

To predict the influence of this structural modulation, the state of LD motifs structurally before and after binding to FAT had to be understood. CD spectra of LD1, LD3 and LD5 peptides showed characteristics of random coil (Fig 2a, S3 Table) thus validating that the LD1, LD3 and LD5 motifs could exist as unfolded effector binding sites (not available for interaction) in our study and could fold upon undergoing allosteric changes after binding to their respective targets.

Validation of protein-protein interaction study using bio-layer interferometry studies

Bio-layer interferometry studies were performed to further validate the interaction studies and also to get insights into the binding affinities. Here apart from constructs B2 and C35, three other constructs that include different lengths of the regulatory region along with the C35 region were used for the studies, namely—Construct C35_1(54–189); Construct C35_2 (79–189) and Construct C35_3 (105–189) (See Fig 3b and S4 Fig). As seen in Fig 3c and Table 1, B2 shows maximum binding with KD value in the nano-molar range and the curves fit into a 1:1 binding model. C35 shows negligible binding and the rest of the constructs show binding lower than B2 with KD values in micro-molar range and the curves fit into a 2:1 binding model (See S5 Fig).

According to previous reports, FAK has to bind to both LD2 and LD4, failing which phosphorylation during signalling is reduced [8], which is observed in case of cancer [3], thus resulting in abnormal functioning of paxillin. We investigated this by analysing B2, which showed higher interaction than B1, despite missing the regulatory region of LD4 (Fig 1b). Similarly, C2 showed activity despite missing regulatory regions for both LD2 and LD4 and the presence of masking regions (similar activity observed in C1). This suggests that the masking region that is located between LD2 and LD4 is flexible to mask only a single LD motif but not both (Fig 3a). Interestingly, paxillin mutations associated with lung cancer were observed in the unstructured segments, particularly the regulatory region of LD2 and masking region of LD4 [3]. We hypothesize that these mutations prevent proper functioning of the regulatory regions, thus resulting in masking of either of the LD motifs causing abnormal functioning of paxillin. Evidence that these regions regulate FAT-paxillin binding was further provided in our study in the form of the bio-layer interferometry results; where C35 did not show any binding, but the constructs that included different lengths of the regulatory region along with the C35 region showed binding with KD values in the micro-molar range. This suggests that the LD2 region in these constructs is not masked, since it is seen in previous studies that the KD value for FAT binding to a single LD motif of paxillin is in micro-molar range. It also suggests that the region between residues 105–131 is sufficient for preventing the masking of LD2 region, thus allowing interaction with FAT (See illustrations in Fig 3b). Except B2 (that had a 1:1 binding stoichiometry and higher binding affinity), all other constructs (C35_1, C35_2, C35_3) showed a 2:1 binding stoichiometry. This suggests that both LD motifs of B2 engage both the FAT HP sites thus resulting in higher affinity; whereas in the other 3 constructs (C35_1, C35_2, C35_3), each FAT HP site (HP1 and HP2) interacts with individual molecules thus giving a 2:1 stoichiometry. This is in agreement with previous studies where both the LD motifs were found to interact with both HP1 and HP2 hydrophobic patches of FAT [16]. The higher affinity of B2 to FAT could be due to presence of both LD2 and LD4; the proposed intra-molecular regulatory regions could also play a role in the increased affinity. Therefore, we understand that the abnormal modulation in cancer involves redirection of FAK to a single LD motif; and targeting drugs for re-establishing the function at regulatory regions could be critical.

Unlike many existing techniques like array based yeast two hybrid assay, phage display method and tandem affinity purification; the strategy used here (combination of cell-free expression, filter based solubility assay and interaction study in HTP format) facilitated quick identification of the role of unstructured regions involved in paxillin-FAT interaction in HTP format. Particularly, in paxillin-FAK interactions, which determine focal adhesion and cellular signalling, we understood the structural masking and unmasking behaviour of unstructured segments in paxillin to determine FAK interaction. The structure of paxillin is not yet elucidated due to difficulties with respect to its disordered nature. In this study, the templates that we generated using the high throughput dissection strategy allowed us to analyze various regions of paxillin, with respect to structure, solubility and function. To our knowledge, this study is the first report of switch on and off mechanisms working together in controlling allosteric modulation/auto-inhibition in a human hub protein. As many eukaryotic proteins are disordered, our study opens avenues for analyzing novel modulations at allosteric sites using appropriate interaction studies, which could lead to identification of new drug target sites. In this regard, we hope the above strategy will be instrumental in understanding mechanisms of other disordered proteins as well, in days rather than months. This strategy could also be used as an initial screening method for techniques like SAXS, smFRET and others.

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long noncoding RNAs

Larry H. Bernstein, MD, FCAP, Curator

LPBI

 

What are lncRNAs?

Advances in RNA sequencing technologies have revealed the complexity of our genome. Non-coding RNAs make up the majority (98%) of the transcriptome, and several different classes of regulatory RNA with important functions are being discovered. Understanding the significance of this RNA world is one of the most important challenges facing biology today, and the non-coding RNAs within it represent a gold mine of potential new biomarkers and drug targets.
lncRNA sequences

Long non-coding RNAs (lncRNAs) are a large and diverse class of transcribed RNA molecules with a length of more than 200 nucleotides that do not encode proteins (or lack > 100 amino acid open reading frame). lncRNAs are thought to encompass nearly 30,000 different transcripts in humans, hence lncRNA transcripts account for the major part of the non-coding transcriptome. lncRNA discovery is still at a preliminary stage. There are many specialized lncRNA databases, which are organized and centralized throughRNAcentral.

lncRNAs can be transcribed as whole or partial natural antisense transcripts (NAT) to coding genes, or located between genes or within introns. Some lncRNAs originate from pseudogenes (Milligan & Lipovich, 2015). lncRNAs may be classified into different subtypes (Antisense, Intergenic, Overlapping, Intronic, Bidirectional, and Processed) according to the position and direction of transcription in relation to other genes (Peschansky & Wahlestedt, 2014, Mattick & Rinn, 2015).
lncRNA expression

Gene expression profiling and in situ hybridization studies have revealed that lncRNA expression is developmentally regulated, can be tissue- and cell-type specific, and can vary spatially, temporally, or in response to stimuli. Many lncRNAs are expressed in a more tissue-specific fashion and with greater variation between tissues compared to protein-coding genes (Derrien et al., 2012).

In general, the expression level of lncRNA is at least one order of magnitude below that of mRNA. Many lncRNAs are located exclusively in the nucleus, but some are cytoplasmic or are located in both nucleus and cytoplasm.
lncRNA functions

To date, very few lncRNAs have been characterized in detail. However, it is clear that lncRNAs are important regulators of gene expression, and lncRNAs are thought to have a wide range of functions in cellular and developmental processes. lncRNAs may carry out both gene inhibition and gene activation through a range of diverse mechanisms, adding yet another layer of complexity to our understanding of genomic regulation. It is estimated that 25 – 40% of coding genes have overlapping antisense transcription, so the impact of lncRNAs on gene regulation is not to be underestimated.

Figure 1

https://www.exiqon.com/ls/PublishingImages/Figures/lncRNA-s.gif

Overview of some of the functions of long non-coding RNA. (Click for a larger image) lncRNAs are involved in gene regulation through a variety of mechanisms. The process of transcription of the lncRNA itself can be a marker of transcription and the resulting lncRNA can function in transcriptional regulation or in chromatin modification (usually via DNA and protein interactions) both in cis and in trans. lncRNAs can bind to complementary RNA and affect RNA processing, turnover or localization. The interaction of lncRNA with proteins can affect protein function and localization as well as facilitate formation of riboprotein complexes. Some lncRNAs are actually precursors for smaller regulatory RNAs such as microRNAs or piwi RNAs. Figure modified from Wilusz et al. Genes Dev. 2009. 23: 1494-1504. PMID: 19571179.

 

lncRNA mechanisms of gene regulation

lncRNAs are not defined by a common mode of action, and can regulate gene expression and protein synthesis in a number of different ways (Figure 1). Some lncRNAs are relatively highly expressed, and appear to function as scaffolds for specialized subnuclear domains. lncRNA possess secondary structures which facilitate their interactions with DNA, RNA and proteins. lncRNA may also bind to DNA or RNA in a sequence-specific manner. Gene regulation may occur in cis (e.g. in close proximity to the transcribed lncRNA) or in trans (at a distance from the transcription site). In the case of chromatin modulation, the effect of lncRNA is typically gene-specific, exerted at a local level (in cis) however regulation of chromatin can also occur in trans.

A few lncRNAs have had their functions experimentally defined and have been shown to be involved in fundamental processes of gene regulation including:

  • Chromatin modification and structure
  • Direct transcriptional regulation
  • Regulation of RNA processing events such as splicing, editing, localization, translation and turnover/degradation
  • Post-translational regulation of protein activity and localization
  • Facilitation of ribonucleoprotein (RNP) complex formation
  • Modulation of microRNA regulation
  • Gene silencing through production of endogenous siRNA (endo-siRNA)
  • Regulation of genomic imprinting

It has recently been attempted to categorize the various types of molecular mechanisms that may be involved in lncRNA function. lncRNAs may be defined as one or more of the following five archetypes:

  • The Signal archetype: functions as a molecular signal or indicator of transcriptional activity.
  • The Decoy archetype: binds to and titrates away other regulatory RNAs (e.g. microRNAs) or proteins (e.g. transcription factors).
  • The Guide archetype: directs the localization of ribonucleoprotein complexes to specific targets (e.g. chromatin modification enzymes are recruited to DNA).
  • The Scaffold archetype: has a structural role as platform upon which relevant molecular components (proteins and or RNA) can be assembled into a complex or spatial proximity.
  • The Enhancer archetype: controls higher order chromosomal looping in an enhancer-like model.

lncRNA and disease

With such a wide range of functions, it is not surprising that lncRNA play a role in the development and pathophysiology of disease. Interestingly, genome wide association studies have demonstrated that most disease variants are located outside of protein-coding genes.

lncRNAs have been found to be differentially expressed in various types of cancer including leukemia, breast cancer, hepatocellular carcinoma, colon cancer, and prostate cancer. Key oncogenes and tumor suppressors including PTEN and KRAS are now known to be regulated by corresponding lncRNA pseudogenes which also act as competing endogenous RNAs (ceRNAs) or microRNA sponges (Poliseno et al., 2010, Johnsson et al., 2013). This highlights the important role that lncRNAs play in oncogenesis.

Other diseases where lncRNAs are dysregulated include cardiovascular diseases, neurological disorders and immune-mediated diseases and genetic disorders. One of the first lncRNA to be discovered was the Xist lncRNA which plays an important role in X chromosome inactivation (Penny et al., 1996), an extreme case of genomic imprinting. lncRNAs are present at almost all imprinted loci, arguing for an important role for lncRNAs in this form of epigenetic regulation.

lncRNAs represent a gold mine of potential new biomarkers and drug targets, as well as a step change in the way we understand mechanisms of disease.
The challenges of studying lncRNA

Only a relatively small proportion of lncRNAs have so far been investigated and although we can start to classify different types of lncRNA functions, we are still far from being able to predict the function of new lncRNAs. This is mainly due to the fact that unlike protein-coding genes whose sequence motifs are indicative of their function, lncRNA sequences are not usually conserved and they don’t tend to contain conserved motifs. Other differences between lncRNA and mRNA are summarized in Table 1.

The main challenges of working with lncRNA are the fact that they can be present in very low amounts (typically an order of magnitude lower than mRNA expression levels), can overlap with coding transcripts on both strands and are often restricted to the nucleus.

Table 1

mRNA lncRNA
Tissue-specific expression Tissue-specific expression
Form secondary structure Form secondary structure
Undergo post-transcriptional processing, i.e. 5’cap, polyadenylation, splicing Undergo post-transcriptional processing, i.e. 5’cap, polyadenylation, splicing
Important roles in diseases and development Important roles in diseases and development
Protein coding transcript Non-protein coding, regulatory functions
Well conserved between species Poorly conserved between species
Present in both nucleus and cytoplasm Many predominantly nuclear, others nuclear and/or cytoplasmic
Total 20-24,000 mRNAs Currently ~30,000 lncRNA transcripts, predicted 3-100 fold of mRNA in number
Expression level: low to high Expression level: very low to moderate

Similarities and differences (dark) between mRNA and lncRNA

 

ncRNA discovery and profiling using Next Generation Sequencing

Expression profiling is one way to start to uncover the function of lncRNA. Identifying lncRNAs that are differentially expressed during development or in particular situations can shed light on their potential functions. Alternatively, looking for lncRNAs and protein-coding genes whose expression is correlated, can perhaps indicate co-regulation or related functions.

Whole transcriptome RNA sequencing is the method of choice for comprehensive lncRNA expression profiling, including the discovery of novel lncRNAs. Whole transcriptome sequencing enables the characterization of all RNA transcripts, including both the coding mRNA and non-coding RNA larger than 170 nucleotides in length, regardless of whether they are polyadenylated or not.

Exiqon offers a comprehensive whole transcriptome NGS Service including everything from RNA isolation to the final report including advanced data analysis and interpretation.
Advantages of LNA™-enhanced research tools for lncRNA

Exiqon offer a broad range of sensitive and specific tools specifically designed to address the challenges faced when investigating lncRNA expression and function. Exiqon’s tools are based on the Locked nucleic acid (LNA™) technology. LNA™ is a class of high-affinity RNA analogs that exhibit unprecedented thermal stability when hybridized to a complementary DNA or RNA strand. Hence, LNA™ enables superior sensitivity and specificity in any hybridization-based approach. We continue to use the LNA™ technology to develop new and innovative ways to improve our understanding of lncRNAs in this rapidly developing field.

Functional analysis of lncRNAs has been revolutionized by the development of Antisense LNA™ GapmeRs which enable efficient silencing of lncRNA both in vitro and in vivo. Exiqon also offer tools to investigate lncRNA function in other ways, for example using LNA™ oligos to block interactions between lncRNA and DNA, RNA or proteins. ExiLERATE LNA™ qPCR assays have been developed to enable robust detection of even low abundance and challenging lncRNAs by qPCR. Precise subcellular localization of lncRNAs can be studied using LNA™ probes for in situ hybridization.
Silencing lncRNA to disrupt their function

One strategy to study the function of lncRNA is to silence them using specific and potent antisense oligonucleotides (Antisense LNA™ GapmeRs).

The nuclear localization of many lncRNAs has meant that siRNA approaches to knockdown lncRNA, have met with limited success. The double-stranded siRNA duplex has difficulty crossing the nuclear membrane and the passenger strand (non-targeting sequence) of the duplex can often elicit its own effect, confounding interpretation of results.

Antisense LNA™ GapmeRs overcome this challenge by enabling highly efficient RNase H mediated silencing of all lncRNA. RNase H is present both in the cytoplasm and in the nucleus and it has been shown that LNA™ Gapmers offer significantly better knockdown of nuclear targets than siRNA mediated silencing. In addition, the single stranded LNA™ GapmeRs are an advantage for lncRNAs that are transcribed as antisense transcripts to coding genes because there is no second strand that could compromise specificity.

Antisense LNA™ GapmeRs show high potency in a broad range of tissues in vivo when administered systemically without formulation in animal models. This makes LNA™ GapmeRs very promising antisense drugs for lncRNA targets in the future.
Studying lncRNA interactions with DNA, RNA and proteins

The fact that the molecular mechanism of lncRNAs often relies on sequence specific interaction with DNA, RNA or proteins means that it is possible to design highly specific LNA™-oligonucleotides that can be used to inhibit these interactions and thereby reveal the details of how lncRNAs function. Please contact us and our experts can help you with the design of custom LNA™ oligonucleotides for studying lncRNA interactions.
lncRNA analysis by qPCR

Short, high affinity, LNA™-enhanced qPCR primers offer an advantage for the detection of low abundance targets. In addition, the use of LNA™ to adjust primer melting temperature provides greater flexibility in primer design which is important for qPCR analysis of overlapping transcripts. The ExiLERATE LNA™ qPCR System offers a sophisticated primer design tool combined with highly sensitive and specific qPCR assays for any RNA target.

ExiLERATE LNA™ qPCR assays are ideal to monitor the efficiency of LNA™ GapmeR-mediated RNA knockdown. Validated LNA™ qPCR primer sets are available to detect the lncRNA targeted by Antisense LNA™ GapmeR positive controls.

ExiLERATE LNA™ qPCR primer sets also provide a convenient way to validate RNA sequencing data. Our advanced online design algorithm can design LNA™ qPCR assays for novel lncRNA transcripts, isoforms or splice variants. LNA™qPCR assays for multiple lncRNAs can easily be designed using the batch mode function in our online design algorithm.
Subcellular localization of lncRNA expression by in situ hybridization

Understanding the subcellular localization of a lncRNA is important information when starting to hypothesize the potential functions that the lncRNA may be performing. LNA™-enhanced probes for in situ hybridization have increased affinity for their target sequence and offer increased sensitivity and increased signal to noise ratio, which is important for detection of rare targets such as lncRNA.

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The Reconstruction of Life Processes requires both Genomics and Metabolomics to explain Phenotypes and Phylogenetics

Writer and Curator: Larry H. Bernstein, MD, FCAP 

 

phylogenetics

phylogenetics

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This discussion that completes and is an epicrisis (summary and critical evaluation) of the series of discussions that preceded it.

  1. Innervation of Heart and Heart Rate
  2. Action of hormones on the circulation
  3. Allogeneic Transfusion Reactions
  4. Graft-versus Host reaction
  5. Unique problems of perinatal period
  6. High altitude sickness
  7. Deep water adaptation
  8. Heart-Lung-and Kidney
  9. Acute Lung Injury

The concept inherent in this series is that the genetic code is an imprint that is translated into a message.  It is much the same as a blueprint, or a darkroom photographic image that has to be converted to a print. It is biologically an innovation of evolutionary nature because it establishes a simple and reproducible standard for the transcription of the message through the transcription of the message using strings of nucleotides (oligonucleotides) that systematically transfer the message through ribonucleotides that communicate in the cytoplasm with the cytoskeleton based endoplasmic reticulum (ER), composing a primary amino acid sequence.  This process is a quite simple and convenient method of biological activity.  However, the simplicity ends at this step.  The metabolic components of the cell are organelles consisting of lipoprotein membranes and a cytosol which have particularly aligned active proteins, as in the inner membrane of the mitochondrion, or as in the liposome or phagosome, or the structure of the  ER, each of which is critical for energy transduction and respiration, in particular, for the mitochondria, cellular remodeling or cell death, with respect to the phagosome, and construction of proteins with respect to the ER, and anaerobic glycolysis and the hexose monophosphate shunt in the cytoplasmic domain.  All of this refers to structure and function, not to leave out the membrane assigned transport of inorganic, and organic ions (electrolytes and metabolites).

I have identified a specific role of the ER, the organelles, and cellular transactions within and between cells that is orchestrated.  But what I have outlined is a somewhat limited and rigid model that does not reach into the dynamics of cellular transactions.  The DNA has expression that may be old, no longer used messages, and this is perhaps only part of a significant portion of “dark matter”.  There is also nuclear DNA that is enmeshed with protein, mRNA that is a copy of DNA, and mDNA  is copied to ribosomal RNA (rRNA).  There is also rDNA. The classic model is DNA to RNA to protein.  However, there is also noncoding RNA, which plays an important role in regulation of transcription.

This has been discussed in other articles.  But the important point is that proteins have secondary structure through disulfide bonds, which is determined by position of sulfur amino acids, and by van der Waal forces, attraction and repulsion. They have tertiary structure, which is critical for 3-D structure.  When like subunits associate, or dissimilar oligomers, then you have heterodimers and oligomers.  These constructs that have emerged over time interact with metabolites within the cell, and also have an important interaction with the extracellular environment.

When you take this into consideration then a more complete picture emerges. The primitive cell or the multicellular organism lives in an environment that has the following characteristics – air composition, water and salinity, natural habitat, temperature, exposure to radiation, availability of nutrients, and exposure to chemical toxins or to predators.  In addition, there is a time dimension that proceeds from embryonic stage to birth in mammals, a rapid growth phase, a tapering, and a decline.  The time span is determined by body size, fluidity of adaptation, and environmental factors.  This is covered in great detail in this work.  The last two pieces are in the writing stage that completes the series. Much content has already be presented in previous articles.

The function of the heart, kidneys and metabolism of stressful conditions have already been extensively covered in http://pharmaceuticalintelligence.com  in the following and more:

The Amazing Structure and Adaptive Functioning of the Kidneys: Nitric Oxide – Part I

https://pharmaceuticalintelligence.com/2012/11/26/the-amazing-structure-and-adaptive-functioning-of-the-kidneys/

Nitric Oxide and iNOS have Key Roles in Kidney Diseases – Part II

https://pharmaceuticalintelligence.com/2012/11/26/nitric-oxide-and-inos-have-key-roles-in-kidney-diseases/

The pathological role of IL-18Rα in renal ischemia/reperfusion injury – Nature.com

https://pharmaceuticalintelligence.com/2014/10/24/the-pathological-role-of-il-18r%CE%B1-in-renal-ischemiareperfusion-injury-nature-com/

Summary, Metabolic Pathways

https://pharmaceuticalintelligence.com/2014/10/23/summary-metabolic-pathways/

 

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Larry H Bernstein, MD, FCAP, Author and Curator

Chief, Scientific Communication

Leaders in Pharmaceutical Intelligence

with contributions from JEDS Rosalis, Brazil
and Radislov Rosov, Univ of Virginia, VA, USA

A Brief Curation of Proteomics, Metabolomics, and Metabolism

This article is a continuation of a series of elaborations of the recent and
accelerated scientific discoveries that are enlarging the scope of and
integration of biological and medical knowledge leading to new drug
discoveries.  The work that has led us to this point actually has roots
that go back 150 years.  The roots go back to studies in the mid-nineteenth century, with the emergence of microbiology, physiology,
pathology, botany, chemistry and physics, and the laying down of a
mechanistic approach divergent from descriptive observation in the
twentieth century. Medicine took on the obligation to renew the method
of training physicians after the Flexner Report (The Flexner Report of
1910 transformed the nature and process of medical education in America
with a resulting elimination of proprietary schools), funded by the Carnegie
Foundation.  Johns Hopkins University Medical School became the first to
adopt the model, as did Harvard, Yale, University of Chicago, and others.

The advances in biochemistry, genetics and genomics, were large, as was
structural organic chemistry in the remainder of the centrury.  The advances
in applied mathematics and in instrumental analysis opened a new gateway
into the 21st century with the Human Genome Project, the Proteome Library,
Signaling Pathways, and the Metabolomes – human, microbial, and plants.

shall elaborate on how the key processes of life are being elucidated as
these interrelated disciplines converge.  I shall not be covering in great
detail the contribution of the genetic code and transcripton because they
have been covered at great length in this series.

Part I.  The foundation for the emergence of a revitalized molecular
biology 
and biochemistry.

In a series of discussions with Jose des Salles Roselino (Brazil) over a
period of months we have come to an important line of reasoning. DNA
to protein link goes from triplet sequence to amino acid sequence. The
realm of genetics. Further, protein conformation, activity and function
requires that environmental and microenvironmental factors should be
considered (Biochemistry).  This has been opened in several articles
preceding this.

In the cAMP coupled hormonal response the transfer of conformation
from protein to protein is paramount. For instance, if your scheme goes
beyond cAMP, it will show an effect over a self-assembly (inhibitor
protein and protein kinase). Therefore, sequence alone does not
explain conformation, activity and function of regulatory proteins.
Recall that sequence is primar structure, determined by the translation
of the code, but secondary structure is determined by disulfide bonds.
There is another level of structure, tertiary structure, that is molded by
steric influences of near neighbors and by noncovalent attractions
and repulsions.

A few comments ( contributed by Assoc. Prof. JEDS Roselino) are in
order to stress the importance of self-assembly (Prigogine, R. A
Marcus, conformation energy) in a subject that is the best for this
connection. We have to stress again that in the cAMP
coupled hormonal response the transfer of conformation from
protein to protein is paramount. For instance, in case the
reaction sequence follows beyond the production of the
second messenger, as in the case of cAMP, this second
messenger will remove a self-assembly of inhibitor protein
with the enzyme protein kinase. Therefore, sequence alone
does not explain conformation, activity and function of
regulatory proteins. In this case, if this important mechanism
was not ignored, the work of Stanley Prusiner would most
certainly have been recognized earlier, and “rogue” proteins
would not have been seen as so rogue as some assumed.
For the general idea of importance of self-assembly versus
change in covalent modification of proteins (see R. A Kahn
and A. G Gilman (1984) J. Biol. Chem.  259(10), pp 6235-
6240. In this case, trimeric or dimeric G does not matter.
“Signaling transduction tutorial”.
G proteins in the G protein coupled-receptor proteins are
presented following a unidirectional series of arrows.
This is adequate to convey the idea of information being
transferred from outside the cell towards cell´s interior
(therefore, against the dogma that says all information
moves from DNA to RNA to protein.  It is important to
consider the following: The entire process is driven by
a very delicate equilibrium between possible conform-
ational states of the proteins. Empty receptors have very
low affinity for G proteins. On the other hand, hormone
bound receptors have a change in conformation that
allows increasing the affinity for the G-trimer. When
hormone receptors bind to G-trimers two things happen:

  1. Receptors transfer conformation information to
    the G-triplex and
  2. the G-triplex transfers information back to the
    complex hormone-receptor.

In the first case , the dissociated G protein exchanges
GDP for GTP and has its affinity for the cyclase increased,
while by the same interaction receptor releases the
hormone which then places the first required step for the
signal. After this first interaction step, on the second and
final transduction system step is represented by an
opposite arrow. When, the G-protein + GTP complex
interacts with the cyclase two things happen:

  1. It changes the cyclase to an active conformation
    starting the production of cAMP as the single
    arrow of the scheme. However, the interaction
    also causes a backward effect.
  2. It activates the GTPase activity of this subunit
    and the breakdown of GTP to GDP moves this 
    subunit back to the initial trimeric inactive
    state
     of G complex.

This was very well studied when the actions of cholera toxin
required better understanding. Cholera toxin changes the
GTPase subunit by ADP-ribosilation (a covalent and far more
stable change in proteins) producing a permanent conformation
of GTP bound G subunit. This keeps the cyclase in permanent
active conformation because ADP-ribosilation inhibits GTPase
activity required to put an end in the hormonal signal.

The study made while G-proteins were considered a dimer still
holds despite its limited vision of the real complexity of the
transduction system. It was also possible to get this very same
“freezing” in the active state using GTP stable analogues. This
transduction system is one of the best examples of the delicate
mechanisms of conformational interaction of proteins. Further-
more, this system also shows on the opposite side of our
reasoning scheme, how covalent changes are adequate for
more stable changes than those mediated by Van der Wall’s
forces between proteins. Yet, these delicate forces are the
same involved when Sc-Prion transfers its rogue
conformation to c-Prion proteins and other similar events.
The Jacob-Monod Model

A combination of genetic and biochemical experiments in
bacteria led to the initial recognition of

  1. protein-binding regulatory sequences associated with genes and
  2. proteins whose binding to a gene’s regulatory sequences
    either activate or repress its transcription.

These key components underlie the ability of both prokaryotic and
eukaryotic cells to turn genes on and off. The  experimental findings lead to a general model of bacterial transcription control.

Gene control serves to allow a single cell to adjust to changes in its
nutritional environment so that its growth and division can be optimized.
Thus, the prime focus of research has been on genes that encode
inducible proteins whose production varies depending on the nutritional
status of the cells. Its most characteristic and biologically far-reaching
purpose in eukaryotes, distinctive from single cell organisms is the
regulation of a genetic program that underlies embryological
development and tissue differentiation.

The principles of transcription have already been described in this
series under the translation of the genetic code into amino acids
that are the building blocks for proteins.

E.coli can use either glucose or other sugars such as the
disaccharide lactose as the sole source of carbon and energy.
When E. coli cells are grown in a glucose-containing medium,
the activity of the enzymes needed to metabolize lactose is
very low. When these cells are switched to a medium
containing lactose but no glucose, the activities of the lactose-metabolizing enzymes increase. Early studies showed that the
increase in the activity of these enzymes resulted from the
synthesis of new enzyme molecules, a phenomenon termed
induction. The enzymes induced in the presence of lactose
are encoded by the lac operon, which includes two genes, Z
and Y, that are required for metabolism of lactose and a third
gene. The lac Y gene encodes lactose permease, which spans the E. coli cell membrane and uses the energy available from
the electrochemical gradient across the membrane to pump
lactose into the cell. The lac Z gene encodes β-galactosidase,
which splits the disaccharide lactose into the monosaccharides
glucose and galactose, which are further metabolized through
the action of enzymes encoded in other operons. The third
gene encodes thiogalactoside transacetylase.

Synthesis of all three enzymes encoded in the lac operon is rapidly
induced when E. coli cells are placed in a medium containing lactose
as the only carbon source and repressed when the cells are switched
to a medium without lactose. Thus all three genes of the lac operon
are coordinately regulated. The lac operon in E. coli provides one
of the earliest and still best-understood examples of gene control.
Much of the pioneering research on the lac operon was conducted by
Francois Jacob, Jacques Monod, and their colleagues in the 1960s.

Some molecules similar in structure to lactose can induce expression
of the lacoperon genes even though they cannot be hydrolyzed by β-galactosidase. Such small molecules (i.e., smaller than proteins) are
called inducers. One of these, isopropyl-β-D-thiogalactoside,
abbreviated IPTG,is particularly useful in genetic studies of the lac
operon, because it can diffuse into cells and, it is not metabolized.
Insight into the mechanisms controlling synthesis of β-galactosidase
and lactose permease came from the study of mutants in which control
of β-galactosidase expression was abnormal and used a colorimetric
assay for β-galactosidase.

When the cells are exposed to chemical mutagens before plating on
X-gal/glucose plates, rare blue colonies appear, but when cells
from these blue colonies are recovered and grown in media containing
glucose, they overexpress all the genes of the lac operon. These cells
are called constitutive mutants because they fail to repress the lac
operon in media lacking lactose and instead continuously express the
enzymes, and the genes were mapped to a region on the E. coli
chromosome. This led to the conclusion that these cells had a defect
in a protein that normally repressed expression of the lac operon in
the absence of lactose, and that it blocks transcription by binding to
a site on the E. coli genome where transcription of the lac operon is
initiated. In addition, it binds to the lac repressor in the lactose
medium and decreases its affinity for the repressor-binding site
on the DNA causing the repressor to unbind the DNA. Thereby,
transcription of the lac operon is initiated, leading to synthesis of
β-galactosidase, lactose permease, and thiogalactoside
transacetylase.

 regulation of the lac operon by lac repressor

Jacob and Monod model of transcriptional regulation of the lac operon

Next, Jacob and Monod isolated mutants that expressed the lac operon
constitutively even when two copies of the wild-type lacI gene
encoding the lac repressor were present in the same cell, and the
constitutive mutations mapped to one end of the lac operon, as the
model predicted.  Further, there are rare cells that carry a mutation
located at the region, promoter, that block initiation of transcription by
RNA polymerase.

lac I+ gene is trans-acting, & encodes a protein, which binds to a lac operator

 lac I+ gene is trans-acting, & encodes a protein, which
binds to a lac operator

They further demonstrated that the two types of mutations lac I and
lac I+, were cis- and trans-acting, the latter encoding a protein that
binds to the lac operator. The cis-acting Oc mutations prevent
binding of the lac repressor to the operator, and  mutations in the
lac promoter are cis-acting, since they alter the binding site for RNA
polymerase. In general, trans-acting genes that regulate expression
of genes on other DNA molecules encode diffusible products. In
most cases these are proteins, but in some cases RNA molecules
can act in trans to regulate gene expression.

According to the Jacob and Monod model of transcriptional control,
transcription of the lac operon, which encodes three inducible
proteins, is repressed by binding of lac repressor protein to the
operator sequence.

 (Section 10.1Bacterial Gene Control: The Jacob-Monod Model.)
This book is accessible by the search feature.

Comment: This seminal work was done a half century ago. It was a
decade after the Watson-Crick model for DNA. The model is
elaborated for the Eukaryote in the examples that follow.

(The next two articles were called to my attention by R. Bosov at
University of Virginia).

An acetate switch regulates stress erythropoiesis

M Xu,  JS Nagati, Ji Xie, J Li, H Walters, Young-Ah Moon, et al.
Nature Medicine 10 Aug 2014(20): 1018–1026.
http://dx.doi.org:/10.1038/nm.3587

message: 1- ( -CH3 ) = Ln ( (1/sqrt(1-Acetate^2) –
sqrt oxalate))/ Ln(oxygen) – K(o)
rsb5n@virginia.edu

The hormone erythropoietin (EPO), synthesized in the kidney or liver
of adult mammals, controls erythrocyte production and is regulated by
the stress-responsive transcription factor hypoxia-inducible factor-2
(HIF-2).
 HIFα acetylation and efficient HIF-2–dependent EPO
induction during hypoxia requires  the lysine acetyltransferase CREB-binding protein (CBP) . These processes require acetate-dependent
acetyl CoA synthetase 2 (ACSS2) as follows.Acetate levels rise and
ACSS2 is required for HIF-2α acetylation, CBP–HIF-2α complex
formation, CBP–HIF-2α recruitment to the EPO enhancer and induction
of EPO gene expression
 in human Hep3B hepatoma cells and in EPO-generating organs of hypoxic or acutely anemic mice. In acutely anemic
mice, acetate supplementation augments stress erythropoiesis in an
ACSS2-dependent manner. Moreover, in acquired and inherited
chronic anemia mouse models, acetate supplementation increases
EPO expression
 and the resting hematocrit. Thus, a mammalian
stress-responsive acetate switch controls HIF-2 signaling and EPO
induction during pathophysiological states marked by tissue hypoxia.

Figure 1: Acss2 controls HIF-2 signaling in hypoxic cells.
Time course of endogenous HIF-2α acetylation during hypoxia following
immunoprecipitation (IP) of HIF-2α from whole-cell extracts and detection
of acetylated lysines by immunoblotting (IB).
http://www.nature.com/nm/journal/v20/n9/carousel/nm.3587-F1.jpg

Figure 2: Acss2 regulates hypoxia-induced renal Epo expression in mice.
http://www.nature.com/nm/journal/v20/n9/carousel/nm.3587-F2.jpg

Figure 3: Acute anemia induces Acss2-dependent HIF-2 signaling in mice.
http://www.nature.com/nm/journal/v20/n9/carousel/nm.3587-F3.jpg

Figure 4: An acetate switch regulates Cbp–HIF-2 interactions in cells.
(a) HIF-2α acetylation following immunoprecipitation of endogenous
HIF-2α and detection by immunoblotting with antibodies to acetylated
lysine or HIF-2α.
http://www.nature.com/nm/journal/v20/n9/carousel/nm.3587-F4.jpg

Figure 5: Acss2 signaling in cells requires intact HIF-2 acetylation.
http://www.nature.com/nm/journal/v20/n9/carousel/nm.3587-F5.jpg

Figure 6: Acetate facilitates recovery from anemia.

Acetate facilitates recovery from anemia

Acetate facilitates recovery from anemia

(a) Serial hematocrits of CD1 wild-type female mice after PHZ treatment, followed
by once daily per os (p.o.) supplementation with water vehicle (Veh; n = 7 mice),
GTA (n = 6 mice), GTB (n = 8 mice) or GTP (n = 7 mice) (single measurem…

http://www.nature.com/nm/journal/v20/n9/carousel/nm.3587-F6.jpg

see also-.
1. Bunn, H.F. & Poyton, R.O. Oxygen sensing and molecular adaptation to
hypoxia. Physiol. Rev. 76, 839–885 (1996).

  1. .Richalet, J.P. Oxygen sensors in the organism: examples of regulation
    under altitude hypoxia in mammals. Comp. Biochem. Physiol. A Physiol.
    118, 9–14 (1997).
  2. .Koury, M.J. Erythropoietin: the story of hypoxia and a finely regulated
    hematopoietic hormone. Exp. Hematol. 33, 1263–1270 (2005).
  3. Wang, G.L., Jiang, B.H., Rue, E.A. & Semenza, G.L. Hypoxia-inducible
    factor 1 is a basic-helix-loop-helix-PAS heterodimer regulated
    by cellular O2 tension. Proc. Natl. Acad. Sci. USA92, 5510–5514 (1995).
  4. Chen, R. et al. The acetylase/deacetylase couple CREB-binding
    protein/sirtuin 1 controls hypoxia-inducible factor 2 signaling. J. Biol.
    Chem. 287, 30800–30811 (2012).
  5. .Papandreou, I., Cairns, R.A., Fontana, L., Lim, A.L. & Denko, N.C.
    HIF-1 mediates adaptation to hypoxia by actively down-regulating
    mitochondrial oxygen consumption. Cell Metab. 3,187–197 (2006).

14. Kim, J.W., Tchernyshyov, I., Semenza, G.L. & Dang, C.V. HIF-1-
mediated expression of pyruvate dehydrogenase kinase: a metabolic
switch required for cellular adaptation to hypoxia. Cell Metab. 3,
177–185 (2006).

16. Fujino, T., Kondo, J., Ishikawa, M., Morikawa, K. & Yamamoto, T.T.
Acetyl-CoA synthetase 2, a mitochondrial matrix enzyme involved in the
oxidation of acetate. J. Biol. Chem. 276,11420–11426 (2001).

17..Luong, A., Hannah, V.C., Brown, M.S. & Goldstein, J.L. Molecular
characterization of human acetyl-CoA synthetase, an enzyme regulated
by sterol regulatory element-binding proteins. J. Biol. Chem. 275,
26458–26466 (2000).

20 .Wellen, K.E. et al. ATP-citrate lyase links cellular metabolism to
histone acetylation. Science324, 1076–1080 (2009).

24. McBrian, M.A. et al. Histone acetylation regulates intracellular pH.
Mol. Cell 49, 310–321(2013).

Asymmetric mRNA localization contributes to fidelity and sensitivity
of spatially localized systems

Robert J Weatheritt, Toby J Gibson & M Madan Babu
Nature Structural & Molecular Biology 21, 833–839 (2014)
http://www.nature.com/nsmb/journal/v21/n9/abs/nsmb.2876.html 

Although many proteins are localized after translation, asymmetric
protein distribution is also achieved by translation after mRNA localization.
Why are certain mRNA transported to a distal location and translated
on-site? Here we undertake a systematic, genome-scale study of
asymmetrically distributed protein and mRNA in mammalian cells.
Our findings suggest that asymmetric protein distribution by mRNA
localization enhances interaction fidelity and signaling sensitivity
.
Proteins synthesized at distal locations frequently contain intrinsically
disordered segments. These regions are generally rich in assembly-
promoting modules and are often regulated by post-translational
modifications. Such proteins are tightly regulated but display distinct
temporal dynamics upon stimulation with growth factors. Thus, proteins
synthesized on-site may rapidly alter proteome composition and
act as dynamically regulated scaffolds to promote the formation
of reversible cellular assemblies. 
Our observations are consistent
across multiple mammalian species, cell types and developmental stages,
suggesting that localized translation is a recurring feature of cell
signaling and regulation.

Figure 1: Classification and characterization of TAS and DSS proteins.

The two major mechanisms for localizing proteins to distal sites in the cell

The two major mechanisms for localizing proteins to distal sites in the cell

(a)The two major mechanisms for localizing proteins to distal sites in the cell.
(b) Data sets used to identify groups of DSS and TAS transcripts, as well as
DSS and TAS proteins in mouse neuroblastoma cells

http://www.nature.com/nsmb/journal/v21/n9/carousel/nsmb.2876-F1.jpg

Figure 2: Structural analysis of DSS proteins reveals an enrichment
in disordered regions.

Distributions of the various structural properties of the DSS and TAS proteins of the mouse neuroblastoma data sets

Distributions of the various structural properties of the DSS and TAS proteins of the mouse neuroblastoma data sets

(a,b) Distributions of the various structural properties of the DSS and TAS
proteins of the mouse neuroblastoma data sets (a), the mouse pseudopodia,
the rat embryonic sensory neuron data set and the adult sensory neuron data set (b).…

http://www.nature.com/nsmb/journal/v21/n9/carousel/nsmb.2876-F2.jpg

Figure 3: Analysis of DSS proteins reveals an enrichment for linear motifs, phase-
transition (i.e., higher-order assembly) promoting segments and PTM sites that act
as molecular switches.

(a,b) Distributions of the various regulatory and structural properties of the DSS
and TAS proteins of the mouse neuroblastoma data sets
http://www.nature.com/nsmb/journal/v21/n9/carousel/nsmb.2876-F3.jpg

Figure 4: Dynamic regulation of DSS transcripts and proteins.

Dynamic regulation of DSS transcripts and proteins

Dynamic regulation of DSS transcripts and proteins

Genome-wide quantitative measurements of gene expression of DSS (n = 289)
and TAS (n = 1,292) proteins in mouse fibroblast cells. DSS transcripts and
proteins have a lower abundance and shorter half-lives

http://www.nature.com/nsmb/journal/v21/n9/carousel/nsmb.2876-F4.jpg

Figure 5: An overview of the potential advantages conferred by distal-site protein
synthesis, inferred from our analysis.

An overview of the potential advantages conferred by distal-site protein synthesis, inferred from our analysis

An overview of the potential advantages conferred by distal-site protein synthesis, inferred from our analysis

Turquoise and red filled circle represents off-target and correct interaction partners,
respectively. Wavy lines – a disordered region within a distal site synthesis protein.

http://www.nature.com/nsmb/journal/v21/n9/carousel/nsmb.2876-F5.jpg

The identification of asymmetrically localized proteins and transcripts.

The identification of asymmetrically localized proteins and transcripts

The identification of asymmetrically localized proteins and transcripts

An illustrative explanation of the resolution of the study and the concept of asymmetric
localization of proteins and mRNA. In this example, on the left a neuron is divided into
its cell body and axon terminal, and transcriptome/proteo…

http://www.nature.com/nsmb/journal/v21/n9/carousel/nsmb.2876-SF1.jpg

Graphs and boxplots of functional and structural properties for distal site synthesis
(DSS) proteins (red) and transport after synthesis (TAS) proteins (gray).
See Online Methods for details and legend of Figure 2 for a description of boxplots
and statistical tests.
http://www.nature.com/nsmb/journal/v21/n9/carousel/nsmb.2876-SF2.jpg

See also –
1. Martin, K.C. & Ephrussi, A. mRNA localization: gene expression in the spatial
dimension. Cell136, 719–730 (2009).

  1. Scott, J.D. & Pawson, T. Cell signaling in space and time: where proteins come
    together and when they’re apart. Science 326, 1220–1224 (2009).

4..Holt, C.E. & Bullock, S.L. Subcellular mRNA localization in animal cells
and why it matters.Science 326, 1212–1216 (2009).

  1. Jung, H., Gkogkas, C.G., Sonenberg, N. & Holt, C.E. Remote control of
    gene function by local translation. Cell 157, 26–40 (2014). 

Regulation of metabolism by hypoxia-inducible factor 1.   
Semenza GL.    Author information
Cold Spring Harb Symp Quant Biol. 2011;76:347-53.
http://dx.doi.org:/10.1101/sqb.2011.76.010678.

The maintenance of oxygen homeostasis is critical for survival, and the
master regulator of this process in metazoan species is hypoxia-inducible
factor 1 (HIF-1), which

  • controls both O(2) delivery and utilization.

Under conditions of reduced O(2) availability,

  • HIF-1 activates the transcription of genes, whose protein products
  • mediate a switch from oxidative to glycolytic metabolism.

HIF-1 is activated in cancer cells as a result of intratumoral hypoxia
and/or genetic alterations.

In cancer cells, metabolism is reprogrammed to

  • favor glycolysis even under aerobic conditions.

Pyruvate kinase M2 (PKM2) has been implicated in cancer growth and
metabolism, although the mechanism by which it exerts these effects is
unclear. Recent studies indicate that

PKM2 interacts with HIF-1α physically and functionally to

  1. stimulate the binding of HIF-1 at target genes,
  2. the recruitment of coactivators,
  3. histone acetylation, and
  4. gene transcription.

Interaction with HIF-1α is facilitated by

  • hydroxylation of PKM2 at proline-403 and -408 by PHD3.

Knockdown of PHD3

  • decreases glucose transporter 1, lactate dehydrogenase A, and
    pyruvate dehydrogenase kinase 1 expression;
  • decreases glucose uptake and lactate production; and
  • increases O(2) consumption.

The effect of PKM2/PHD3 is not limited to genes encoding metabolic
enzymes because VEGF is similarly regulated.

These results provide a mechanism by which PKM2

  • promotes metabolic reprogramming and

suggest that it plays a broader role in cancer progression than has
previously been appreciated.   PMID: 21785006   

Cadherins

Cadherins are thought to be the primary mediators of adhesion
between the cells
 of vertebrate animals, and also function in cell
adhesion in many invertebrates. The expression of numerous cadherins
during development is highly regulated, and the precise pattern of
cadherin expression plays a pivotal role in the morphogenesis of tissues
and organs. The cadherins are also important in the continued maintenance
of tissue structure and integrity. The loss of cadherin expression appears
to be highly correlated with the invasiveness of some types of tumors. Cadherin adhesion is also dependent on the presence of calcium ions
in the extracellular milieu.

The cadherin protein superfamily, defined as proteins containing a
cadherin-like domain, can be divided into several sub-groups. These include

  • the classical (type I) cadherins, which mediate adhesion at adherens junctions;
  • the highly-related type II cadherins;
  • the desmosomal cadherins found in desmosome junctions;
  • protocadherins, expressed only in the nervous system; and
  • atypical cadherin-like domain containing proteins.

Members of all but the atypical group have been shown to play a role
in intercellular adhesion.

Part II.  PKM2 and regulation of glycolysis

PKM2 regulates the Warburg effect and promotes ​HMGB1
release in sepsis

L Yang, M Xie, M Yang, Y Yu, S Zhu, W Hou, R Kang, …, & D Tang
Nature Communic 14 July 2014; 5(4436)
http://dx.doi.org/doi:10.1038/ncomms5436

Increasing evidence suggests the important role of metabolic reprogramming

  • in the regulation of the innate inflammatory response,

We provide evidence to support a novel role for the

  • ​pyruvate kinase M2 (​PKM2)-mediated Warburg effect,

namely aerobic glycolysis,

  • in the regulation of ​high-mobility group box 1 (​HMGB1) release. ​
  1. PKM2 interacts with ​hypoxia-inducible factor 1α (​HIF1α) and
  2. activates the ​HIF-1α-dependent transcription of enzymes necessary
    for aerobic glycolysis in macrophages.

Knockdown of ​PKM2, ​HIF1α and glycolysis-related genes

  • uniformly decreases ​lactate production and ​HMGB1 release.

Similarly, a potential ​PKM2 inhibitor, ​shikonin,

  1. reduces serum ​lactate and ​HMGB1 levels, and
  2. protects mice from lethal endotoxemia and sepsis.

Collectively, these findings shed light on a novel mechanism for

  • metabolic control of inflammation by
  • regulating ​HMGB1 release and

highlight the importance of targeting aerobic glycolysis in the treatment
of sepsis and other inflammatory diseases.

  1. Glycolytic inhibitor ​2-D G attenuates ​HMGB1 release by activated macrophages.
    http://www.nature.com/ncomms/2014/140714/ncomms5436/carousel/ncomms5436-f1.jpg
  2. Figure 2: Upregulated ​PKM2 promotes aerobic glycolysis and ​HMGB1
    release in activated macrophages.
    http://www.nature.com/ncomms/2014/140714/ncomms5436/carousel/ncomms5436-f2.jpg
  3. Figure 3: ​PKM2-mediated ​HIF1α activation is required for ​HMGB1
    release in activated macrophages.
    http://www.nature.com/ncomms/2014/140714/ncomms5436/carousel/ncomms5436-f3.jpg

 

ERK1/2-dependent phosphorylation and nuclear translocation of
PKM2 promotes the Warburg effect  

W Yang, Y Zheng, Y Xia, Ha Ji, X Chen, F Guo, CA Lyssiotis, & Zhimin Lu
Nature Cell Biology  2012 (27 June 2014); 14: 1295–1304
Corrigendum (January, 2013)  http://dx.doi.org:/10.1038/ncb2629

Pyruvate kinase M2 (PKM2) is upregulated in multiple cancer types and
contributes to the Warburg. We demonstrate that

  • EGFR-activated ERK2 binds directly to PKM2 Ile 429/Leu 431
  • through the ERK2 docking groove
  • and phosphorylates PKM2 at Ser 37, but
  • does not phosphorylate PKM1.

Phosphorylated PKM2 Ser 37

  1. recruits PIN1 for cis–trans isomerization of PKM2, which
  2. promotes PKM2 binding to importin α5
  3. and PKM2 translocates to the nucleus.

Nuclear PKM2 acts as

  • a coactivator of β-catenin to
  • induce c-Myc expression,

This is followed by

  1. the upregulation of GLUT1, LDHA and,
  2. in a positive feedback loop,
  • PTB-dependent PKM2 expression.

Replacement of wild-type PKM2 with

  • a nuclear translocation-deficient mutant (S37A)
  • blocks the EGFR-promoted Warburg effect
    and brain tumour development in mice.

In addition, levels of PKM2 Ser 37 phosphorylation

  • correlate with EGFR and ERK1/2 activity
    in human glioblastoma specimens.

Our findings highlight the importance of

  • nuclear functions of PKM2 in the Warburg effect
    and tumorigenesis.
  1. ERK is required for PKM2 nucleus translocation.
    http://www.nature.com/ncb/journal/v14/n12/carousel/ncb2629-f1.jpg
  2. ERK2 phosphorylates PKM2 Ser 37.
    http://www.nature.com/ncb/journal/v14/n12/carousel/ncb2629-f2.jpg
  3. Figure 3: PKM2 Ser 37 phosphorylation recruits PIN1.
    http://www.nature.com/ncb/journal/v14/n12/carousel/ncb2629-f3.jpg

 Pyruvate kinase M2 activators promote tetramer formation
and suppress tumorigenesis

D Anastasiou, Y Yu, WJ Israelsen, Jian-Kang Jiang, MB Boxer, B Hong, et al.
Nature Chemical Biology  11 Oct 2012; 8: 839–847

Cancer cells engage in a metabolic program to

  • enhance biosynthesis and support cell proliferation.

The regulatory properties of pyruvate kinase M2 (PKM2)

  • influence altered glucose metabolism in cancer.

The interaction of PKM2 with phosphotyrosine-containing proteins

  • inhibits PTM2 enzyme activity and
  • increases the availability of glycolytic metabolites
  • supporting cell proliferation.

This suggests that high pyruvate kinase activity may suppress
tumor growth
.

  1. expression of PKM1,  the pyruvate kinase isoform with high
    constitutive activity, or
  2. exposure to published small-molecule PKM2 activators
  • inhibits the growth of xenograft tumors.

Structural studies reveal that

  • small-molecule activators bind PKM2
  • at the subunit interaction interface,
  • a site that is distinct from that of the
    • endogenous activator fructose-1,6-bisphosphate (FBP).

However, unlike FBP,

  • binding of activators to PKM2 promotes
  • a constitutively active enzyme state that is resistant to inhibition
  • by tyrosine-phosphorylated proteins.

These data support the notion that small-molecule activation of PKM2
can interfere with anabolic metabolism

  1. PKM1 expression in cancer cells impairs xenograft tumor growth.
    http://www.nature.com/nchembio/journal/v8/n10/carousel/nchembio.1060-F1.jpg
  2. TEPP-46 and DASA-58 isoform specificity in vitro and in cells.
    TEPP-46 and DASA-58 isoform specificity in vitro and in cells.

    TEPP-46 and DASA-58 isoform specificity in vitro and in cells.

    (a) Structures of the PKM2 activators TEPP-46 and DASA-58. (b) Pyruvate kinase (PK) activity in purified recombinant human
    PKM1 or PKM2 expressed in bacteria in the presence of increasing
    concentrations of TEPP-46 or DASA-58. M1, PKM1;…
    http://www.nature.com/nchembio/journal/v8/n10/carousel/nchembio.1060-F2.jpg

  3. Activators promote PKM2 tetramer formation and prevent
    inhibition by phosphotyrosine signaling.
Activators promote PKM2 tetramer formation and prevent inhibition by phosphotyrosine signaling.

Activators promote PKM2 tetramer formation and prevent inhibition by phosphotyrosine signaling.

Sucrose gradient ultracentrifugation profiles of purified recombinant
PKM2 (rPKM2) and the effects of FBP and TEPP-46 on PKM2 subunit stoichiometry.
http://www.nature.com/nchembio/journal/v8/n10/carousel/nchembio.1060-F3.jpg

Figure 5: Metabolic effects of cell treatment with PKM2 activators.
(a) Effects of TEPP-46, DASA-58 (both used at 30 μM) or PKM1
expression on the doubling time of H1299 cells under normoxia
(21% O2) or hypoxia (1% O2). (b) Effects of DASA-58 on lactate
production from glucose. The P value shown was ca…
http://www.nature.com/nchembio/journal/v8/n10/carousel/nchembio.1060-F5.jpg

EGFR has a tumour-promoting role in liver macrophages during
hepatocellular carcinoma formation

H Lanaya, A Natarajan, K Komposch, L Li, N Amberg, …, & Maria Sibilia
Nature Cell Biology 31 Aug 2014   http://dx.doi.org:/10.1038/ncb3031

Tumorigenesis has been linked with macrophage-mediated chronic
inflammation and diverse signaling pathways, including the ​epidermal
growth factor receptor (​EGFR) pathway. ​EGFR is expressed in liver
macrophages in both human HCC and in a mouse HCC model. Mice
lacking ​EGFR in macrophages show impaired hepatocarcinogenesis,
Mice lacking ​EGFR in hepatocytes develop HCC owing to increased
hepatocyte damage and compensatory proliferation. EGFR is required
in liver macrophages to transcriptionally induce ​interleukin-6 following
interleukin-1 stimulation, which triggers hepatocyte proliferation and HCC.
Importantly, the presence of ​EGFR-positive liver macrophages in HCC
patients is associated with poor survival. This study demonstrates a

  • tumour-promoting mechanism for ​EGFR in non-tumour cells,
  • which could lead to more effective precision medicine strategies.
  1. HCC formation in mice lacking ​EGFRin hepatocytes or all liver cells.
    http://www.nature.com/ncb/journal/vaop/ncurrent/carousel/ncb3031-f1.jpg

2. EGFR expression in Kupffer cells/liver macrophages promotes HCC development.

EGFR c2a expression in Kupffer cells.liver macrophages promotes HCC development.

EGFR c2a expression in Kupffer cells.liver macrophages promotes HCC development.

http://www.nature.com/ncb/journal/vaop/ncurrent/carousel/ncb3031-f2.jpg

Hypoxia-inducible factor 1 activation by aerobic glycolysis implicates
the Warburg effect in carcinogenesis
.

Lu H1, Forbes RA, Verma A.
J Biol Chem. 2002 Jun 28;277(26):23111-5. Epub 2002 Apr 9

Cancer cells display high rates of aerobic glycolysis, a phenomenon
known historically as the Warburg effect. Lactate and pyruvate, the end
products of glycolysis, are highly produced by cancer cells even in the
presence of oxygen
.

Hypoxia-induced gene expression in cancer cells

  • has been linked to malignant transformation.

Here we provide evidence that lactate and pyruvate

  • regulate hypoxia-inducible gene expression
  • independently of hypoxia
  • by stimulating the accumulation of hypoxia-inducible Factor 1alpha
    (HIF-1alpha).

In human gliomas and other cancer cell lines,

  • the accumulation of HIF-1alpha protein under aerobic conditions
  • requires the metabolism of glucose to pyruvate that
  1. prevents the aerobic degradation of HIF-1alpha protein,
  2. activates HIF-1 DNA binding activity, and
  3. enhances the expression of several HIF-1-activated genes
  4. erythropoietin,
  5. vascular endothelial growth factor,
  6. glucose transporter 3, and
  7. aldolase A.

Our findings support a novel role for pyruvate in metabolic signaling
and suggest a mechanism by which

  • high rates of aerobic glycolysis
  • can promote the malignant transformation and
  • survival of cancer cells.PMID: 11943784

Part IV. Transcription control and innate immunity

 c-Myc-induced transcription factor AP4 is required for
host protection mediated by CD8+ T cells

C Chou, AK Pinto, JD Curtis, SP Persaud, M Cella, Chih-Chung Lin, … & T Egawa Nature Immunology 17 Jun 2014;   http://dx.doi.org:/10.1038/ni.2943

The transcription factor c-Myc is essential for

  • the establishment of a metabolically active and proliferative state
  • in T cells after priming,

We identified AP4 as the transcription factor

  • that was induced by c-Myc and
  • sustained activation of antigen-specific CD8+ T cells.

Despite normal priming,

  • AP4-deficient CD8+ T cells
  • failed to continue transcription of a broad range of
    c-Myc-dependent targets.

Mice lacking AP4 specifically in CD8+ T cells showed

  • enhanced susceptibility to infection with West Nile virus.

Genome-wide analysis suggested that

  • many activation-induced genes encoding molecules
  • involved in metabolism were shared targets of
  • c-Myc and AP4.

Thus, AP4 maintains c-Myc-initiated cellular activation programs

  • in CD8+ T cells to control microbial infection.
  1. AP4 is regulated post-transcriptionally in CD8+ T cells.

Microarray analysis of transcription factor–encoding genes with a difference
in expression of >1.8-fold in activated CD8+ T cells treated for 12 h with
IL-2 (100 U/ml; + IL-2) relative to their expression in activated CD8+ T cells…
http://www.nature.com/ni/journal/vaop/ncurrent/carousel/ni.2943-F1.jpg

2. AP4 is required for the population expansion of antigen specific
CD8+ T cells following infection with LCMV-Arm.

Expression of CD4, CD8α and KLRG1 (a) and binding of an
H-2Db–gp(33–41) tetramer and expression of CD8α, KLRG1 and
CD62L (b) in splenocytes from wild-type (WT) and Tfap4−/− mice,
assessed by flow cytometry 8 d after infection
http://www.nature.com/ni/journal/vaop/ncurrent/carousel/ni.2943-F2.jpg

3. AP4 is required for the sustained clonal expansion of CD8+ T cells
but  not for their initial proliferation.
http://www.nature.com/ni/journal/vaop/ncurrent/carousel/ni.2943-F3.jpg

  1. AP4 is essential for host protection against infection with WNV, in
    a CD8+ T cell–intrinsic manner.
AP4 is essential for host protection against infection with WNV, in a CD8+ T cell–intrinsic manner.

AP4 is essential for host protection against infection with WNV, in a CD8+ T cell–intrinsic manner.

  •  Survival of Tfap4F/FCre− control mice (Cre−; n = 16) and
  • Tfap4F/FCD8-Cre+ mice (CD8-Cre+; n = 22) following infection with WNV.
    (b,c) Viral titers in the brain (b) and spleen (c) of Tfap4F/F Cre− and Tfap4F/F
    CD8-Cre+ mice  on day 9…
    http://www.nature.com/ni/journal/vaop/ncurrent/carousel/ni.2943-F4.jpg

AP4 is essential for the sustained expression of genes that are targets of c-Myc.

Normalized signal intensity (NSI) of endogenous transcripts in
Tfap4+/+ and Tfap4−/− OT-I donor T cells adoptively transferred into
host mice and assessed on day 4 after infection of the host with LM-OVA
(top), and that of ERCC controls
http://www.nature.com/ni/journal/vaop/ncurrent/carousel/ni.2943-F6.jpg

Sustained c-Myc expression ‘rescues’ defects of Tfap4−/− CD8+ T cells.
http://www.nature.com/ni/journal/vaop/ncurrent/carousel/ni.2943-F7.jpg

AP4 and c-Myc have distinct biological functions.
http://www.nature.com/ni/journal/vaop/ncurrent/carousel/ni.2943-SF7.jpg

Mucosal memory CD8+ T cells are selected in the periphery
by an MHC class I molecule

Y Huang, Y Park, Y Wang-Zhu, …A Larange, R Arens, & H Cheroutre

Nature Immunology 2 Oct 2011; 12: 1086–1095
http://dx.doi.org:/10.1038/ni.2106

The presence of immune memory at pathogen-entry sites is a prerequisite
for protection. We show that the non-classical major histocompatibility
complex (MHC) class I molecule

  • thymus leukemia antigen (TL),
  • induced on dendritic cells interacting with CD8αα on activated CD8αβ+ T cells,
  • mediated affinity-based selection of memory precursor cells.

Furthermore, constitutive expression of TL on epithelial cells

  • led to continued selection of mature CD8αβ+ memory T cells.

The memory process driven by TL and CD8αα

  • was essential for the generation of CD8αβ+ memory T cells in the intestine and
  • the accumulation of highly antigen-sensitive CD8αβ+ memory T cells
  • that form the first line of defense at the largest entry port for pathogens.

The metabolic checkpoint kinase mTOR is essential for IL-15 signaling during the development and activation of NK cells.

Marçais A, Cherfils-Vicini J, Viant C, Degouve S, Viel S, Fenis A, Rabilloud J,
Mayol K, Tavares A, Bienvenu J, Gangloff YG, Gilson E, Vivier E,Walzer T.
Nat Immunol. 2014 Aug; 15(8):749-757. Epub 2014 Jun 29
http://dx.doi.org:/10.1038/ni.2936  .    PMID: 24973821

Interleukin 15 (IL-15) controls

  • both the homeostasis and the peripheral activation of natural killer (NK) cells.

We found that the metabolic checkpoint kinase

  • mTOR was activated and boosted bioenergetic metabolism
  • after exposure of NK cells to high concentrations of IL-15,

whereas low doses of IL-15 triggered

  • only phosphorylation of the transcription factor STAT5.

mTOR

  • stimulated the growth and nutrient uptake of NK cells and
  • positively fed back on the receptor for IL-15.

This process was essential for

  • sustaining NK cell proliferation during development and
  • the acquisition of cytolytic potential during inflammation
    or viral infection.

The mTORC1 inhibitor rapamycin 

  • inhibited NK cell cytotoxicity both in mice and humans;
    • this probably contributes to the immunosuppressive
      activity of this drug in different clinical settings.

The Critical Role of IL-15-PI3K-mTOR Pathway in Natural Killer Cell
Effector Functions.
Nandagopal NAli AKKomal AKLee SH.   Author information
Front Immunol. 2014 Apr 23; 5:187. eCollection 2014.
http://dx.doi.org:/10.3389/fimmu.2014.00187

Natural killer (NK) cells were so named for their uniqueness in killing
certain tumor and virus-infected cells without prior sensitization.
Their functions are modulated in vivo by several soluble immune mediators;

  • interleukin-15 (IL-15) being the most potent among them in
    enabling NK cell homeostasis, maturation, and activation.

During microbial infections,

  • NK cells stimulated with IL-15 display enhanced cytokine responses.

This priming effect has previously been shown with respect to increased
IFN-γ production in NK cells

  • upon IL-12 and IL-15/IL-2 co-stimulation.
  • we explored if this effect of IL-15 priming 
  • can be extended to various other cytokines and
  • observed enhanced NK cell responses to stimulation
    • with IL-4, IL-21, IFN-α, and IL-2 in addition to IL-12.
  • we also observed elevated IFN-γ production in primed NK cells

Currently, the fundamental processes required for priming and

  • whether these signaling pathways work collaboratively or
    independently 

    • for NK cell functions are poorly understood.

We examined IL-15 effects on NK cells in which

  • the pathways emanating from IL-15 receptor activation
    • were blocked with specific inhibitors
    • To identify the key signaling events for NK cell priming,

Our results demonstrate that

the PI3K-AKT-mTOR pathway is critical for cytokine responses
in IL-15 primed NK cells. 

This pathway is also implicated in a broad range of

  • IL-15-induced NK cell effector functions such as
    • proliferation and cytotoxicity.

Likewise, NK cells from mice

  • treated with rapamycin to block the mTOR pathway
  • displayed defects in proliferation, and IFN-γ and granzyme B productions
  • resulting in elevated viral burdens upon murine cytomegalovirus infection.

Taken together, our data demonstrate

  • the requirement of PI3K-mTOR pathway
    • for enhanced NK cell functions by IL-15, thereby
  • coupling the metabolic sensor mTOR to NK cell anti-viral responses.

KEYWORDS: IL-15; JAK–STAT pathway; mTOR pathway; natural killer cells; signal transduction

Part V. Predicting Therapeutic Targets 

New discovery approach accelerates identification of potential cancer treatments
 Laura Williams, Univ. of Michigan   09/30/2014
http://www.rdmag.com/news/2014/09/new-discovery-approach-accelerates-identification-potential-cancer-treatments

Researchers at the Univ. of Michigan have described a new approach to
discovering potential cancer treatments that

  • requires a fraction of the time needed for more traditional methods.

They used the platform to identify

  • a novel antibody that is undergoing further investigation as a potential
    treatment for breast, ovarian and other cancers.

In research published online in the Proceedings of the National Academy
of Sciences
, researchers in the laboratory of Stephen Weiss at the U-M Life
Sciences Institute detail an approach

  • that replicates the native environment of cancer cells and
  • increases the likelihood that drugs effective against the growth of
    tumor cells in test tube models
  • will also stop cancer from growing in humans.

The researchers have used their method

  • to identify an antibody that stops breast cancer tumor growth in animal models, and
  • they are investigating the antibody as a potential treatment in humans.

“Discovering new targets for cancer therapeutics is a long and tedious undertaking, and

  • identifying and developing a potential drug to specifically hit that
    target without harming healthy cells is a daunting task,” Weiss said.
  • “Our approach allows us to identify potential therapeutics
    • in a fraction of the time that traditional methods require.”

The researchers began by

  • creating a 3-D “matrix” of collagen, a connective tissue molecule very similar to that found
    • surrounding breast cancer cells in human patients.
  • They then embedded breast cancer cells into the collagen matrix,
    • where the cells grew as they would in human tissue.

The investigators then injected the cancer-collagen tissue composites into mice that then

  • recognize the human cancer cells as foreign tissue.
    • Much in the way that our immune system generates antibodies
      to fight infection,
  • the mice began to generate thousands of antibodies directed against
    the human cancer cells.
  • These antibodies were then tested for the ability to stop the growth
    of the human tumor cells.

“We create an environment in which cells cultured in the laboratory ‘think’
they are growing in the body and then

  • rapidly screen large numbers of antibodies to see if any exert
    anti-cancer effects,” Weiss said.
  • “This allows us to select promising antibodies very quickly and then

They discovered a particular antibody, 4C3, which was able to

  • almost completely stop the proliferation of the breast cancer cells.

They then identified the molecule on the cancer cells that the antibody targets.

The antibody can be further engineered to generate

  • humanized monoclonal antibodies for use in patients

“We still need to do a lot more work to determine how effective 4C3 might be as a
treatment for breast and other cancers, on its own or in conjunction with other
therapies,” Weiss said. “But we have enough data to warrant further pursuit,
and are expanding our efforts to use this discovery platform to find similarly promising antibodies.”

Source: Univ. of Michigan

  1. Jose Eduardo de Salles Roselino

    Larry,
    I think you have made a great effort in order to connect basic ideas of metabolic regulation with those of gene expression control “modern” mechanisms.
    Yet, I do not think that at this stage it will be clear for all readers. At least, for the great majority of the readers. The most important factor I my opinion, is derived from the fact that modern readers considers that metabolic regulation deals with so called “housekeeping activities” of the cell. Something that is of secondary, tertiary or even less level of relevance.
    My idea, that you have mentioned in the text when you write at the beginning, the word biochemistry, in order to resume it, derives from the reading of What is life together with Prof. Leloir . For me and also, for him, biochemistry comprises a set of techniques and also a framework of reasoning about scientific results. As a set of techniques, Schrodinger has considered that it will lead to better understanding of genetics and of physiology as a two legs structure supporting the future progress related to his time (mid-forties). For Leloir, the key was the understanding of chemical reactivity and I agree with him. However, as I was able to talk and discuss it with him in detail, we should also take into account levels of stabilities of macromolecules and above all, regulation of activities and function (this is where) Pasteur effect that I was studying in Leloir´s lab at that time, 1970-72, gets into the general picture.
    Regulation for complex living beings , that also have cancer cell as a great topic of research problem can be understood through the understanding of two quite different results when opposition with lack of regulation is taken into account or experimentally elicited. The most clearly line of experiments can follow the Pasteur Effect as the intracellular result best seen when aerobiosis is compared with anaerobiosis as conditions in which maintenance of ATP levels and required metabolic regulation (Energy charge D.E, Atkinson etc) is studied. Another line of experiments is one that takes into account the extracellular result or for instance the homeostatic regulation of blood glucose levels. The blood glucose level is the most conspicuous and related to Pasteur Effect regulatory event that can be studied in the liver taking into account both final results tested or compared regarding its regulation, ATP levels maintenance (intracellular) and blood glucose maintenance (extracellular).
    My key idea is to consider that the same factors that elicits fast regulatory responses also elicits the slow energetic expensive regulatory responses. The biologic logic behind this common root is the ATP economy. In case, the regulatory stimulus fades out quickly the fast regulatory responses are good enough to maintain life and the time requiring, energetic costly responses will soon be stopped cutting short the ATP expenditure. In case, the stimulus last for long periods of time the fast responses are replaced by adaptive responses that in general will follow the line of cell differentiation mechanisms with changes in gene expression etc.
    The change from fast response mechanisms to long lasting developmentally linked ones is not sharp. Therefore, somehow, cancer cells becomes trapped into a metastable regulatory mechanism that prevents cell differentiation and reinforces those mechanisms linked to its internal regulatory goals. This metastable mechanism takes advantage from the fact that other cells, tissues and organs will take good care of homeostatic mechanisms that provide for their nutritional needs. In the case of my Hepatology work you will see a Piruvate kinase that does not responds to homeostatic signals .

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