Publications by authors named "Eileen M Lafer"

47 Publications

Liquid-like protein interactions catalyse assembly of endocytic vesicles.

Nat Cell Biol 2021 Apr 5;23(4):366-376. Epub 2021 Apr 5.

Department of Biomedical Engineering, The University of Texas at Austin, Austin, TX, USA.

During clathrin-mediated endocytosis, dozens of proteins assemble into an interconnected network at the plasma membrane. As initiators of endocytosis, Eps15 and Fcho1/2 concentrate downstream components, while permitting dynamic rearrangement within the budding vesicle. How do initiator proteins meet these competing demands? Here we show that Eps15 and Fcho1/2 rely on weak, liquid-like interactions to catalyse endocytosis. In vitro, these weak interactions promote the assembly of protein droplets with liquid-like properties. To probe the physiological role of these liquid-like networks, we tuned the strength of initiator protein assembly in real time using light-inducible oligomerization of Eps15. Low light levels drove liquid-like assemblies, restoring normal rates of endocytosis in mammalian Eps15-knockout cells. By contrast, initiator proteins formed solid-like assemblies upon exposure to higher light levels, which stalled vesicle budding, probably owing to insufficient molecular rearrangement. These findings suggest that liquid-like assembly of initiator proteins provides an optimal catalytic platform for endocytosis.
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http://dx.doi.org/10.1038/s41556-021-00646-5DOI Listing
April 2021

Clathrin senses membrane curvature.

Biophys J 2021 Mar 30;120(5):818-828. Epub 2021 Jan 30.

Department of Biomedical Engineering, The University of Texas at Austin, Austin, Texas; Institute for Cellular and Molecular Biology, The University of Texas at Austin, Austin, Texas. Electronic address:

The ability of proteins to assemble at sites of high membrane curvature is essential to diverse membrane remodeling processes, including clathrin-mediated endocytosis. Multiple adaptor proteins within the clathrin pathway have been shown to sense regions of high membrane curvature, leading to local recruitment of the clathrin coat. Because clathrin triskelia do not bind to the membrane directly, it has remained unclear whether the clathrin coat plays an active role in sensing membrane curvature or is passively recruited by adaptor proteins. Using a synthetic tag to assemble clathrin directly on membrane surfaces, here we show that clathrin is a strong sensor of membrane curvature, comparable with previously studied adaptor proteins. Interestingly, this sensitivity arises from clathrin assembly rather than from the properties of unassembled triskelia, suggesting that triskelia have preferred angles of interaction, as predicted by earlier structural data. Furthermore, when clathrin is recruited by adaptors, its curvature sensitivity is amplified by 2- to 10-fold, such that the resulting protein complex is up to 100 times more likely to assemble on a highly curved surface compared with a flatter one. This exquisite sensitivity points to a synergistic relationship between the coat and its adaptor proteins, which enables clathrin to pinpoint sites of high membrane curvature, an essential step in ensuring robust membrane traffic. More broadly, these findings suggest that protein networks, rather than individual protein domains, are likely the most potent drivers of membrane curvature sensing.
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http://dx.doi.org/10.1016/j.bpj.2020.12.035DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8008260PMC
March 2021

Chaperone proteins as ameliorators of α-synuclein-induced synaptic pathologies: insights into Parkinson's disease.

Neural Regen Res 2021 Jun;16(6):1198-1199

The Eugene Bell Center for Regenerative Biology & Tissue Engineering, Marine Biological Laboratory, Woods Hole, MA, USA.

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http://dx.doi.org/10.4103/1673-5374.300431DOI Listing
June 2021

Hsc70 Ameliorates the Vesicle Recycling Defects Caused by Excess α-Synuclein at Synapses.

eNeuro 2020 Jan/Feb;7(1). Epub 2020 Jan 31.

The Eugene Bell Center for Regenerative Biology and Tissue Engineering Marine Biological Laboratory, Woods Hole, MA 02543

α-Synuclein overexpression and aggregation are linked to Parkinson's disease (PD), dementia with Lewy bodies (DLB), and several other neurodegenerative disorders. In addition to effects in the cell body, α-synuclein accumulation occurs at presynapses where the protein is normally localized. While it is generally agreed that excess α-synuclein impairs synaptic vesicle trafficking, the underlying mechanisms are unknown. We show here that acute introduction of excess human α-synuclein at a classic vertebrate synapse, the lamprey reticulospinal (RS) synapse, selectively impaired the uncoating of clathrin-coated vesicles (CCVs) during synaptic vesicle recycling, leading to an increase in endocytic intermediates and a severe depletion of synaptic vesicles. Furthermore, human α-synuclein and lamprey γ-synuclein both interact with Hsc70, the chaperone protein that uncoats CCVs at synapses. After introducing excess α-synuclein, Hsc70 availability was reduced at stimulated synapses, suggesting Hsc70 sequestration as a possible mechanism underlying the synaptic vesicle trafficking defects. In support of this hypothesis, increasing the levels of exogenous Hsc70 along with α-synuclein ameliorated the CCV uncoating and vesicle recycling defects. These experiments identify a reduction in Hsc70 availability at synapses, and consequently its function, as the mechanism by which α-synuclein induces synaptic vesicle recycling defects. To our knowledge, this is the first report of a viable chaperone-based strategy for reversing the synaptic vesicle trafficking defects associated with excess α-synuclein, which may be of value for improving synaptic function in PD and other synuclein-linked diseases.
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http://dx.doi.org/10.1523/ENEURO.0448-19.2020DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7031854PMC
January 2020

Molecular Mechanisms of Membrane Curvature Sensing by a Disordered Protein.

J Am Chem Soc 2019 07 20;141(26):10361-10371. Epub 2019 Jun 20.

Department of Biomedical Engineering , The University of Texas at Austin , Austin , Texas 78712 , United States.

The ability of proteins to sense membrane curvature is essential for the initiation and assembly of curved membrane structures. Established mechanisms of curvature sensing rely on proteins with specific structural features. In contrast, it has recently been discovered that intrinsically disordered proteins, which lack a defined three-dimensional fold, can also be potent sensors of membrane curvature. How can an unstructured protein sense the structure of the membrane surface? Many disordered proteins that associate with membranes have two key physical features: a high degree of conformational entropy and a high net negative charge. Binding of such proteins to membrane surfaces results simultaneously in a decrease in conformational entropy and an increase in electrostatic repulsion by anionic lipids. Here we show that each of these effects gives rise to a distinct mechanism of curvature sensing. Specifically, as the curvature of the membrane increases, the steric hindrance between the disordered protein and membrane is reduced, leading to an increase in chain entropy. At the same time, increasing membrane curvature increases the average separation between anionic amino acids and lipids, creating an electrostatic preference for curved membranes. Using quantitative imaging of membrane vesicles, our results demonstrate that long disordered amino acid chains with low net charge sense curvature predominately through the entropic mechanism. In contrast, shorter, more highly charged amino acid chains rely largely on the electrostatic mechanism. These findings provide a roadmap for predicting and testing the curvature sensitivity of the large and diverse set of disordered proteins that function at cellular membranes.
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http://dx.doi.org/10.1021/jacs.9b03927DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6610580PMC
July 2019

The Physics of Entropic Pulling: A Novel Model for the Hsp70 Motor Mechanism.

Int J Mol Sci 2019 May 11;20(9). Epub 2019 May 11.

Department of Biochemistry and Structural Biology, University of Texas Health Science Center, San Antonio, TX 78229-3900, USA.

Hsp70s use ATP to generate forces that disassemble protein complexes and aggregates, and that translocate proteins into organelles. Entropic pulling has been proposed as a novel mechanism, distinct from the more familiar power-stroke and Brownian ratchet models, for how Hsp70s generate these forces. Experimental evidence supports entropic pulling, but this model may not be well understood among scientists studying these systems. In this review we address persistent misconceptions regarding the dynamics of proteins in solution that contribute to this lack of understanding, and we clarify the basic physics of entropic pulling with some simple analogies. We hope that increased understanding of the entropic pulling mechanism will inform future efforts to characterize how Hsp70s function as motors, and how they coordinate with their regulatory cochaperones in mechanochemical cycles that transduce the energy of ATP hydrolysis into physical changes in their protein substrates.
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http://dx.doi.org/10.3390/ijms20092334DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6539501PMC
May 2019

BAR scaffolds drive membrane fission by crowding disordered domains.

J Cell Biol 2019 02 30;218(2):664-682. Epub 2018 Nov 30.

Department of Biomedical Engineering, University of Texas at Austin, Austin, TX

Cellular membranes are continuously remodeled. The crescent-shaped bin-amphiphysin-rvs (BAR) domains remodel membranes in multiple cellular pathways. Based on studies of isolated BAR domains in vitro, the current paradigm is that BAR domain-containing proteins polymerize into cylindrical scaffolds that stabilize lipid tubules. But in nature, proteins that contain BAR domains often also contain large intrinsically disordered regions. Using in vitro and live cell assays, here we show that full-length BAR domain-containing proteins, rather than stabilizing membrane tubules, are instead surprisingly potent drivers of membrane fission. Specifically, when BAR scaffolds assemble at membrane surfaces, their bulky disordered domains become crowded, generating steric pressure that destabilizes lipid tubules. More broadly, we observe this behavior with BAR domains that have a range of curvatures. These data suggest that the ability to concentrate disordered domains is a key driver of membrane remodeling and fission by BAR domain-containing proteins.
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http://dx.doi.org/10.1083/jcb.201807119DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6363457PMC
February 2019

Synergy between intrinsically disordered domains and structured proteins amplifies membrane curvature sensing.

Nat Commun 2018 10 8;9(1):4152. Epub 2018 Oct 8.

Department of Biomedical Engineering, The University of Texas at Austin, Austin, TX, 78712, USA.

The ability of proteins to sense membrane curvature is essential to cellular function. All known sensing mechanisms rely on protein domains with specific structural features such as wedge-like amphipathic helices and crescent-shaped BAR domains. Yet many proteins that contain these domains also contain large intrinsically disordered regions. Here we report that disordered domains are themselves potent sensors of membrane curvature. Comparison of Monte Carlo simulations with in vitro and live-cell measurements demonstrates that the polymer-like behavior of disordered domains found in endocytic proteins drives them to partition preferentially to convex membrane surfaces, which place fewer geometric constraints on their conformational entropy. Further, proteins containing both structured curvature sensors and disordered regions are more than twice as curvature sensitive as their respective structured domains alone. These findings demonstrate an entropic mechanism of curvature sensing that is independent of protein structure and illustrate how structured and disordered domains can synergistically enhance curvature sensitivity.
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http://dx.doi.org/10.1038/s41467-018-06532-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6175956PMC
October 2018

Anti-HIV IgM protects against mucosal SHIV transmission.

AIDS 2018 07;32(11):F5-F13

Department of Virology and Immunology, Texas Biomedical Research Institute.

Objective: Worldwide, most new HIV infections occur through mucosal exposure. Immunoglobulin M (IgM) is the first antibody class generated in response to infectious agents; IgM is present in the systemic circulation and in mucosal fluids as secretory IgM. We sought to investigate for the first time the role of IgM in preventing AIDS virus acquisition in vivo.

Design: Recombinant polymeric monoclonal IgM was generated from the neutralizing monoclonal IgG1 antibody 33C6-IgG1, tested in vitro, and given by passive intrarectal immunization to rhesus macaques 30 min before intrarectal challenge with simian-human immunodeficiency virus (SHIV) that carries an HIV-1 envelope gene.

Results: In vitro, 33C6-IgM captured virions more efficiently and neutralized the challenge SHIV with a 50% inhibitory molar concentration (IC50) that was 1 log lower than that for 33C6-IgG1. The IgM form also exhibited significantly higher affinity and avidity compared with 33C6-IgG1. After intrarectal administration, 33C6-IgM prevented viremia in four out of six rhesus macaques after high-dose intrarectal SHIV challenge. Five out of six rhesus macaques given 33C6-IgG1 were protected at a five times higher molar concentration compared with the IgM form; all untreated controls became highly viremic. Rhesus macaques passively immunized with 33C6-IgM with breakthrough infection had notably early development of autologous neutralizing antibody responses.

Conclusion: Our primate model data provide the first proof-of-concept that mucosal IgM can prevent mucosal HIV transmission and have implications for HIV prevention and vaccine development.
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http://dx.doi.org/10.1097/QAD.0000000000001857DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6380498PMC
July 2018

Clathrin coat controls synaptic vesicle acidification by blocking vacuolar ATPase activity.

Elife 2018 04 13;7. Epub 2018 Apr 13.

Synaptic Vesicle Dynamics Group, European Neuroscience Institute, University Medical Center Göttingen, Göttingen, Germany.

Newly-formed synaptic vesicles (SVs) are rapidly acidified by vacuolar adenosine triphosphatases (vATPases), generating a proton electrochemical gradient that drives neurotransmitter loading. Clathrin-mediated endocytosis is needed for the formation of new SVs, yet it is unclear when endocytosed vesicles acidify and refill at the synapse. Here, we isolated clathrin-coated vesicles (CCVs) from mouse brain to measure their acidification directly at the single vesicle level. We observed that the ATP-induced acidification of CCVs was strikingly reduced in comparison to SVs. Remarkably, when the coat was removed from CCVs, uncoated vesicles regained ATP-dependent acidification, demonstrating that CCVs contain the functional vATPase, yet its function is inhibited by the clathrin coat. Considering the known structures of the vATPase and clathrin coat, we propose a model in which the formation of the coat surrounds the vATPase and blocks its activity. Such inhibition is likely fundamental for the proper timing of SV refilling.
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http://dx.doi.org/10.7554/eLife.32569DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5935483PMC
April 2018

Membrane fission by protein crowding.

Proc Natl Acad Sci U S A 2017 04 3;114(16):E3258-E3267. Epub 2017 Apr 3.

Department of Biomedical Engineering, The University of Texas at Austin, Austin, TX 78712;

Membrane fission, which facilitates compartmentalization of biological processes into discrete, membrane-bound volumes, is essential for cellular life. Proteins with specific structural features including constricting rings, helical scaffolds, and hydrophobic membrane insertions are thought to be the primary drivers of fission. In contrast, here we report a mechanism of fission that is independent of protein structure-steric pressure among membrane-bound proteins. In particular, random collisions among crowded proteins generate substantial pressure, which if unbalanced on the opposite membrane surface can dramatically increase membrane curvature, leading to fission. Using the endocytic protein epsin1 N-terminal homology domain (ENTH), previously thought to drive fission by hydrophobic insertion, our results show that membrane coverage correlates equally with fission regardless of the hydrophobicity of insertions. Specifically, combining FRET-based measurements of membrane coverage with multiple, independent measurements of membrane vesiculation revealed that fission became spontaneous as steric pressure increased. Further, fission efficiency remained equally potent when helices were replaced by synthetic membrane-binding motifs. These data challenge the view that hydrophobic insertions drive membrane fission, suggesting instead that the role of insertions is to anchor proteins strongly to membrane surfaces, amplifying steric pressure. In line with these conclusions, even green fluorescent protein (GFP) was able to drive fission efficiently when bound to the membrane at high coverage. Our conclusions are further strengthened by the finding that intrinsically disordered proteins, which have large hydrodynamic radii yet lack a defined structure, drove fission with substantially greater potency than smaller, structured proteins.
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http://dx.doi.org/10.1073/pnas.1616199114DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5402459PMC
April 2017

Binding Properties of the Transforming Growth Factor-β Coreceptor Betaglycan: Proposed Mechanism for Potentiation of Receptor Complex Assembly and Signaling.

Biochemistry 2016 12 2;55(49):6880-6896. Epub 2016 Dec 2.

Department of Structural Biology, University of Pittsburgh , Pittsburgh, Pennsylvania 15261, United States.

Transforming growth factor (TGF) β1, β2, and β3 (TGF-β1-TGF-β3, respectively) are small secreted signaling proteins that each signal through the TGF-β type I and type II receptors (TβRI and TβRII, respectively). However, TGF-β2, which is well-known to bind TβRII several hundred-fold more weakly than TGF-β1 and TGF-β3, has an additional requirement for betaglycan, a membrane-anchored nonsignaling receptor. Betaglycan has two domains that bind TGF-β2 at independent sites, but how it binds TGF-β2 to potentiate TβRII binding and how the complex with TGF-β, TβRII, and betaglycan undergoes the transition to the signaling complex with TGF-β, TβRII, and TβRI are not understood. To investigate the mechanism, the binding of the TGF-βs to the betaglycan extracellular domain, as well as its two independent binding domains, either directly or in combination with the TβRI and TβRII ectodomains, was studied using surface plasmon resonance, isothermal titration calorimetry, and size-exclusion chromatography. These studies show that betaglycan binds TGF-β homodimers with a 1:1 stoichiometry in a manner that allows one molecule of TβRII to bind. These studies further show that betaglycan modestly potentiates the binding of TβRII and must be displaced to allow TβRI to bind. These findings suggest that betaglycan functions to bind and concentrate TGF-β2 on the cell surface and thus promote the binding of TβRII by both membrane-localization effects and allostery. These studies further suggest that the transition to the signaling complex is mediated by the recruitment of TβRI, which simultaneously displaces betaglycan and stabilizes the bound TβRII by direct receptor-receptor contact.
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http://dx.doi.org/10.1021/acs.biochem.6b00566DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5551644PMC
December 2016

Instability of the Human Cytochrome P450 Reductase A287P Variant Is the Major Contributor to Its Antley-Bixler Syndrome-like Phenotype.

J Biol Chem 2016 09 5;291(39):20487-502. Epub 2016 Aug 5.

From the Department of Biochemistry, University of Texas Health Science Center, San Antonio, Texas 78229,

Human NADPH-cytochrome P450 oxidoreductase (POR) gene mutations are associated with severe skeletal deformities and disordered steroidogenesis. The human POR mutation A287P presents with disordered sexual development and skeletal malformations. Difficult recombinant expression and purification of this POR mutant suggested that the protein was less stable than WT. The activities of cytochrome P450 17A1, 19A1, and 21A2, critical in steroidogenesis, were similar using our purified, full-length, unmodified A287P or WT POR, as were those of several xenobiotic-metabolizing cytochromes P450, indicating that the A287P protein is functionally competent in vitro, despite its functionally deficient phenotypic behavior in vivo Differential scanning calorimetry and limited trypsinolysis studies revealed a relatively unstable A287P compared with WT protein, leading to the hypothesis that the syndrome observed in vivo results from altered POR protein stability. The crystal structures of the soluble domains of WT and A287P reveal only subtle differences between them, but these differences are consistent with the differential scanning calorimetry results as well as the differential susceptibility of A287P and WT observed with trypsinolysis. The relative in vivo stabilities of WT and A287P proteins were also examined in an osteoblast cell line by treatment with cycloheximide, a protein synthesis inhibitor, showing that the level of A287P protein post-inhibition is lower than WT and suggesting that A287P may be degraded at a higher rate. Current studies demonstrate that, unlike previously described mutations, A287P causes POR deficiency disorder due to conformational instability leading to proteolytic susceptibility in vivo, rather than through an inherent flavin-binding defect.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5034044PMC
http://dx.doi.org/10.1074/jbc.M116.716019DOI Listing
September 2016

Clathrin-coat disassembly illuminates the mechanisms of Hsp70 force generation.

Nat Struct Mol Biol 2016 09 1;23(9):821-9. Epub 2016 Aug 1.

Department of Biochemistry and Center for Biomedical Neuroscience, University of Texas Health Science Center at San Antonio, San Antonio, Texas, USA.

Hsp70s use ATP hydrolysis to disrupt protein-protein associations and to move macromolecules. One example is the Hsc70- mediated disassembly of the clathrin coats that form on vesicles during endocytosis. Here, we exploited the exceptional features of these coats to test three models-Brownian ratchet, power-stroke and entropic pulling-proposed to explain how Hsp70s transform their substrates. Our data rule out the ratchet and power-stroke models and instead support a collision-pressure mechanism whereby collisions between clathrin-coat walls and Hsc70s drive coats apart. Collision pressure is the complement to the pulling force described in the entropic pulling model. We also found that self-association augments collision pressure, thereby allowing disassembly of clathrin lattices that have been predicted to be resistant to disassembly. These results illuminate how Hsp70s generate the forces that transform their substrates.
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http://dx.doi.org/10.1038/nsmb.3272DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5016234PMC
September 2016

Intrinsically disordered proteins drive membrane curvature.

Nat Commun 2015 Jul 24;6:7875. Epub 2015 Jul 24.

1] Department of Biomedical Engineering, The University of Texas at Austin, 107 W Dean Keeton, Austin, Texas 78712, USA [2] Institute for Cellular and Molecular Biology, The University of Texas at Austin, 107 W Dean, Keeton,Texas 78712, USA.

Assembly of highly curved membrane structures is essential to cellular physiology. The prevailing view has been that proteins with curvature-promoting structural motifs, such as wedge-like amphipathic helices and crescent-shaped BAR domains, are required for bending membranes. Here we report that intrinsically disordered domains of the endocytic adaptor proteins, Epsin1 and AP180 are highly potent drivers of membrane curvature. This result is unexpected since intrinsically disordered domains lack a well-defined three-dimensional structure. However, in vitro measurements of membrane curvature and protein diffusivity demonstrate that the large hydrodynamic radii of these domains generate steric pressure that drives membrane bending. When disordered adaptor domains are expressed as transmembrane cargo in mammalian cells, they are excluded from clathrin-coated pits. We propose that a balance of steric pressure on the two surfaces of the membrane drives this exclusion. These results provide quantitative evidence for the influence of steric pressure on the content and assembly of curved cellular membrane structures.
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http://dx.doi.org/10.1038/ncomms8875DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4515776PMC
July 2015

The role of molecular chaperones in clathrin mediated vesicular trafficking.

Front Mol Biosci 2015 19;2:26. Epub 2015 May 19.

Department of Biochemistry and Center for Biomedical Neuroscience, University of Texas Health Science Center at San Antonio San Antonio, TX, USA.

The discovery that the 70 kD "uncoating ATPase," which removes clathrin coats from vesicles after endocytosis, is the constitutively expressed Hsc70 chaperone was a surprise. Subsequent work, however, revealed that uncoating is an archetypal Hsp70 reaction: the cochaperone auxilin, which contains a clathrin binding domain and an Hsc70 binding J domain, recruits Hsc70(*)ATP to the coat and, concomitant with ATP hydrolysis, transfers it to a hydrophobic Hsc70-binding element found on a flexible tail at the C-terminus of the clathrin heavy chain. Release of clathrin in association with Hsc70(*)ADP follows, and the subsequent, persistent association of clathrin with Hsc70 is important to prevent aberrant clathrin polymerization. Thus, the two canonical functions of Hsp70-dissociation of existing protein complexes or aggregates, and binding to a protein to inhibit its inappropriate aggregation-are recapitulated in uncoating. Association of clathrin with Hsc70 in vivo is regulated by Hsp110, an Hsp70 NEF that is itself a member of the Hsp70 family. How Hsp110 activity is itself regulated to make Hsc70-free clathrin available for endocytosis is unclear, though at synapses it's possible that the influx of calcium that accompanies depolarization activates the Ca(++)/calmodulin dependent calcineurin phosphatase which then dephosphorylates and activates Hsp110 to stimulate ADP/ATP exchange and release clathrin from Hsc70(*)ADP:clathrin complexes.
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http://dx.doi.org/10.3389/fmolb.2015.00026DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4436892PMC
June 2015

Nuclear Magnetic Resonance Structural Mapping Reveals Promiscuous Interactions between Clathrin-Box Motif Sequences and the N-Terminal Domain of the Clathrin Heavy Chain.

Biochemistry 2015 Apr 16;54(16):2571-80. Epub 2015 Apr 16.

Department of Biochemistry and Center for Biomedical Neuroscience, University of Texas Health Science Center at San Antonio, San Antonio, Texas 78229, United States.

The recruitment and organization of clathrin at endocytic sites first to form coated pits and then clathrin-coated vesicles depend on interactions between the clathrin N-terminal domain (TD) and multiple clathrin binding sequences on the cargo adaptor and accessory proteins that are concentrated at such sites. Up to four distinct protein binding sites have been proposed to be present on the clathrin TD, with each site proposed to interact with a distinct clathrin binding motif. However, an understanding of how such interactions contribute to clathrin coat assembly must take into account observations that any three of these four sites on clathrin TD can be mutationally ablated without causing loss of clathrin-mediated endocytosis. To take an unbiased approach to mapping binding sites for clathrin-box motifs on clathrin TD, we used isothermal titration calorimetry (ITC) and nuclear magnetic resonance spectroscopy. Our ITC experiments revealed that a canonical clathrin-box motif peptide from the AP-2 adaptor binds to clathrin TD with a stoichiometry of 3:1. Assignment of 90% of the total visible amide resonances in the TROSY-HSQC spectrum of (13)C-, (2)H-, and (15)N-labeled TD40 allowed us to map these three binding sites by analyzing the chemical shift changes as clathrin-box motif peptides were titrated into clathrin TD. We found that three different clathrin-box motif peptides can each simultaneously bind not only to the previously characterized clathrin-box site but also to the W-box site and the β-arrestin splice loop site on a single TD. The promiscuity of these binding sites can help explain why their mutation does not lead to larger effects on clathrin function and suggests a mechanism by which clathrin may be transferred between different proteins during the course of an endocytic event.
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http://dx.doi.org/10.1021/acs.biochem.5b00065DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4429812PMC
April 2015

A parametrically constrained optimization method for fitting sedimentation velocity experiments.

Biophys J 2014 Apr;106(8):1741-50

The University of Texas Health Science Center at San Antonio, Department of Biochemistry, San Antonio, Texas. Electronic address:

A method for fitting sedimentation velocity experiments using whole boundary Lamm equation solutions is presented. The method, termed parametrically constrained spectrum analysis (PCSA), provides an optimized approach for simultaneously modeling heterogeneity in size and anisotropy of macromolecular mixtures. The solutions produced by PCSA are particularly useful for modeling polymerizing systems, where a single-valued relationship exists between the molar mass of the growing polymer chain and its corresponding anisotropy. The PCSA uses functional constraints to identify this relationship, and unlike other multidimensional grid methods, assures that only a single molar mass can be associated with a given anisotropy measurement. A description of the PCSA algorithm is presented, as well as several experimental and simulated examples that illustrate its utility and capabilities. The performance advantages of the PCSA method in comparison to other methods are documented. The method has been added to the UltraScan-III software suite, which is available for free download from http://www.ultrascan.uthscsa.edu.
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http://dx.doi.org/10.1016/j.bpj.2014.02.022DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4008825PMC
April 2014

A role for an Hsp70 nucleotide exchange factor in the regulation of synaptic vesicle endocytosis.

J Neurosci 2013 May;33(18):8009-21

Eugene Bell Center for Regenerative Biology and Tissue Engineering, Marine Biological Laboratory, Woods Hole, Massachusetts 02543, USA.

Neurotransmission requires a continuously available pool of synaptic vesicles (SVs) that can fuse with the plasma membrane and release their neurotransmitter contents upon stimulation. After fusion, SV membranes and membrane proteins are retrieved from the presynaptic plasma membrane by clathrin-mediated endocytosis. After the internalization of a clathrin-coated vesicle, the vesicle must uncoat to replenish the pool of SVs. Clathrin-coated vesicle uncoating requires ATP and is mediated by the ubiquitous molecular chaperone Hsc70. In vitro, depolymerized clathrin forms a stable complex with Hsc70*ADP. This complex can be dissociated by nucleotide exchange factors (NEFs) that release ADP from Hsc70, allowing ATP to bind and induce disruption of the clathrin:Hsc70 association. Whether NEFs generally play similar roles in vesicle trafficking in vivo and whether they play such roles in SV endocytosis in particular is unknown. To address this question, we used information from recent structural and mechanistic studies of Hsp70:NEF and Hsp70:co-chaperone interactions to design a NEF inhibitor. Using acute perturbations at giant reticulospinal synapses of the sea lamprey (Petromyzon marinus), we found that this NEF inhibitor inhibited SV endocytosis. When this inhibitor was mutated so that it could no longer bind and inhibit Hsp110 (a NEF that we find to be highly abundant in brain cytosol), its ability to inhibit SV endocytosis was eliminated. These observations indicate that the action of a NEF, most likely Hsp110, is normally required during SV trafficking to release clathrin from Hsc70 and make it available for additional rounds of endocytosis.
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http://dx.doi.org/10.1523/JNEUROSCI.4505-12.2013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3707978PMC
May 2013

Unraveling protein-protein interactions in clathrin assemblies via atomic force spectroscopy.

Methods 2013 Mar 25;59(3):316-27. Epub 2012 Dec 25.

Laboratory of Cellular Imaging and Macromolecular Biophysics, National Institute of Biomedical Imaging and Bioengineering, National Institutes of Health, DHHS, Bethesda, MD 20892, United States.

Atomic force microscopy (AFM), single molecule force spectroscopy (SMFS), and single particle force spectroscopy (SPFS) are used to characterize intermolecular interactions and domain structures of clathrin triskelia and clathrin-coated vesicles (CCVs). The latter are involved in receptor-mediated endocytosis (RME) and other trafficking pathways. Here, we subject individual triskelia, bovine-brain CCVs, and reconstituted clathrin-AP180 coats to AFM-SMFS and AFM-SPFS pulling experiments and apply novel analytics to extract force-extension relations from very large data sets. The spectroscopic fingerprints of these samples differ markedly, providing important new information about the mechanism of CCV uncoating. For individual triskelia, SMFS reveals a series of events associated with heavy chain alpha-helix hairpin unfolding, as well as cooperative unraveling of several hairpin domains. SPFS of clathrin assemblies exposes weaker clathrin-clathrin interactions that are indicative of inter-leg association essential for RME and intracellular trafficking. Clathrin-AP180 coats are energetically easier to unravel than the coats of CCVs, with a non-trivial dependence on force-loading rate.
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http://dx.doi.org/10.1016/j.ymeth.2012.12.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3608793PMC
March 2013

Mechanical stress-activated integrin α5β1 induces opening of connexin 43 hemichannels.

Proc Natl Acad Sci U S A 2012 Feb 13;109(9):3359-64. Epub 2012 Feb 13.

Department of Biochemistry, Cardiology Division, University of Texas Health Science Center, San Antonio, TX 78229, USA.

The connexin 43 (Cx43) hemichannel (HC) in the mechanosensory osteocytes is a major portal for the release of factors responsible for the anabolic effects of mechanical loading on bone formation and remodeling. However, little is known about how the Cx43 molecule responds to mechanical stimulation leading to the opening of the HC. Here, we demonstrate that integrin α5β1 interacts directly with Cx43 and that this interaction is required for mechanical stimulation-induced opening of the Cx43 HC. Direct mechanical perturbation via magnetic beads or conformational activation of integrin α5β1 leads to the opening of the Cx43 HC, and this role of the integrin is independent of its association with an extracellular fibronectin substrate. PI3K signaling is responsible for the shear stress-induced conformational activation of integrin α5β1 leading to the opening of the HC. These results identify an unconventional function of integrin that acts as a mechanical tether to induce opening of the HC and provide a mechanism connecting the effect of mechanical forces directly to anabolic function of the bone.
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http://dx.doi.org/10.1073/pnas.1115967109DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3295295PMC
February 2012

Peptide from the C-terminal domain of tissue inhibitor of matrix metalloproteinases-2 (TIMP-2) inhibits membrane activation of matrix metalloproteinase-2 (MMP-2).

Matrix Biol 2011 Sep 4;30(7-8):404-12. Epub 2011 Aug 4.

Department of Periodontics, University of Texas Health Science Center at San Antonio, San Antonio, TX 78229-3900, USA.

Cellular activation of latent matrix metalloproteinase-2 (proMMP-2) requires formation of a cell membrane-associated activation complex that involves specific binding between the hemopexin domain of proMMP-2 (PEX) and the C-terminal domain of tissue inhibitor of matrix metalloproteinases-2 (C-TIMP-2). In this study, we tested the feasibility of inhibiting activation of proMMP-2 by exogenous inhibitors, which block the binding between PEX and TIMP-2. The recombinant C-TIMP-2 and synthetic peptides from C-TIMP-2 were used as inhibitors for proMMP-2 activation. Recombinant C-TIMP-2 bound specifically to both the catalytically inactive MMP-2(E404A) and the C-terminal domain of MMP-2 (PEX) in a concentration dependent manner with apparent K(d) of 3.9×10(-7)M and 1.7×10(-7)M, respectively. Moreover, C-TIMP-2 competed the binding between MMP-2(E404A) and full-length TIMP-2. Finally, activity assays showed that addition of C-TIMP-2 to HT-1080 fibrosarcoma cells inhibited proMMP-2 activation in a concentration-dependent manner. We then designed a synthetic peptide, P175L, consisting of 20 residues from the PEX-binding tail region of C-TIMP-2. P175L bound PEX and inhibited cell membrane-mediated activation of proMMP-2 in a concentration dependent manner. Deletion of the last 9 tail residues of C-TIMP-2 in P175L abrogated the inhibitory activities of the peptide showing that these residues were essential for function. Overall, these experiments have demonstrated that proMMP-2 activation can be inhibited by exogenous inhibitors which points to a potential strategy for MMP-2 specific inhibition.
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http://dx.doi.org/10.1016/j.matbio.2011.07.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3191288PMC
September 2011

Dynamic interactions between clathrin and locally structured elements in a disordered protein mediate clathrin lattice assembly.

J Mol Biol 2010 Nov 25;404(2):274-90. Epub 2010 Sep 25.

Department of Biochemistry, University of Texas Health Science Center at San Antonio, San Antonio, TX 78229, USA.

Assembly of clathrin lattices is mediated by assembly/adaptor proteins that contain domains that bind lipids or membrane-bound cargo proteins and clathrin binding domains (CBDs) that recruit clathrin. Here, we characterize the interaction between clathrin and a large fragment of the CBD of the clathrin assembly protein AP180. Mutational, NMR chemical shift, and analytical ultracentrifugation analyses allowed us to precisely define two clathrin binding sites within this fragment, each of which is found to bind weakly to the N-terminal domain of the clathrin heavy chain (TD). The locations of the two clathrin binding sites are consistent with predictions from sequence alignments of previously identified clathrin binding elements and, by extension, indicate that the complete AP180 CBD contains ∼12 degenerate repeats, each containing a single clathrin binding site. Sequence and circular dichroism analyses have indicated that the AP180 CBD is predominantly unstructured and our NMR analyses confirm that this is largely the case for the AP180 fragment characterized here. Unexpectedly, unlike the many proteins that undergo binding-coupled folding upon interaction with their binding partners, the AP180 fragment is similarly unstructured in its bound and free states. Instead, we find that this fragment exhibits localized β-turn-like structures at the two clathrin binding sites both when free and when bound to clathrin. These observations are incorporated into a model in which weak binding by multiple, pre-structured clathrin binding elements regularly dispersed throughout a largely unstructured CBD allows efficient recruitment of clathrin to endocytic sites and dynamic assembly of the clathrin lattice.
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http://dx.doi.org/10.1016/j.jmb.2010.09.044DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2981644PMC
November 2010

Polycomb group targeting through different binding partners of RING1B C-terminal domain.

Structure 2010 Aug;18(8):966-75

Department of Biochemistry, University of Texas Health Science Center at San Antonio, MSC 7760, 7703 Floyd Curl Drive, San Antonio, TX 78229-3990, USA.

RING1B, a Polycomb Group (PcG) protein, binds methylated chromatin through its association with another PcG protein called Polycomb (Pc). However, RING1B can associate with nonmethylated chromatin suggesting an alternate mechanism for RING1B interaction with chromatin. Here, we demonstrate that two proteins with little sequence identity between them, the Pc cbox domain and RYBP, bind the same surface on the C-terminal domain of RING1B (C-RING1B). Pc cbox and RYBP each fold into a nearly identical, intermolecular beta sheet with C-RING1B and a loop structure which are completely different in the two proteins. Both the beta sheet and loop are required for stable binding and transcription repression. Further, a mutation engineered to disrupt binding on the Drosophila dRING1 protein prevents chromatin association and PcG function in vivo. These results suggest that PcG targeting to different chromatin locations relies, in part, on binding partners of C-RING1B that are diverse in sequence and structure.
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http://dx.doi.org/10.1016/j.str.2010.04.013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4445678PMC
August 2010

Ternary complex of transforming growth factor-beta1 reveals isoform-specific ligand recognition and receptor recruitment in the superfamily.

J Biol Chem 2010 May 5;285(19):14806-14. Epub 2010 Mar 5.

Structural Immunology Section, Laboratory of Immunogenetics, NIAID, National Institutes of Health, Rockville, Maryland 20852, USA.

Transforming growth factor (TGF)-beta1, -beta2, and -beta3 are 25-kDa homodimeric polypeptides that play crucial nonoverlapping roles in embryogenesis, tissue development, carcinogenesis, and immune regulation. Here we report the 3.0-A resolution crystal structure of the ternary complex between human TGF-beta1 and the extracellular domains of its type I and type II receptors, TbetaRI and TbetaRII. The TGF-beta1 ternary complex structure is similar to previously reported TGF-beta3 complex except with a 10 degrees rotation in TbetaRI docking orientation. Quantitative binding studies showed distinct kinetics between the receptors and the isoforms of TGF-beta. TbetaRI showed significant binding to TGF-beta2 and TGF-beta3 but not TGF-beta1, and the binding to all three isoforms of TGF-beta was enhanced considerably in the presence of TbetaRII. The preference of TGF-beta2 to TbetaRI suggests a variation in its receptor recruitment in vivo. Although TGF-beta1 and TGF-beta3 bind and assemble their ternary complexes in a similar manner, their structural differences together with differences in the affinities and kinetics of their receptor binding may underlie their unique biological activities. Structural comparisons revealed that the receptor-ligand pairing in the TGF-beta superfamily is dictated by unique insertions, deletions, and disulfide bonds rather than amino acid conservation at the interface. The binding mode of TbetaRII on TGF-beta is unique to TGF-betas, whereas that of type II receptor for bone morphogenetic protein on bone morphogenetic protein appears common to all other cytokines in the superfamily. Further, extensive hydrogen bonds and salt bridges are present at the high affinity cytokine-receptor interfaces, whereas hydrophobic interactions dominate the low affinity receptor-ligand interfaces.
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http://dx.doi.org/10.1074/jbc.M109.079921DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2863181PMC
May 2010

AFM visualization of clathrin triskelia under fluid and in air.

FEBS Lett 2010 Jan;584(1):44-8

Laboratory of Bioengineering and Physical Science, National Institute of Biomedical Imaging and Bioengineering, National Institutes of Health, DHHS, Bethesda, MD 20892, USA.

Atomic force microscopy (AFM) is used to characterize the structure and interactions of clathrin triskelia. Time sequence images of individual, wet triskelia resting on mica surfaces clearly demonstrate conformational fluctuations of the triskelia. AFM of dried samples yields images having nanometric resolution comparable to that obtainable by electron microscopy of shadowed samples. Increased numbers of triskelion dimers and assembly intermediates, as well as structures having dimensions similar to those of clathrin cages, are observed when the triskelia were immersed in a low salt, low pH buffer. These entities have been quantified by AFM protein volume computation.
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http://dx.doi.org/10.1016/j.febslet.2009.11.039DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2801348PMC
January 2010

ATP-induced conformational changes in Hsp70: molecular dynamics and experimental validation of an in silico predicted conformation.

Biochemistry 2009 Dec;48(48):11470-7

Department of Chemistry, University of Nevada, Reno, Nevada 89557-0216, USA.

The 70 kDa heat shock proteins (Hsp70s) play important roles in preventing the misfolding of proteins and repairing damage under stress by coupling ATP binding and hydrolysis to protein substrate release and binding, respectively. ATP binding is believed to induce closing of the Hsp70 nucleotide binding domain (NBD) around the nucleotide. We report here a combined computational-experimental study of this open-closed transition. All-atom molecular dynamics simulations were performed for isolated open state NBDs with and without bound ATP. The nucleotide-free NBD samples a wide range of open configurations exhibiting flexible rearrangements of its four subdomains (IA-IIB). In contrast, the ATP-bound Hsp70 NBD closes to a range of configurations that is substantially more closed than the conformation observed in crystals of ATP-complexed NBDs. The close approach of subdomains IB and IIB observed in the simulations results in a strong coordination of the fluorescence probe Trp90 of IB with Arg261 of IIB, a feature not seen in the crystal structures. To determine if this computationally observed conformation occurs in solution, we constructed an R261A mutant. The mutation was found to increase the K(m) and k(cat) for ATP and to significantly reduce the extent of the fluorescence quench observed upon ATP binding. Our results thus account for the previously unexplained ATP-driven change in Trp90 fluorescence seen in the isolated NBD.
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http://dx.doi.org/10.1021/bi901256yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2787669PMC
December 2009

Structural transitions of the RING1B C-terminal region upon binding the polycomb cbox domain.

Biochemistry 2008 Aug 11;47(31):8007-15. Epub 2008 Jul 11.

Department of Biochemistry, University of Texas Health Science Center at San Antonio, MSC 7760, 7703 Floyd Curl Drive, San Antonio, Texas 78229-3900, USA.

Polycomb group (PcG) proteins are required for maintaining cell identity and stem cell self-renewal. RING1B and Polycomb (Pc) are two components of a multiprotein complex called polycomb repression complex 1 (PRC1) that is essential for establishing and maintaining long-term repressed gene states. Here we characterize the interaction between the C-terminal region of RING1B (C-RING1B) and the Pc cbox domain. The C-RING1B-cbox interaction displays a 1:1 stoichiometry with dissociation constants ranging from 9.2 to 180 nM for the different Pc orthologues. NMR analysis of C-RING1B alone reveals line broadening. However, when it is in complex with the cbox domain, there is a striking change to the NMR spectrum indicative of conformational tightening. This conformational change may arise from the organization of the C-RING1B subdomains. The C-terminal regions of all PcG RING1 proteins are composed of two stretches of conserved sequences separated by a variable linker sequence. While the entire C-RING1B region is required for cbox binding, the N- and C-terminal halves of C-RING1B can be separated and are able to interact, suggesting the presence of an intramolecular interaction within C-RING1B. The flexibility within the C-RING1B structure allowing transitions between the intramolecular bound and unbound states may cause the broadened peaks of the C-RING1B NMR spectrum. Binding the cbox domain stabilizes C-RING1B, whereby broadening is eliminated. The presence of flexible regions could allow C-RING1B to bind a variety of different factors, ultimately recruiting RING1B and its associated PcG proteins to different genomic loci.
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http://dx.doi.org/10.1021/bi800857fDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4442619PMC
August 2008