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Acetylation and Deacetylation of non-Histone Proteins

Author and Curator: Larry H Bernstein, MD, FCAP 

 

Acetylation and Deacetylation of non-histone proteins

MA Glozak, N Sengupta, X Zhang, E Seto
Gene 2005; 363(19): 15-23     http://dx.doi.org/10.1016/j.gene.2005.09.010

Since the first report of p53 as a non-histone target of a histone acetyltransferase (HAT), there has been a rapid proliferation in the description of new non-histone targets of HATs. Of these,

  • transcription factors comprise the largest class of new targets.

The substrates for HATs extend to

  1. cytoskeletal proteins,
  2. molecular chaperones and
  3. nuclear import factors.

Deacetylation of these non-histone proteins by histone deacetylases (HDACs) opens yet another exciting new field of discovery in

  • the role of the dynamic acetylation and deacetylation on cellular function.

This review will focus on these non-histone targets of HATs and HDACs and the consequences of their modification.

Abbreviations:

HAT, histone acetyltransferase; HDAC, histone deacetylase; TSA, trichostatin A; CtBP, C-terminal binding protein; YY1, yin yang 1; HMG, high mobility group; NR, nuclear receptor; AR, androgen receptor; ER α, estrogen receptor α; SHP, short heterodimer partner; EKLF, erythroid Kruppel like factor; Rb, retinoblastoma; GR, glucocorticoid receptor; HDV, hepatitis delta virus; L-HDAg, large delta antigen; S-HDAg, small delta antigen

Keywords  HATs; HDACs; Post-translational modification

Histone deacetylases (EC 3.5.1.98, HDAC) are a class of enzymes that

This is important because DNA is wrapped around histones, and

  • DNA expression is regulated by acetylation and de-acetylation.

Its action is opposite to that of histone acetyltransferase. HDAC proteins are now also called

  • lysine deacetylases (KDAC),
  • to describe their function rather than their target, which also
  • includes non-histone proteins

Histone modification

Histone tails are normally positively charged due to

These positive charges help the histone tails to

  • interact with and bind to the negatively charged phosphate groups on the DNA backbone.

Acetylation, which occurs normally in a cell,

  1. neutralizes the positive charges on the histone by changing amines into amides and
  2. decreases the ability of the histones to bind to DNA.

This decreased binding

Histone deacetylases

  1. remove those acetyl groups,
  2. increasing the positive charge of histone tails and
  3. encouraging high-affinity binding between the histones and DNA backbone.

The increased DNA binding

  1. condenses DNA structure,
  2. preventing transcription.

Histone deacetylase is involved in a series of pathways within the living system. According to the Kyoto Encyclopedia of Genes and Genomes (KEGG), these are:

Histone acetylation plays an important role in the regulation of gene expression.

Hyperacetylated chromatin is

  • transcriptionally active, and

hypoacetylated chromatin

  • is silent.

A study on mice found that a

  • specific subset of mouse genes (7%) was
    • deregulated in the absence of HDAC1.[10]

Their study also found a

  • regulatory crosstalk between HDAC1 and HDAC2 and suggest
    • a novel function for HDAC1 as a transcriptional coactivator.

HDAC1 expression was found to be

  1. increased in the prefrontal cortex of schizophrenia subjects,[11]
  2. negatively correlating with the expression of GAD67 mRNA.

Non-histone effects

It is a mistake to regard HDACs solely in the context of regulating gene transcription by modifying histones and chromatin structure, although

  • that appears to be the predominant function.

The function, activity, and stability of proteins can be controlled by

Protein phosphorylation is perhaps the most widely studied and understood modification in which

  1. certain amino acid residues are phosphorylated by the action of protein kinases or
  2. dephosphorylated by the action of phosphatases.

The acetylation of lysine residues is emerging as an analogous mechanism, in which

    • non-histone proteins are acted on by acetylases and deacetylases.[12]

It is in this context that HDACs are being found to interact with a variety of non-histone proteins

some of these are transcription factors and co-regulators, some are not. Note the following four examples:

  • HDAC6 is associated with aggresomes.Misfolded protein aggregates are
    • tagged by ubiquitination and removed from the cytoplasm by dynein motors via the microtubule network to an organelle termed the aggresome.
    • HDAC 6 binds polyubiquitinated misfolded proteins and links to dynein motors, thereby
    • allowing the misfolded protein cargo to be physically transported to chaperones and proteasomes for subsequent destruction.[13]
  • PTEN is an important phosphatase involved in cell signaling via phosphoinositols and the AKT/PI3 kinase pathway.
    • PTEN is subject to complex regulatory control via phosphorylation, ubiquitination, oxidation and acetylation.
    • Acetylation of  PTEN by the histone acetyltransferase p300/CBP-associated factor (PCAF) can repress its activity; on the converse,
    • deacetylation of  PTEN by SIRT1 deacetylase and, by HDAC1, can stimulate its activity.[14][15]
  • APE1/Ref-1 (APEX1) is a multifunctional protein possessing both
    • DNA repair activity (on abasic and single-strand break sites) and
    • transcriptional regulatory activity associated with oxidative stress.
    • APE1/Ref-1isacetylatedbyPCAF; on the converse,
      • it is stably associated with and deacetylated by Class I HDACs.
    • The acetylation state of APE1/Ref-1 does not appear to affect its DNA repair activity, but it does
      • regulate its transcriptional activity such as
      • its ability to bind to the PTH promoter and initiate transcription of the parathyroid hormone gene.[16][17]
  • NF-κB is a key transcription factor and
    • effector molecule involved in responses to cell stress, consisting of a p50/p65 heterodimer.
    • The p65 subunit is controlled by acetylation via PCAF and by deacetylation via HDAC3 and HDAC6.[18]

HDAC inhibitors

Main article: Histone deacetylase inhibitor

Histone deacetylase inhibitors (HDIs) have a long history of use in psychiatry and neurology as mood stabilizers and anti-epileptics,

In more recent times, HDIs are being studied as

  1. a mitigator or treatment for neurodegenerative diseases.[19][20]
  2. there has been an effort to develop HDIs for cancer therapy.[21][22]

The exact mechanisms by which the compounds may work are unclear, but

  • epigenetic pathways are proposed.[23] In addition, a clinical trial is studying valproic acid effects on the latent pools of HIV in infected persons.[24]

HDIs are currently being investigated as chemosensitizers for

  • cytotoxic chemotherapy or radiation therapy, or in association with DNA methylation inhibitors based on in vitro synergy.[25]

Recent research has focused on developing isoform selective HDIs which can aid in elucidating role of

  1. individual HDAC isoforms and device strategy for effective treatment of
  2. diseases related to relevant HDAC isoform.[26][27][28]

HDAC inhibitors have effects on non-histone proteins that are related to acetylation. HDIs can

  1. alter the degree of acetylation of these molecules and, therefore,
  2. increase or repress their activity.

For the four examples given above (see Function) on HDACs acting on non-histone proteins, in each of those instances

HDIs have been shown to alter the activity of many transcription factors, including

ACTR, cMyb, E2F1, EKLF, FEN 1, GATA, HNF-4, HSP90, Ku70, NFκB, PCNA, p53, RB, Runx, SF1 Sp3, STAT, TFIIE, TCF, YY1.[29][30]

To carry out gene expression, a cell must control the coiling and uncoiling of DNA around histones. This is accomplished with the assistance of histone acetyl transferases (HAT), which

  1. acetylate the lysine residues in core histones leading to
    • a less compact and more transcriptionally active chromatin, and, on the converse,
  2. the actions of histone deacetylases (HDAC), which
    • remove the acetyl groups from the lysine residues
    • leading to the formation of a condensed and transcriptionally silenced chromatin.

Reversible modification of the terminal tails of core histones constitutes

HDAC inhibitors (HDI) block this action and

  • can result in hyperacetylation of histones, thereby affecting gene expression.[5][6][7]

The histone deacetylase inhibitors are a new class of cytostatic agents that inhibit the proliferation of tumor cells in culture and in vivo

  1. by inducing cell cycle arrest,
  2. differentiation
  3. and/or apoptosis.

Histone deacetylase inhibitors exert their anti-tumour effects via

  1. the induction of expression changes of oncogenes or tumour suppressor, through
  2. modulating that the acetylation/deactylation of histones and/or non-histone proteins such as transcription factors[8].

Histone acetylation and deacetylation play important roles in the modulation of chromatin topology and the regulation of gene transcription.

Histone deacetylase inhibition induces

  • the accumulation of hyperacetylated nucleosome core histones in most regions of chromatin

but affects the expression of only a small subset of genes, leading to transcriptional activation of some genes, but repression of an equal or larger number of other genes.

Non-histone proteins such as transcription factors are also targets for acetylation with varying functional effects. Acetylation

  • enhances the activity of some transcription factors such as the tumor suppressor p53 and
  • the erythroid differentiation factor GATA-1
  • but may repress transcriptional activity of others including T cell factor and the co-activator ACTR.

Recent studies […] have shown that the estrogen receptor alpha (ERalpha) can be hyperacetylated

  1. in response to histone deacetylase inhibition,
  2. suppressing ligand sensitivity and regulating transcriptional activation by histone deacetylase inhibitors.[9]

Conservation of the acetylated ER-alpha motif in other nuclear receptors suggests that

  • acetylation may play an important regulatory role in diverse nuclear receptor signaling functions.

A number of structurally diverse histone deacetylase inhibitors have shown potent antitumor efficacy with little toxicity in vivo in animal models. Several compounds are currently in early phase clinical development as potential treatments for solid and hematological cancers both as monotherapy and in combination with cytotoxics and differentiation agents.”[10]

HDIs MI  ·  Granger, A.; Abdullah, I.; Huebner, F.; Stout, A.; Wang, T.; Huebner, T.; Epstein, J. A.; Gruber, P. J. (2008). “Histone deacetylase inhibition reduces myocardial ischemia-reperfusion injury in mice”. The FASEB Journal 22 (10): 3549–60. http://dx.doi.org/10.1096/fj.08-108548. PMC 2537432. PMID 18606865.

 

Protein Acetylation: Much More than Histone Acetylation

By Tom Brock, Ph.D.

Just last decade, everyone was excited about the Human Genome Project,  and the gene was king. Today, epigenetics is reminding us that

  • non-genetic factors are important in shaping gene expression and development.

Similarly, where phosphorylation once seemed the primary way to modulate proteins,

  • epigenetics has re-introduced us to acetylation as an important force in defining protein function.

In particular, the acetylation of histones has moved to center stage, even though it was described over 45 years ago. Research on histone acetylation has

  • led to a resurgence in the interest in enzymatically-mediated acetylation of other proteins.

This article examines acetylation as a post-translational modification of proteins that impacts gene expression and plays a role in epigenetics.

The Basics

Acetylation refers to the addition of an acetyl group (CH3CO) to organic compounds. Proteins can be acetylated by both enzymatic and non-enzymatic processes.

One group of acetyltransferases commonly catalyze the transfer of an acetyl group from acetyl-CoA to the terminal amine on the side chain of lysine residues (Figure 1).

These enzymes are commonly called HATs, because their best-known substrates have been histones.

However, the nomenclature is being revised to lysine acetyltransferases (KATs), reflecting their ability to acetylate lysine (denoted ‘K’) on many proteins.

1 The KATs are numerous, with many assigned, based on structural similarities, to either

  1. the GNAT (Gcn5-related N-acetyltransferases) superfamily or
  2. the MYST (MOZ, YBF2/Sas3, Sas2, Tip60) family.

Other important KATs include

  1. p300 (E1A-associated protein 300 kDa),
  2. CBP (cAMP response element binding (CREB)-binding protein), and
  3. TAFII 250 (TATA-binding protein associated factor II 250).

The conversion of the positively charged lysine to acetyl-lysine, like the addition of negative phosphates to uncharged amino acids during phosphorylation,

alters protein structure and interactions with other biomolecules. For example, acetylation of  histones typically

  1. promotes the recruitment of effector proteins,
  2. relaxation of chromatin conformation, and
  3. an increase in transcription.

Like phosphorylation,

  • acetylation is reversible.

Histone deacetylases (HDACs, a.k.a. KDACs) are a smaller group of evolutionarily conserved enzymes.

The human class I HDACs are

  • homologous to the yeast enzyme Rpd3 and include HDAC1, 2, 3, and 8.

Class II HDACs are

  • homologous to yeast HDA1 and are divided into class IIa (HDAC4, 5, 7, 9) and class IIb (HDAC6 and 10) based on structure.
  • The human class III HDACs include the sirtuin family of NAD+-dependent protein deacetylases.
  • The novel HDAC11 has a distinct structure and is a class IV HDAC.

The HDACs often participate in the formation of transcriptional repressor complexes, inducing

  • chromatin compaction through histone deacetylation, and silencing gene expression.

A Diversity of Partners

A great resource for the research scientist is the National Center for Biotechnology Information (NCBI), your tax dollars at work compiling information about everything molecular. This site should be your first stopping point when trying to learn authoritative information about a new protein or gene that you’re studying. Information at this site helps to underscore two points about KATs and deacetylases: they are social enzymes, always interacting with other proteins, and they are promiscuous, binding to an astounding array of partners. Take, for example, the KAT known commonly as p300. At the NCBI gene link, entering ‘human p300’ finds the gene EP300 (KAT3B), with a summary stating that it associates with the adenovirus protein E1A, acetylates histones, binds CREB, and is a co-activator of HIF-1α (hypoxia-inducible factor 1α). Further down, we find that it binds three different proteins produced by the lentivirus human immunodeficiency virus (HIV)-1. Then, impressively, is a list of over two hundred proteins that have been documented to directly interact with p300 (with links to references and other interactome datasets included). Similarly, the deacetylase HDAC1 is summarized as a histone deacetylase that also interacts with retinoblastoma tumor-suppressor to control cell growth and, together with metastasis-associated protein-2, deacetylates the tumor suppressor p53. Like p300, HDAC1 has an amazing list of partners: it interacts with some 300 proteins, with over 125 of these documented as direct binding partners.

The abundance of protein partners, for both KATs and HDACs, suggests that these enzymes tend to form multimeric complexes. In fact, such complexes serve the critical purpose of positioning the (de)acetylases at specific sites to perform their functions. Certainly, KATs can directly acetylate substrates in vitro. However, KAT activity in vivo is regulated, at least in part, by where it is positioned. For example, the classical model for activation of PPARs (peroxisome proliferator-activated receptors) posits that this receptor heterodimerizes at specific response elements with RXR (retinoid X receptor). In the absence of ligand, the unactivated heterodimer binds co-repressor proteins, such as nuclear receptor co-repressors (NCoR), G-protein pathways suppressor 2 (GPS2), and HDACs (Figure 2). The HDACs help prevent expression of PPAR-specific genes by keeping the neighboring histones deacetylated. The appearance of a ligand for PPAR causes dissociation of the co-repressor proteins followed by the recruitment of co-activators, including PPAR co-activator (PGC-1), CREB binding protein (CBP), and p300. Formation of the PPAR activation complex leads to histone acetylation by CBP and p300, giving rise to altered expression of genes involved in fatty acid metabolism, lipid homeostasis, and adipocyte differentiation. In this example, ligand binding to its receptor causes a large scale switch from a cluster of proteins serving various roles in preventing transcription to a different group designed to facilitate gene transcription.

Acetylation Patterns

In its simplest form acetylation is merely another form of post-translational modification of proteins. A good example is the acetylation of tubulin, which can be deacetylated by HDAC6 or SIRT2. Acetylation of this key microtubule component appears to alter its affinity for kinesin-1 and redirect motor-based trafficking of vesicles.2,3 In short, acetylation changes protein function by adjusting protein-protein interactions. The net ‘global’ acetylation, in this case, may be determined by the balance of overall KAT and HDAC activities.

More commonly, acetylation is targeted to specific proteins and, possibly, specific lysine residues on those protein targets. One way that this can be achieved is by the formation of protein complexes containing either KATs or HDACs, as in the PPAR case described above. The assembly of the complex serves to place the KATs/HDACs near histones, transcription factors, or other targets. Histones, assembled as an octamer core surrounded by DNA, have amino termini that are freely exposed (Figure 3). Positively-charged lysine residues on these tails interact electrostatically with negatively-charged phosphate groups along the DNA backbone. Acetylation reduces these interactions and loosens the DNA, facilitating transcription. Bear in mind that, while it is generally true that histone acetylation increases transcriptional activation, there are exceptions. For example, acetylation of estrogen receptor-α suppresses ligand sensitivity and reduces ligand-induced transcriptional activity.4,5

References

1. Glozak, M.A., Sengubpta, N., Zhang, X., et al. Gene 363, 15-23 (2005).

2. Hammond, J.W., Cai, D., and Verhey, K.J. Curr. Opin. Cell Biol. 20, 71-76 (2008).

3. Gao, Y., Hubber, C.C., and Yao, T.P. J. Biol. Chem. epub ahead of print (2010).

4. Wang, C., Fu, M., Angeletti, R.H., et al. J. Biol. Chem. 276, 18375-18383 (2001).

5. Popov, V.M., Wang, C., Shirley, L.A., et al. Steroids 72, 221-230 (2007).

6. Mellert, H.S. and McMahon, S.B. Trends Biochem. Sci. 34, 571-578 (2009).

7. Yang, X.J. and Seto, E. Mol. Cell 31, 49-461 (2008).

8. Wilson, A.J., Byun, D.S., Popova, N., et al. J. Biol. Chem. 281, 13548-13558 (2006).

9. Vincent, A. and Van Seuningen, I. Differentiation 78, 99-107 (2009).

10. Li, Z., Chen, L., Kabra, N., et al. J. Biol. Chem. 284, 10361-10366 (2009).

From Protein Acetylation: Much More than Histone Acetylation by Brock, T.G.

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PTM modifications

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Analysis of S-nitrosylated Proteins

Author and Curator: Larry H Bernstein, MD, FCAP

 

The biotin switch method for the detection of S-nitrosylated proteins.

Jaffrey S. R. and Snyder S. H.
Sci STKE. 2001, pl1.

 

  • apoptosis thymocytes induced by GSNO.

Meanwhile, the level of the protein S-nitrosylation

  • was inhibited by the NOS inhibitor L-NMMA, which is consistent with
  • the protection of L-NMMA from apoptosis.

We also found that proteins with

  • moderate molecular weight (20-60 kDa)
  • are more sensitive to GSNO S-nitrosylation.

Thymocytes of 3-4 week-old inbred male/female Balb/c mice (11.5-13.0 g), were obtained by gently pressing the thymus against a nylon net  submerged in  SWIM’S S-77 medium without fetal calf serum (FCS) and sulphydryl from cysteine. The suspension was put in Ficoll and prepared by density gradient centrifugation  at 410 g.

Thymocytes were resuspended in S-77 medium (1×10  cells/ml) and cultured at 37°C, 5% CO2. The viability of untreated thymocytes were identified by trypan blue exclusion assay or/and MoFlo high performance cell sorter (MoFlo cell sorter) to be always greater than about 96% during the 3-hour real-time process (data not shown).

GSNO is the nitroso derivative of glutathione (GSH) and must be freshly synthesized right before the experiments. To prepare 30 mM GSNO, GSH was dissolved in 0.1 N HCl to the final concentration of 60 mM, and then mixed with equal molar amount of sodium nitrite. During the preparation, the mixture was protected from light. The final GSNO was characterized using ultraviolet-spectroscopy [7].

Thymocytes were treated with GSNO for 3 hrs in S-77 medium and then washed with PBS. The washed cells were stained by double labeled Hoechst 33342 (final concentration 10 µM) and PI (5 µg/ml) for 20 min, and subjected to MoFlo high-performance cell sorter (Dako, USA) using excitation /emission equal to 351/460 nm for Hoechst 33342 and 488/630 nm for PI respectively.

Biotin-switch method and western blotting-detected S-nitrosated proteins

The analysis of S-nitrosylated proteins was described previously [8, 9]. After the exposure to 0.3 mM GSNO for 3 h, cells were washed three times with ice-cold PBS, and lysed in HEN buffer (250 mM Hepes-NaOH pH 7.7, 1 mM EDTA, 0.1 mM Neocupeoine) containing 0.5% NP-40 for 30 min on ice and centrifuged at 10,000 g for 10 min. One volume of the supernatant was incubated with four volumes of blocking buffer (9 volumes of HEN buffer plus one volume 25% SDS, 20 mM MMTS) at 50 °C for 20 min with frequent vortexing. MMTS was then removed by protein precipitation with ten volumes of pre-chilled acetone. After SDS-PAGE sample buffer was added, the samples were resolved by SDS-PAGE and transferred for immunoblotting with streptravidin-HRP. S-nitrosated bovine serum albumin (BSA) was used as a positive control.

In order to gain reasonable data of PCR amplification, two controls were set up in parallel. One of them sets water as the template, the other of them sets un-reversed transcribed total RNA as the template. Every samples were serially diluted 10 times for a calibration curve. The housekeeping gene beta-actin was set in this study. The primers were used :

beta-actin-FP: 5’-GAG ACC TTC AAC ACC CCA GCC-3’,
beta-actin-RP: 5’-AAT GTC ACG CAC GAT TTC CC-3’;
p53-FP: 5’ACG TGC CCT GTG CAG TTG T-3’,
p53-RP: 5’GGA TAG GTC GGC GGT TCA T-3’;
ING1-FP: 5’-CTC CAG GGC TTT GTC CAT-3’,
ING1-RP 5’-GCA ACC AGG TCT CCT ACG-3’;
bax-FP: 5’-GTA GAA GAG GGC AAC CAC G-3’,
bax-RP :5’CCA GGA TGC GTC CAC CAA-3’,
All raw data of Real time PCR in this study were obtained from software of Gene Amp 5700 Sequence Detection System.

 

Reference

Nitric oxide: NO apoptosis or turning it ON? Cell Death Differ 2003, 10:864-869.

Hess DT, Matsumoto A, Kim SO, Marshall HE, Stamler JS: Protein S-nitrosylation: purview and parameters. Nat Rev Mol Cell Biol 2005,  6:150-166.

Messmer UK, Reed UK, Brune B: Bcl-2 protects macrophages from nitric oxide-induced apoptosis. J Biol Chem 1996, 271:20192-20197.

Brune B, Nicoletti I, Migliorati G, Pagliacci MC, Grignani F, Riccardi C: A rapid and simple method for measuring thymocyte apoptosis by propidium iodide staining and flow cytometry. J Immunol Methods 1991, 139:271-279.

Lin DY, Ma WY, Duan SJ, Zhang Y, Du LY: Real-time imaging of viable-apoptotic switch in GSNO-induced mouse thymocyte apoptosis. Apoptosis 2006, 11:1289-1298.

Fehsel K, Kroncke KD, Meyer KL, Huber H, Wahn V, Kolb-Bachofen V: Nitric oxide induces apoptosis in mouse thymocytes. J Immunol 1995, 155:2858-2865.

Gabor G, Allon N: Spectrofluorometric method for NO determination. Anal Biochem 1994, 220:16-19.

Jaffrey SR, Snyder SH: The biotin switch method for the detection of S-nitrosylated proteins. Sci STKE 2001, 2001:PL1.

Sumbayev VV, Budde A, Zhou J, Brune B: HIF-1 alpha protein as a target for S-nitrosation. FEBS Lett 2003, 535:106-112.

 

SJ Williams:

There are two very good volumes of Methods in Enzymology (Volumes 528 (2013) and Volume 437 (2008) which deal with methods to quantitate nitric oxide modifications in cells,(including whole cell imaging). These methods usually have delt with the reversible nitrosylation reaction however the irreversible covalent modification (highlighted in our Nitric oxide ebook) is quite difficult to measure yet is a very biologically relevant modification.

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A Protease for ‘Middle-down’ Proteomics

Author and Reporter: Ritu Saxena, Ph.D.

Neil Kelleher and his research team at Northwestern University have developed a method for enzymatic proteolysis large peptides for mass spectrometry–based proteomics using a protease OmpT. The method was published in a recent issue of the journal Nature. http://www.ncbi.nlm.nih.gov/pubmed/22706673

Proteomics is defined as the study of the structure and function of proteins. Proteomic technologies will play an important role in drug discovery, diagnostics and molecular medicine because is the link between genes, proteins and disease. As researchers study defective proteins that cause particular diseases, their findings will help develop new drugs that either alter the shape of a defective protein or mimic a missing one. http://www.ama-assn.org/ama/pub/physician-resources/medical-science/genetics-molecular-medicine/current-topics/proteomics.page Proteomics, although refers to the study of the structure and function of proteins, it is often specifically used for protein purification and mass spectrometry.

‘Bottom-up’ and ‘Top-down’ are the two main strategies for proteomic studies using mass spectrometry. In Bottom-up proteomics referred to as the more common method, proteins are broken down into smaller pieces through enzymatic digestion followed by characterization into amino acid sequences and post translational modifications prior to analysis by mass spectrometry. By identifying and sequencing these smaller pieces, researchers can then determine the identity of the protein they make up. In Top-down proteomics, on the other hand, the process of proteolysis is skipped and it focuses on complete characterization of intact proteins and their post-translational modifications (PTMs).

“Although both the top-down and bottom-up approaches continue to mature, they each have limitations. The tryptic peptides used in the bottom-up approach are the primary unit of measurement, but their relatively small size (typically ~8–25 residues long) leads to problems such as sample complex­ity, difficulties in assigning peptides to specific gene products rather than protein groups, and loss of single and combinato­rial PTM information. The top-down approach handles these issues by characterizing intact proteins, but its success declines in the high-mass region. Therefore, a hybrid approach based on 2–20 kDa peptides could unite positive aspects of both bottom-up and top-down proteomics” says Kelleher et al in the research article.

The hybrid approach, referred to as ‘middle-down’ proteomics would enable the analysis of complex mixtures pre-sorted by protein size. Previously research efforts ‘middle-down’ proteomics included exploring the restricted proteolysis with enzyme alternatives to Trypsin and chemical methods (such as microwave-assisted acid hydrolysis), However, these methods generated peptides that were marginally longer than those produced by trypsin digestion. For the current study, Kelleher adds “We established an OmpT-based middle-down platform to analyze complex mixtures pre-sorted by protein size. After inte­grating the data from the middle-down workflow that was applied to ~20–100-kDa proteins fractionated from the HeLa cell proteome, we identified 3,697 unique peptides (average size: 6.3 kDa) from 1,038 unique proteins (26% average sequence coverage) at an esti­mated 1% false discovery rate”.

OmpT, a protease derived from Escherichia coli K12 outer membrane belongs to the novel omptin protease family10 and is known to cleave between two consecutive basic amino acid residues (Lys/Arg-Lys/Arg). The authors developed OmpT into an efficient rea­gent to generate >2-kDa peptides for middle-down proteomics, thus, utilizing OmpT to achieve robust, yet restricted, proteolysis of a complex genome. http://www.ncbi.nlm.nih.gov/pubmed/22706673

Researcher Kelleher and his team have been in news earlier for their work on ‘top-down’ proteomics when his team developed a new method that could separate and identify thousands of protein molecules quickly. In the first large-scale demonstration of the top-down method, the researchers were able to identify more than 3,000 protein forms created from 1,043 genes from human HeLa cells. The study was published in last year in the October issue of the journal Nature. http://www.ncbi.nlm.nih.gov/pubmed?term=22037311

Thus, Kelleher and his group was able to demonstrate that OmpT-based proteomic approach has a robust and restricted proteolysis capacity making it an attractive option for mass-spectrometry-based analysis of primary structure of protein.

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