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In this lesson we will go over the biochemical makeup and formation of various actin containing cellular structures involved in cellular motility, structure, as well as the dynamics of muscular contraction. The lesson had been put on your Canvas and I am emailing you the Google Docs version. If you are having problems downloading you can download here (I believe maybe the Canvas version had problems with embedding videos properly so that is why I am sending you also by email)
After opening the powerpoint (or Google Doc) please review with the following notes which highlight some concepts as well as some reviews and reminders of past lectures. It may be handy to also have lecture 5 handy if you need to refer to it. In between some sections there will be polls (really multiple choice quizzes DON’T WORRY you will not be graded on them but they are for your benefit. There will also be a section under Comments all the way at the end and at the last quiz where you can also ask questions.
In addition you can also leave comments at the very bottom which can be answered.
Slide 2 of lesson 6 is a refresher of the end of our last lecture, talking about Actin Binding Regulatory Proteins.
The picture above shows a brief review of some of the structures and actin binding proteins involved in helping to form these actin filament structures (like filamin in cross linked structures, profilin which binds the actin monomers [G-actin] and helps with addition of these monomers to the leading plus end.
*** Remember G-actin (Globular Actin) is the monomer and F-actin (filamentious actin) is the polymerized actin strand [filament]
Also remember from the last lecture that G-Actin as monomer has affinity for ATP {Adenosine triphosphate} and these G-Actin-ATP will be able to polymerize to form the F-Actin form. Also F-actin can then hydrolyze the ATP to ADP and inorganic phosphate. At this point the actin-ADP unit looses affinity for the remaining F-Actin chain and depolymerization can occur
An event referred to as TREADMILLING or when the G actin units are removed from minus end and added to the plus (or growing barbed) end
Also remember that there is a critical concentration of G-Actin-ATP needed for bypassing the lag phase of nucleation before the elongation phase and the rate of addition to the plus end is faster than addition to minus end and greater than the rate of depolymerization at the minus end
Cell Structures That Involve Actin (see links for more information)
Nucleating proteins Arp (actin related protein and Formins
Arp ====> formation of lamellipodia
Formins ====> formation of stress fibers
Process involving formins starts with a signaling event by activation of a G-protein, the GTP binding protein Rho
Rho is a subfamily member of the Ras superfamily. The Rho family consists of cdc42, rac1, and RhoA (we will discuss at a later date). Rho acts like G proteins, as a molecular switch.
Note that just like the Ras member of G-proteins and the Ras GTP/GDP cycle, the Rho activation, deactivation cycle also depends on GEFs [Guanine nucleotide exchange factors] and GAPs [GTPase activating proteins] and also GDIs [guanine nucleotide dissociation inhibitors which we will discuss later but involved in preventing Rho diffusion in the cell, acting as a tether].
Myosin and Motor (muscle) Function; Neuromuscular junctions, the sarcoplasmic reticulum and Ohhh the plethora of signaling events
In this section, from slides 29 to 54, we talk about myosin and the interactions between myosin and actin in formation of the contractile unit of the muscle (skeletal).
We also talk about some familiar signaling events, in particular the neuromuscular junction.
At this junction is a special type of acetylcholine receptor
Remember we talked about two types of acetylcholine receptors:
muscarinic receptors – typical GPCRs that tranduce the signal via Gi or Gq depending on the muscarinic subtype
nicotinic receptors – these are ligand {receptor} operated channels and when activated opens a Na+ channel which leads to depolarization
Now the depolarization activates another set of channels, the voltage operated calcium channels so we have two types of ion channels: Receptor {ligand} operated channels and Voltage operated channels. These are sometimes abbreviated as ROCs and VOCs.
The unit of the myofibril on the contactile unit of the skeletal muscle is the sarcomere and upon the calcium transient, the sarcomere shortens with the two z-disks moving closer to each other as shown in the video in the lecture.
Also briefly review the introduction part on microtubules. We will finish that next week. Note that the microtubule is comprised of the protein tubulin, which is another GTP binding protein.
For other articles and more information please see
Poll for Students of #TUBiol3373: Cell Signaling and Motility class
Author: Stephen J. Williams, PhD
As your instructor of your Cell Signaling and Motility class, I am conducting this poll to try to understand how useful the supplemental information of this website (https://pharmaceuticalintelligence.com/) and online journal was toward your needs in the course Cell Signaling and Motility. In addition, I would like to determine the ease of use and to find needed information from this site in order for you to complete projects as well as find extra study materials. In addition, I am trying to understand if students might use this for other topics as well as the utility of the information in this site/journal for continued learning.
All answers are completely confidential and participation in this poll is voluntary, and no other information is collected.
Stephen J. Williams, PhD
Do you find using an open access, curated information platform like this site more useful than using multiple sources to find useful extra study/presentation material?
Do you find using a web based platform such as a site like this an easier communication platform for posting lecture notes/added information than a platform like Canvas?
There is a good reference to read on The Hallmarks of Cancer published first in 2000 and then updated with 2 new hallmarks in 2011 (namely the ability of cancer cells to reprogram their metabolism and 2. the ability of cancer cells to evade the immune system)
Please also go to other articles on this site which are relevant to this lecture. You can use the search box in the upper right hand corner of the Home Page or these are few links you might find interesting
Lesson 8 Cell Signaling and Motility: Lesson and Supplemental Information on Cell Junctions and ECM: #TUBiol3373
Curator: Stephen J. Williams, Ph.D.
Please click on the following link for the PowerPoint Presentation for Lecture 8 on Cell Junctions and the Extracellular Matrix: (this is same lesson from 2018 so don’t worry that file says 2018)
(for this lesson pay attention to the part that shows how Receptor Tyrosine Kinase activation (RTK) can lead to signaling to an integrin and also how the thrombin receptor leads to cellular signals both to GPCR (G-protein coupled receptors like the thrombin receptor, the ADP receptor; but also the signaling cascades that lead to integrin activation of integrins leading to adhesion to insoluble fibrin mesh of the newly formed clot and subsequent adhesion of platelets, forming the platelet plug during thrombosis.)
Lesson 5 Cell Signaling And Motility: Cytoskeleton & Actin: Curations and Articles of reference as supplemental information: #TUBiol3373
Curator: Stephen J. Williams, Ph.D.
Cell motility or migration is an essential cellular process for a variety of biological events. In embryonic development, cells migrate to appropriate locations for the morphogenesis of tissues and organs. Cells need to migrate to heal the wound in repairing damaged tissue. Vascular endothelial cells (ECs) migrate to form new capillaries during angiogenesis. White blood cells migrate to the sites of inflammation to kill bacteria. Cancer cell metastasis involves their migration through the blood vessel wall to invade surrounding tissues.
Please Click on the Following Powerpoint Presentation for Lesson 4 on the Cytoskeleton, Actin, and Filaments
This article, constitutes a broad, but not complete review of the emerging discoveries of the critical role of calcium signaling on cell motility and, by extension, embryonic development, cancer metastasis, changes in vascular compliance at the junction between the endothelium and the underlying interstitial layer. The effect of calcium signaling on the heart in arrhtmogenesis and heart failure will be a third in this series, while the binding of calcium to troponin C in the synchronous contraction of the myocardium had been discussed by Dr. Lev-Ari in Part I.
Universal MOTIFs essential to skeletal muscle, smooth muscle, cardiac syncytial muscle, endothelium, neovascularization, atherosclerosis and hypertension, cell division, embryogenesis, and cancer metastasis. The discussion will be presented in several parts:
1. Biochemical and signaling cascades in cell motility
2. Extracellular matrix and cell-ECM adhesions
3. Actin dynamics in cell-cell adhesion
4. Effect of intracellular Ca++ action on cell motility
5. Regulation of the cytoskeleton
6. Role of thymosin in actin-sequestration
7. T-lymphocyte signaling and the actin cytoskeleton
(This article has a great 3D visualization of a microtuble structure as well as description of genetic diseases which result from mutations in tubulin and effects on intracellular trafficking of proteins.
A latticework of tiny tubes called microtubules gives your cells their shape and also acts like a railroad track that essential proteins travel on. But if there is a glitch in the connection between train and track, diseases can occur. In the November 24, 2015 issue of PNAS, Tatyana Polenova, Ph.D., Professor of Chemistry and Biochemistry, and her team at the University of Delaware (UD), together with John C. Williams, Ph.D., Associate Professor at the Beckman Research Institute of City of Hope in Duarte, California, reveal for the first time — atom by atom — the structure of a protein bound to a microtubule. The protein of focus, CAP-Gly, short for “cytoskeleton-associated protein-glycine-rich domains,” is a component of dynactin, which binds with the motor protein dynein to move cargoes of essential proteins along the microtubule tracks. Mutations in CAP-Gly have been linked to such neurological diseases and disorders as Perry syndrome and distal spinal bulbar muscular dystrophy.
Today’s lesson 3 explains how extracellular signals are transduced (transmitted) into the cell through receptors to produce an agonist-driven event (effect). This lesson focused on signal transduction from agonist through G proteins (GTPases), and eventually to the effectors of the signal transduction process. Agonists such as small molecules like neurotransmitters, hormones, nitric oxide were discussed however later lectures will discuss more in detail the large growth factor signalings which occur through receptor tyrosine kinases and the Ras family of G proteins as well as mechanosignaling through Rho and Rac family of G proteins.
Transducers: The Heterotrimeric G Proteins (GTPases)
An excellent review of heterotrimeric G Proteins found in the brain is given by
Cyclic AMP is an important second messenger. It forms, as shown, when the membrane enzyme adenylyl cyclase is activated (as indicated, by the alpha subunit of a G protein).
The cyclic AMP then goes on the activate specific proteins. Some ion channels, for example, are gated by cyclic AMP. But an especially important protein activated by cyclic AMP is protein kinase A, which goes on the phosphorylate certain cellular proteins. The scheme below shows how cyclic AMP activates protein kinase A.
Updated 7/15/2019
Additional New Studies on Regulation of the Beta 2 Adrenergic Receptor
We had discussed regulation of the G protein coupled beta 2 adrenergic receptor by the B-AR receptor kinase (BARK)/B arrestin system which uncouples and desensitizes the receptor from its G protein system. In an article by Xiangyu Liu in Science in 2019, the authors describe another type of allosteric modulation (this time a POSITIVE allosteric modulation) in the intracellular loop 2. See below:
Mechanism of β2AR regulation by an intracellular positive allosteric modulator
Xiangyu Liu1,*, Ali Masoudi2,*, Alem W. Kahsai2,*, Li-Yin Huang2, Biswaranjan Pani2, Dean P. Staus2, Paul J. Shim2, Kunio Hirata3,4, Rishabh K. Simhal2, Allison M. Schwalb2, Paula K. Rambarat2, Seungkirl Ahn2, Robert J. Lefkowitz2,5,6,†, Brian Kobilka1
Positive reinforcement in a GPCR
Many drug discovery efforts focus on G protein–coupled receptors (GPCRs), a class of receptors that regulate many physiological processes. An exemplar is the β2-adrenergic receptor (β2AR), which is targeted by both blockers and agonists to treat cardiovascular and respiratory diseases. Most GPCR drugs target the primary (orthosteric) ligand binding site, but binding at allosteric sites can modulate activation. Because such allosteric sites are less conserved, they could possibly be targeted more specifically. Liu et al. report the crystal structure of β2AR bound to both an orthosteric agonist and a positive allosteric modulator that increases receptor activity. The structure suggests why the modulator compound is selective for β2AR over the closely related β1AR. Furthermore, the structure reveals that the modulator acts by enhancing orthosteric agonist binding and stabilizing the active conformation of the receptor.
Abstract
Drugs targeting the orthosteric, primary binding site of G protein–coupled receptors are the most common therapeutics. Allosteric binding sites, elsewhere on the receptors, are less well-defined, and so less exploited clinically. We report the crystal structure of the prototypic β2-adrenergic receptor in complex with an orthosteric agonist and compound-6FA, a positive allosteric modulator of this receptor. It binds on the receptor’s inner surface in a pocket created by intracellular loop 2 and transmembrane segments 3 and 4, stabilizing the loop in an α-helical conformation required to engage the G protein. Structural comparison explains the selectivity of the compound for β2– over the β1-adrenergic receptor. Diversity in location, mechanism, and selectivity of allosteric ligands provides potential to expand the range of receptor drugs.
Recent structures of GPCRs bound to allosteric modulators have revealed that receptor surfaces are decorated with diverse cavities and crevices that may serve as allosteric modulatory sites (1). This substantiates the notion that GPCRs are structurally plastic and can be modulated by a variety of allosteric ligands through distinct mechanisms (2-7). Most of these structures have been solved with negative allosteric modulators (NAMs), which stabilize receptors in their inactive states (1). To date, only a single structure of an active GPCR bound to a small-molecule positive allosteric modulator (PAM) has been reported, namely, the M2 muscarinic acetylcholine receptor with LY2119620 (8). Thus, mechanisms of PAMs and their potential binding sites remain largely unexplored.
Fig 1. Structure of the active state T4L-B2AR in complex with the orthosteric agonist BI-167107, nanobody 689, and compound 6FA. (A) The chemical structure of compound-6FA (Cmpd-6FA). (B) Isoproterenol (ISO) competition binding with 125I-cyanopindolol (CYP) to the β2AR reconstituted in nanodisks in the presence of vehicle (0.32% dimethylsulfoxide; DMSO), Cmpd-6, or Cmpd-6FA at 32 μM. Values were normalized to percentages of the maximal 125I-CYP binding level obtained from a one-site competition binding–log IC50 (median inhibitory concentration) curve fit. Binding curves were generated by GraphPad Prism. Points on curves represent mean ± SEM obtained from five independent experiments performed in duplicate. (C) Analysis of Cmpd-6FA interaction with the BI-167107–bound β2AR by ITC. Representative thermogram (inset) and binding isotherm, of three independent experiments, with the best titration curve fit are shown. Summary of thermodynamic parameters obtained by ITC: binding affinity (KD = 1.2 ± 0.1 μM), stoichiometry (N = 0.9 ± 0.1 sites), enthalpy (ΔH = 5.0 ± 1.2 kcal mol−1), and entropy (ΔS =13 ± 2.0 cal mol−1 deg−1). (D) Side view of T4L-β2AR bound to the orthosteric agonist BI-167107, nanobody 6B9 (Nb6B9), and Cmpd-6FA. The gray box indicates the membrane layer as defined by the OPM database. (E) Close-up view of Cmpd-6FA binding site. Covering Cmpd-6FA is 2Fo– Fc electron density contoured at 1.0 σ (green mesh).From Science 28 Jun 2019:
Vol. 364, Issue 6447, pp. 1283-1287
Fig 3. Fig. 3Mechanism of allosteric activation of the β2AR by Cmpd-6FA.
(A) Superposition of the inactive β2AR bound to the antagonist carazolol (PDB code: 2RH1) and the active β2AR bound to the agonist BI-167107, Cmpd-6FA, and Nb6B9. Close-up view of the Cmpd-6FA binding site is shown. The residues of the inactive (yellow) and active (blue) β2AR are depicted, and the hydrogen bond formed between Asp1303.49and Tyr141ICL2 in the active state is indicated by a black dashed line. (B) Topography of Cmpd-6FA binding surface on the active β2AR (left, blue) and the corresponding surface of the inactive β2AR (right, yellow) with Cmpd-6FA (orange sticks) docked on top. Molecular surfaces are of only those residues involved in interaction with Cmpd-6FA. Steric clash between Cmpd-6FA and the surface of inactive β2AR is represented by a purple asterisk. (C) Overlay of the β2AR bound to BI-167107, Nb6B9, and Cmpd-6FA with the β2AR–Gscomplex (PDB code: 3SN6). The inset shows the position of Phe139ICL2 relative to the α subunit of Gs. (D) Superposition of the active β2AR bound to the agonist BI-167107, Nb6B9, and Cmpd-6FA (blue) with the inactive β2AR bound to carazolol (yellow) (PDB code: 2RH1) as viewed from the cytoplasm. For clarity, Nb6B9 and the orthosteric ligands are omitted. The arrows indicate shifts in the intracellular ends of the TM helices 3, 5, and 6 upon activation and their relative distances.
Allosteric sites may not face the same evolutionary pressure as do orthosteric sites, and thus are more divergent across subtypes within a receptor family (24–26). Therefore, allosteric sites may provide a greater source of specificity for targeting GPCRs.
D. M. Thal, A. Glukhova, P. M. Sexton, A. Christopoulos, Structural insights into G-protein-coupled receptor allostery. Nature 559, 45–53 (2018). doi:10.1038/s41586-018-0259-zpmid:29973731CrossRefPubMedGoogle Scholar
D. Wacker, R. C. Stevens, B. L. Roth, How Ligands Illuminate GPCR Molecular Pharmacology. Cell 170, 414–427 (2017).
D. P. Staus, R. T. Strachan, A. Manglik, B. Pani, A. W. Kahsai, T. H. Kim, L. M. Wingler, S. Ahn, A. Chatterjee, A. Masoudi, A. C. Kruse, E. Pardon, J. Steyaert, W. I. Weis, R. S. Prosser, B. K. Kobilka, T. Costa, R. J. Lefkowitz, Allosteric nanobodies reveal the dynamic range and diverse mechanisms of G-protein-coupled receptor activation. Nature 535, 448–452 (2016). doi:10.1038/nature18636pmid:27409812CrossRefPubMedGoogle Scholar
A. Manglik, T. H. Kim, M. Masureel, C. Altenbach, Z. Yang, D. Hilger, M. T. Lerch, T. S. Kobilka, F. S. Thian, W. L. Hubbell, R. S. Prosser, B. K. Kobilka, Structural Insights into the Dynamic Process of β2-Adrenergic Receptor Signaling. Cell 161, 1101–1111 (2015). doi:10.1016/j.cell.2015.04.043pmid:25981665CrossRefPubMedGoogle Scholar
5, L. Ye, N. Van Eps, M. Zimmer, O. P. Ernst, R. S. Prosser, Activation of the A2A adenosine G-protein-coupled receptor by conformational selection. Nature 533, 265–268 (2016). doi:10.1038/nature17668pmid:27144352CrossRefPubMedGoogle Scholar
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Additional information on Nitric Oxide as a Cellular Signal
Nitric oxide is actually a free radical and can react with other free radicals, resulting in a very short half life (only a few seconds) and so in the body is produced locally to its site of action (i.e. in endothelial cells surrounding the vascular smooth muscle, in nerve cells). In the late 1970s, Dr. Robert Furchgott observed that acetylcholine released a substance that produced vascular relaxation, but only when the endothelium was intact. This observation opened this field of research and eventually led to his receiving a Nobel prize. Initially, Furchgott called this substance endothelium-derived relaxing factor (EDRF), but by the mid-1980s he and others identified this substance as being NO.
Nitric oxide is implicated in many pathologic processes as well. Nitric oxide post translational modifications have been attributed to nitric oxide’s role in pathology however, although the general mechanism by which nitric oxide exerts its physiological effects is by stimulation of soluble guanylate cyclase to produce cGMP, these post translational modifications can act as a cellular signal as well. For more information of NO pathologic effects and how NO induced post translational modifications can act as a cellular signal see the following:
BIO 3096, Cell Structure and Function (Minimum Grade of C- | May not be taken concurrently).
Description:
The communication among cells is essential for the regulation of the development of an organism and for the control of its physiology and homeostasis. Aberrant cellular signaling events are often associated with human pathological conditions, such as cancer, neurological disorders, cardiovascular diseases and so on. The full characterization of cell signaling systems may provide useful insights into the pathogenesis of several human maladies.
Text:
Molecular Biology of the Cell 6th Edition, Alberts et al. Garland Science. This textbook is available at the Temple Bookstore.
Grading:
The final grade will be based on the score of four examinations that include both group and individuals assignment. Each exam accounts for 25% of the final grade. There will be no make-up tests during the course. If you have a documented medical excuse and you contact me as soon as possible after the emergency, I will arrange a make-up exam. Complaints regarding the grading will not be considered later than two weeks after the test is returned.
Blackboard:
Announcements will be readily posted on Blackboard. It is your responsibility to check Blackboard periodically.
Attendance: Lecture attendance is mandatory. In addition, punctuality is expected.
Disabilities: Students with documented disabilities who need particular accommodation should contact me privately as soon as possible.
Honesty and Civility:
Students must follow the Temple’s Code of Conduct (see http://www.temple.edu/assistance/udc/coc.htm). This Code of Conduct prohibits: 1. Academic dishonesty and impropriety, including plagiarism and cheating. 2. Interfering or attempting to interfere with or disrupting the conduct of classes or any other activity of the University.”
This policy sets the parameters for freedom to learn and freedom to teach, which constitute the pillars of academia.
SCHEDULE
This schedule is a general outline, which may be eventually modified. Changes will be announced in advance. Please, always check Blackboard and your email.
Date
Topic
Jan 13
Introduction (course overview and discussion of syllabus). General concepts: Eukaryotic and prokaryotic cell; DNA, RNA and proteins: Protein synthesis
Jan 20
Martin Luther King, Jr. Day (no classes held)
Jan 27
DNA analysis, RNA analysis; Proteins analysis; Microscopy.
Feb 3
Signaling: general concepts; Introduction to G-proteins; signaling via G-proteins (1)
Feb 10
Exam 1: In class presentation (group assignment)
Feb 17
Signaling via G-proteins (2); tyrosine kinase receptors signaling; Ras-MAPK pathway.
Medical consequences of aberrant signaling pathways; production of small molecules for protein kinases In cancer therapy.
Study days
May 4
Exam 4: In class presentation (group assignment)
Below is Powerpoint presentations for Lesson 1 and Lesson 2. Please check for UPDATES on this page for additional supplemental information for these Lessons including articles from this Online Access Journal
The following articles and curations discuss about the new paradigm how we now envision DNA, in particular how we now understand that the important parts of the genome are not just the exons which code for proteins but also the intronic DNA, which contains all the regulatory elements such as promoters, lncDNA, miRNA sequences etc. These are good reads for your presentations.
And on How the Cell Creates Diversity post the Genetic Code by Use of Post Translational Modifications to Bring Diversity to Protein Structure/Function
Curation of selected topics and articles on Role of G-Protein Coupled Receptors in Chronic Disease as supplemental information for #TUBiol3373
Curator: Stephen J. Williams, PhD
Below is a series of posts and articles related to the role of G protein coupled receptors (GPCR) in various chronic diseases. This is only a cursory collection and by no means represents the complete extensive literature on pathogenesis related to G protein function or alteration thereof. However it is important to note that, although we think of G protein signaling as rather short lived, quick, their chronic activation may lead to progression of various disease. As to whether disease onset, via GPCR, is a result of sustained signal, loss of desensitization mechanisms, or alterations of transduction systems is an area to be investigated.
Inflammatory and infectious factors are present in diseased airways that interact with G-protein coupled receptors (GPCRs), such as purinergic receptors and bradykinin (BK) receptors, to stimulate phospholipase C [PLC]. This is followed by the activation of inositol 1,4,5-trisphosphate (IP3)-dependent activation of IP3 channel receptors in the ER, which results in channel opening and release of stored Ca2+ into the cytoplasm. When ER Ca2+ stores are depleted a pathway for Ca2+ influx across the plasma membrane is activated. This has been referred to as “capacitative Ca2+ entry”, and “store-operated calcium entry” (3). In the next step PLC mediated Ca2+ i is mobilized as a result of GPCR activation by inflammatory mediators, which triggers cytokine production by Ca2+ i-dependent activation of the transcription factor nuclear factor kB (NF-kB) in airway epithelia.
Larry H. Bernstein, MD, FCAP, Curator discusses findings from a research team at University of California at San Diego (UCSD) which the neuropeptide hormone corticotropin-releasing factor (CRF) as having an important role in the etiology of Alzheimer’s Disease (AD). CRF activates the CRF receptor (a G stimulatory receptor). It was found inhibition of the CRF receptor prevented cognitive impairment in a mouse model of AD. Furthermore researchers at the Flanders Interuniversity Institute for Biotechnology found the loss of a protein called G protein-coupled receptor 3 (GPR3) may lower the amyloid plaque aggregation, resulting in improved cognitive function. Additionally inhibition of several G-protein coupled receptors alter amyloid precursor processing, providing a further mechanism of the role of GPCR in AD (see references in The role of G protein-coupled receptors in the pathology of Alzheimer’s disease by Amantha Thathiah and Bart De Strooper Nature Reviews Feb 2011; 12: 73-87 and read post).
Further curations and references of G proteins and chronic disease can be found at the Open Access journal https://pharmaceuticalintelligence.com using the search terms “GCPR” and “disease” in the Search box in the upper right of the home page.
Remember our lessons on the importance of signal termination. The CANONICAL WNT signaling (that is the β-catenin dependent signaling)
is terminated by the APC-driven degradation complex. This leads to the signal messenger β-catenin being degraded by the proteosome. Other examples of growth factor signaling that is terminated by a proteosome-directed include the Hedgehog signaling system, which is involved in growth and differentiation as well as WNTs and is implicated in various cancers.
A good article on the Hedgehog signaling pathway is found here:
All images in use for this article are under copyrights with Shutterstock.com
Cancer is expressed through a series of transformations equally involving metabolic enzymes and glucose, fat, and protein metabolism, and gene transcription, as a result of altered gene regulatory and transcription pathways, and also as a result of changes in cell-cell interactions. These are embodied in the following series of graphics.
Figure 1: Sonic_hedgehog_pathway
The Voice of Dr. Larry
The figure shows a modification of nuclear translocation by Sonic hedgehog pathway. The hedgehog proteins have since been implicated in the development of internal organs, midline neurological structures, and the hematopoietic system in humans. The Hh signaling pathway consists of three main components: the receptor patched 1 (PTCH1), the seven transmembrane G-protein coupled receptor smoothened (SMO), and the intracellular glioma-associated oncogene homolog (GLI) family of transcription factors.5The GLI family is composed of three members, including GLI1 (gene activating), GLI2 (gene activating and repressive), and GLI3 (gene repressive).6 In the absence of an activating signal from either Shh, Ihh or Dhh, PTCH1 exerts an inhibitory effect on the signal transducer SMO, preventing any downstream signaling from occurring.7 When Hh ligands bind and activate PTCH1, the inhibition on SMO is released, allowing the translocation of SMO into the cytoplasm and its subsequent activation of the GLI family of transcription factors.
Finally, termination of Hh signaling is also important for controlling the duration of pathway activity.Hh induced ubiquitination and degradation of Ci/Gli is the most well-established mechanism for limiting signal duration, and inhibiting this process can lead to cell patterning disruption and excessive cell proliferation (Di Marcotullio et al. 2006; Huntzicker et al. 2006; Kent et al. 2006; Zhang et al. 2006a; Di Marcotullio et al. 2007; Ou et al. 2007). In addition to Ci/Gli, a growing body of evidence suggests that ubiquitination also plays critical roles in regulating other Hh signaling components including Ptc, Smo, and Sufu. Thus, ubiquitination serves as a general mechanism in the dynamic regulation of the Hh pathway.
Overview of Hedgehog signaling showing the signal termination by ubiquitnation and subsequent degradation of the Gli transcriptional factors. obtained from Oncotarget 5(10):2881-911 · May 2014. GSK-3B as a Therapeutic Intervention in Cancer
Note that in absence of Hedgehog ligands Ptch inhibits Smo accumulation and activation but upon binding of Hedgehog ligands (by an autocrine or paracrine fashion) Ptch is now unable to inhibit Smo (evidence exists that Ptch is now targeted for degradation) and Smo can now inhibit Sufu-dependent and GSK-3B dependent induced degradation of Gli factors Gli1 and Gli2. Also note the Gli1 and Gli2 are transcriptional activators while Gli3 is a transcriptional repressor.
UPDATED 4/16/2019
Please click on the followinglinks for the Powerpoint presentation for lesson 9. In addition click on the mp4 links to download the movies so you can view them in Powerpoint slide 22:
Tumorigenic but noninvasive MCF-7 cells motility on an extracellular matrix derived from normal (3DCntrol) or tumor associated (TA) fibroblasts. Note that TA ECM is “soft” and not organized and tumor cells appear to move randomly if much at all.
Movie 2:
Note that these tumorigenic and invasive MDA-MB-231 breast cancer cells move in organized patterns on organized ECM derived from Tumor Associated (TA) fibroblasts than from the ‘soft’ or unorganized ECM derived from normal (3DCntrl) fibroblasts
The following contain curations of scientific articles from the site https://pharmaceuticalintelligence.com intended as additional reference material to supplement material presented in the lecture.
Wnts are a family of lipid-modified secreted glycoproteins which are involved in:
And in pathologic processes such as oncogenesis (refer toWnt/β-catenin Signaling [7.10]) and to your Powerpoint presentation
The curation Wnt/β-catenin Signaling is a comprehensive review of canonical and noncanonical Wnt signaling pathways
To review:
Activating the canonical Wnt pathway frees B-catenin from the degradation complex, resulting in B-catenin translocating to the nucleus and resultant transcription of B-catenin/TCF/LEF target genes.
Fig. 1 Canonical Wnt/FZD signaling pathway. (A) In the absence of Wnt signaling, soluble β-catenin is phosphorylated by a degradation complex consisting of the kinases GSK3β and CK1α and the scaffolding proteins APC and Axin1. Phosphorylated β-catenin is targeted for proteasomal degradation after ubiquitination by the SCF protein complex. In the nucleus and in the absence of β-catenin, TCF/LEF transcription factor activity is repressed by TLE-1; (B) activation of the canonical Wnt/FZD signaling leads to phosphorylation of Dvl/Dsh, which in turn recruits Axin1 and GSK3β adjacent to the plasma membrane, thus preventing the formation of the degradation complex. As a result, β-catenin accumulates in the cytoplasm and translocates into the nucleus, where it promotes the expression of target genes via interaction with TCF/LEF transcription factors and other proteins such as CBP, Bcl9, and Pygo.
NOTE: In the canonical signaling, the Wnt signal is transmitted via the Frizzled/LRP5/6 activated receptor to INACTIVATE the degradation complex thus allowing free B-catenin to act as the ultimate transducer of the signal.
Remember, as we discussed, the most frequent cancer-related mutations of WNT pathway constituents is in APC.
This shows how important the degradation complex is in controlling canonical WNT signaling.
Other cell signaling systems are controlled by protein degradation:
1. Question: In cell involving G-proteins, the signal can be terminated by desensitization mechanisms. How is both the canonical and noncanonical Wnt signal eventually terminated/desensitized?
We also discussed the noncanonical Wnt signaling pathway (independent of B-catenin induced transcriptional activity). Note that the canonical and noncanonical involve different transducers of the signal.
Noncanonical WNT Signaling
Note: In noncanonical signaling the transducer is a G-protein and second messenger system is IP3/DAG/Ca++ and/or kinases such as MAPK, JNK.
Depending on the different combinations of WNT ligands and the receptors, WNT signaling activates several different intracellular pathways (i.e. canonical versus noncanonical)
In addition different Wnt ligands are expressed at different times (temporally) and different cell types in development and in the process of oncogenesis.
The following paper on Wnt signaling in ovarian oncogenesis shows how certain Wnt ligands are expressed in normal epithelial cells but the Wnt expression pattern changes upon transformation and ovarian oncogenesis. In addition, differential expression of canonical versus noncanonical WNT ligands occur during the process of oncogenesis (for example below the authors describe the noncanonical WNT5a is expressed in normal ovarian epithelia yet WNT5a expression in ovarian cancer is lower than the underlying normal epithelium. However the canonical WNT10a, overexpressed in ovarian cancer cells, serves as an oncogene, promoting oncogenesis and tumor growth.
Epithelial ovarian cancer (EOC) remains the most lethal gynecological malignancy in the US. Thus, there is an urgent need to develop novel therapeutics for this disease. Cellular senescence is an important tumor suppression mechanism that has recently been suggested as a novel mechanism to target for developing cancer therapeutics. Wnt5a is a non-canonical Wnt ligand that plays a context-dependent role in human cancers. Here, we investigate the role of Wnt5a in regulating senescence of EOC cells. We demonstrate that Wnt5a is expressed at significantly lower levels in human EOC cell lines and in primary human EOCs (n = 130) compared with either normal ovarian surface epithelium (n = 31; p = 0.039) or fallopian tube epithelium (n = 28; p < 0.001). Notably, a lower level of Wnt5a expression correlates with tumor stage (p = 0.003) and predicts shorter overall survival in EOC patients (p = 0.003). Significantly, restoration of Wnt5a expression inhibits the proliferation of human EOC cells both in vitro and in vivo in an orthotopic EOC mouse model. Mechanistically, Wnt5a antagonizes canonical Wnt/β-catenin signaling and induces cellular senescence by activating the histone repressor A (HIRA)/promyelocytic leukemia (PML) senescence pathway. In summary, we show that loss of Wnt5a predicts poor outcome in EOC patients and Wnt5a suppresses the growth of EOC cells by triggering cellular senescence. We suggest that strategies to drive senescence in EOC cells by reconstituting Wnt5a signaling may offer an effective new strategy for EOC therapy.
Oncol Lett. 2017 Dec;14(6):6611-6617. doi: 10.3892/ol.2017.7062. Epub 2017 Sep 26.
Ovarian cancer is one of the five most malignant types of cancer in females, and the only currently effective therapy is surgical resection combined with chemotherapy. Wnt family member 10A (Wnt10a) has previously been identified to serve an oncogenic function in several tumor types, and was revealed to have clinical significance in renal cell carcinoma; however, there is still only limited information regarding the function of Wnt10a in the carcinogenesis of ovarian cancer. The present study identified increased expression levels of Wnt10a in two cell lines, SKOV3 and A2780, using reverse transcription-polymerase chain reaction. Functional analysis indicated that the viability rate and migratory ability of SKOV3 cells was significantly inhibited following Wnt10a knockdown using short interfering RNA (siRNA) technology. The viability rate of SKOV3 cells decreased by ~60% compared with the control and the migratory ability was only ~30% of that in the control. Furthermore, the expression levels of β-catenin, transcription factor 4, lymphoid enhancer binding factor 1 and cyclin D1 were significantly downregulated in SKOV3 cells treated with Wnt10a-siRNA3 or LGK-974, a specific inhibitor of the canonical Wnt signaling pathway. However, there were no synergistic effects observed between Wnt10a siRNA3 and LGK-974, which indicated that Wnt10a activated the Wnt/β-catenin signaling pathway in SKOV3 cells. In addition, using quantitative PCR, Wnt10a was overexpressed in the tumor tissue samples obtained from 86 patients with ovarian cancer when compared with matching paratumoral tissues. Clinicopathological association analysis revealed that Wnt10a was significantly associated with high-grade (grade III, P=0.031) and late-stage (T4, P=0.008) ovarian cancer. Furthermore, the estimated 5-year survival rate was 18.4% for patients with low Wnt10a expression levels (n=38), whereas for patients with high Wnt10a expression (n=48) the rate was 6.3%. The results of the present study suggested that Wnt10a serves an oncogenic role during the carcinogenesis and progression of ovarian cancer via the Wnt/β-catenin signaling pathway.
Targeting the Wnt Pathway includes curations of articles related to the clinical development of Wnt signaling inhibitors as a therapeutic target in various cancers including hepatocellular carcinoma, colon, breast and potentially ovarian cancer.
2. Question: Given that different Wnt ligands and receptors activate differentsignaling pathways, AND WNT ligands can be deferentially and temporally expressed in various tumor types and the process of oncogenesis, how would you approach a personalized therapy targeting the WNT signaling pathway?
3. Question: What are the potential mechanisms of either intrinsic or acquired resistance to Wnt ligand antagonists being developed?
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