Publications by authors named "Rachel E Klevit"

97 Publications

Mediator subunit Med15 dictates the conserved "fuzzy" binding mechanism of yeast transcription activators Gal4 and Gcn4.

Nat Commun 2021 04 13;12(1):2220. Epub 2021 Apr 13.

Department of Biochemistry, University of Washington, Seattle, WA, USA.

The acidic activation domain (AD) of yeast transcription factor Gal4 plays a dual role in transcription repression and activation through binding to Gal80 repressor and Mediator subunit Med15. The activation function of Gal4 arises from two hydrophobic regions within the 40-residue AD. We show by NMR that each AD region binds the Mediator subunit Med15 using a "fuzzy" protein interface. Remarkably, comparison of chemical shift perturbations shows that Gal4 and Gcn4, two intrinsically disordered ADs of different sequence, interact nearly identically with Med15. The finding that two ADs of different sequence use an identical fuzzy binding mechanism shows a common sequence-independent mechanism for AD-Mediator binding, similar to interactions within a hydrophobic cloud. In contrast, the same region of Gal4 AD interacts strongly with Gal80 via a distinct structured complex, implying that the structured binding partner of an intrinsically disordered protein dictates the type of protein-protein interaction.
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http://dx.doi.org/10.1038/s41467-021-22441-4DOI Listing
April 2021

Toggle switch residues control allosteric transitions in bacterial adhesins by participating in a concerted repacking of the protein core.

PLoS Pathog 2021 Apr 7;17(4):e1009440. Epub 2021 Apr 7.

Department of Microbiology, University of Washington, Seattle, Washington, United States of America.

Critical molecular events that control conformational transitions in most allosteric proteins are ill-defined. The mannose-specific FimH protein of Escherichia coli is a prototypic bacterial adhesin that switches from an 'inactive' low-affinity state (LAS) to an 'active' high-affinity state (HAS) conformation allosterically upon mannose binding and mediates shear-dependent catch bond adhesion. Here we identify a novel type of antibody that acts as a kinetic trap and prevents the transition between conformations in both directions. Disruption of the allosteric transitions significantly slows FimH's ability to associate with mannose and blocks bacterial adhesion under dynamic conditions. FimH residues critical for antibody binding form a compact epitope that is located away from the mannose-binding pocket and is structurally conserved in both states. A larger antibody-FimH contact area is identified by NMR and contains residues Leu-34 and Val-35 that move between core-buried and surface-exposed orientations in opposing directions during the transition. Replacement of Leu-34 with a charged glutamic acid stabilizes FimH in the LAS conformation and replacement of Val-35 with glutamic acid traps FimH in the HAS conformation. The antibody is unable to trap the conformations if Leu-34 and Val-35 are replaced with a less bulky alanine. We propose that these residues act as molecular toggle switches and that the bound antibody imposes a steric block to their reorientation in either direction, thereby restricting concerted repacking of side chains that must occur to enable the conformational transition. Residues homologous to the FimH toggle switches are highly conserved across a diverse family of fimbrial adhesins. Replacement of predicted switch residues reveals that another E. coli adhesin, galactose-specific FmlH, is allosteric and can shift from an inactive to an active state. Our study shows that allosteric transitions in bacterial adhesins depend on toggle switch residues and that an antibody that blocks the switch effectively disables adhesive protein function.
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http://dx.doi.org/10.1371/journal.ppat.1009440DOI Listing
April 2021

BRCA1/BARD1 site-specific ubiquitylation of nucleosomal H2A is directed by BARD1.

Nat Struct Mol Biol 2021 03 15;28(3):268-277. Epub 2021 Feb 15.

Department of Biochemistry, University of Washington, Seattle, WA, USA.

Mutations in the E3 ubiquitin ligase RING domains of BRCA1/BARD1 predispose carriers to breast and ovarian cancers. We present the structure of the BRCA1/BARD1 RING heterodimer with the E2 enzyme UbcH5c bound to its cellular target, the nucleosome, along with biochemical data that explain how the complex selectively ubiquitylates lysines 125, 127 and 129 in the flexible C-terminal tail of H2A in a fully human system. The structure reveals that a novel BARD1-histone interface couples to a repositioning of UbcH5c compared to the structurally similar PRC1 E3 ligase Ring1b/Bmi1 that ubiquitylates H2A Lys119 in nucleosomes. This interface is sensitive to both H3 Lys79 methylation status and mutations found in individuals with cancer. Furthermore, NMR reveals an unexpected mode of E3-mediated substrate regulation through modulation of dynamics in the C-terminal tail of H2A. Our findings provide insight into how E3 ligases preferentially target nearby lysine residues in nucleosomes by a steric occlusion and distancing mechanism.
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http://dx.doi.org/10.1038/s41594-020-00556-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8007219PMC
March 2021

Who with whom: functional coordination of E2 enzymes by RING E3 ligases during poly-ubiquitylation.

EMBO J 2020 11 5;39(22):e104863. Epub 2020 Oct 5.

Department of Biochemistry, School of Medicine, University of Washington, Seattle, WA, USA.

Protein modification with poly-ubiquitin chains is a crucial process involved in a myriad of cellular pathways. Chain synthesis requires two steps: substrate modification with ubiquitin (priming) followed by repetitive ubiquitin-to-ubiquitin attachment (elongation). RING-type E3 ligases catalyze both reactions in collaboration with specific priming and elongating E2 enzymes. We provide kinetic insight into poly-ubiquitylation during protein quality control by showing that priming is the rate-determining step in protein degradation as directed by the yeast ERAD RING E3 ligases, Hrd1 and Doa10. Doa10 cooperates with the dedicated priming E2, Ubc6, while both E3s use Ubc7 for elongation. Here, we provide direct evidence that Hrd1 uses Ubc7 also for priming. We found that Ubc6 has an unusually high basal activity that does not require strong stimulation from an E3. Doa10 exploits this property to pair with Ubc6 over Ubc7 during priming. Our work not only illuminates the mechanisms of specific E2/E3 interplay in ERAD, but also offers a basis to understand how RING E3s may have properties that are tailored to pair with their preferred E2s.
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http://dx.doi.org/10.15252/embj.2020104863DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7667886PMC
November 2020

UbcH5 Interacts with Substrates to Participate in Lysine Selection with the E3 Ubiquitin Ligase CHIP.

Biochemistry 2020 06 27;59(22):2078-2088. Epub 2020 May 27.

Department of Biochemistry, Medical College of Wisconsin, Milwaukee, Wisconsin 53226, United States.

The E3 ubiquitin ligase C-terminus of Hsc70 interacting protein (CHIP) plays a critical role in regulating the ubiquitin-dependent degradation of misfolded proteins. CHIP mediates the ubiquitination of the α-amino-terminus of substrates with the E2 Ube2w and facilitates the ubiquitination of lysine residues with the E2 UbcH5. While it is known that Ube2w directly interacts with the disordered regions at the N-terminus of its substrates, it is unclear how CHIP and UbcH5 mediate substrate lysine selection. Here, we have decoupled the contributions of the E2, UbcH5, and the E3, CHIP, in ubiquitin transfer. We show that UbcH5 selects substrate lysine residues independent of CHIP, and that CHIP participates in lysine selection by fine-tuning the subset of substrate lysines that are ubiquitinated. We also identify lysine 128 near the C-terminus of UbcH5 as a critical residue for the efficient ubiquitin transfer by UbcH5 in both the presence and absence of CHIP. Together, these data demonstrate an important role of the UbcH5/substrate interactions in mediating the efficient ubiquitin transfer by the CHIP/UbcH5 complex.
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http://dx.doi.org/10.1021/acs.biochem.0c00084DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7780428PMC
June 2020

Legionella effector MavC targets the Ube2N~Ub conjugate for noncanonical ubiquitination.

Nat Commun 2020 05 12;11(1):2365. Epub 2020 May 12.

Department of Chemistry, Purdue University, West Lafayette, IN, 47907, USA.

The bacterial effector MavC modulates the host immune response by blocking Ube2N activity employing an E1-independent ubiquitin ligation, catalyzing formation of a γ-glutamyl-ε-Lys (Gln40-Lys92) isopeptide crosslink using a transglutaminase mechanism. Here we provide biochemical evidence in support of MavC targeting the activated, thioester-linked Ube2N~ubiquitin conjugate, catalyzing an intramolecular transglutamination reaction, covalently crosslinking the Ube2N and Ub subunits effectively inactivating the E2~Ub conjugate. Ubiquitin exhibits weak binding to MavC alone, but shows an increase in affinity when tethered to Ube2N in a disulfide-linked substrate that mimics the charged E2~Ub conjugate. Crystal structures of MavC in complex with the substrate mimic and crosslinked product provide insights into the reaction mechanism and underlying protein dynamics that favor transamidation over deamidation, while revealing a crucial role for the structurally unique insertion domain in substrate recognition. This work provides a structural basis of ubiquitination by transglutamination and identifies this enzyme's true physiological substrate.
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http://dx.doi.org/10.1038/s41467-020-16211-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7217864PMC
May 2020

Peeking from behind the veil of enigma: emerging insights on small heat shock protein structure and function.

Authors:
Rachel E Klevit

Cell Stress Chaperones 2020 07 8;25(4):573-580. Epub 2020 Apr 8.

Department of Biochemistry, University of Washington School of Medicine, Seattle, WA, 98195, USA.

This is a short paper on new ways to think about the structure and function of small heat shock proteins (sHSPs), perhaps the most enigmatic family among protein chaperones. The goal is to incorporate new observations regarding the disordered regions of small heat shock proteins (sHSPs) into the large body of structural information on the conserved structural alpha-crystallin domains (ACD) that define the sHSP family. Disordered regions (N-terminal region and C-terminal region or NTR and CTR, respectively) represent over 50% of the sHSP sequence space in the human genome and are refractory to traditional structural biology approaches, posing a roadblock on the path towards a mechanistic understanding of how sHSPs function. A model in which an ACD dimer serves as a template that presents three grooves into which other proteins or other segments of sHSPs can bind is presented. Short segments within the disordered regions are observed to bind into the ACD grooves. There are more binding segments than there are grooves, and each binding event is weak and transient, creating a dynamic equilibrium of tethered and untethered disordered regions. The ability of an NTR to be in dynamic equilibrium between tethered/sequestered and untethered states suggests several mechanistic alternatives that need not be mutually exclusive. New ways of thinking about (and approaching) the intrinsic properties of sHSPs may finally allow the veil of enigma to be removed from sHSPs.
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http://dx.doi.org/10.1007/s12192-020-01092-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7332599PMC
July 2020

Release of a disordered domain enhances HspB1 chaperone activity toward tau.

Proc Natl Acad Sci U S A 2020 02 23;117(6):2923-2929. Epub 2020 Jan 23.

Department of Biochemistry, School of Medicine, University of Washington, Seattle, WA 98195;

Small heat shock proteins (sHSPs) are a class of ATP-independent molecular chaperones that play vital roles in maintaining protein solubility and preventing aberrant protein aggregation. They form highly dynamic, polydisperse oligomeric ensembles and contain long intrinsically disordered regions. Experimental challenges posed by these properties have greatly impeded our understanding of sHSP structure and mechanism of action. Here we characterize interactions between the human sHSP HspB1 (Hsp27) and microtubule-associated protein tau, which is implicated in multiple dementias, including Alzheimer's disease. We show that tau binds both to a well-known binding groove within the structured alpha-crystallin domain (ACD) and to sites within the enigmatic, disordered N-terminal region (NTR) of HspB1. However, only interactions involving the NTR lead to productive chaperone activity, whereas ACD binding is uncorrelated with chaperone function. The tau-binding groove in the ACD also binds short hydrophobic regions within HspB1 itself, and HspB1 mutations that disrupt these intrinsic ACD-NTR interactions greatly enhance chaperone activity toward tau. This leads to a mechanism in which the release of the disordered NTR from a binding groove on the ACD enhances chaperone activity toward tau. The study advances understanding of the mechanisms by which sHSPs achieve their chaperone activity against amyloid-forming clients and how cells defend against pathological tau aggregation. Furthermore, the resulting mechanistic model points to ways in which sHSP chaperone activity may be increased, either by native factors within the cell or by therapeutic intervention.
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http://dx.doi.org/10.1073/pnas.1915099117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7022203PMC
February 2020

Indirect sexual selection drives rapid sperm protein evolution in abalone.

Elife 2019 12 23;8. Epub 2019 Dec 23.

Department of Genome Sciences, University of Washington, Seattle, United States.

Sexual selection can explain the rapid evolution of fertilization proteins, yet sperm proteins evolve rapidly even if not directly involved in fertilization. In the marine mollusk abalone, sperm secrete enormous quantities of two rapidly evolving proteins, lysin and sp18, that are stored at nearly molar concentrations. We demonstrate that this extraordinary packaging is achieved by associating into Fuzzy Interacting Transient Zwitterion (FITZ) complexes upon binding the intrinsically disordered FITZ Anionic Partner (FITZAP). FITZ complexes form at intracellular ionic strengths and, upon exocytosis into seawater, lysin and sp18 are dispersed to drive fertilization. NMR analyses revealed that lysin uses a common molecular interface to bind both FITZAP and its egg receptor VERL. As sexual selection alters the lysin-VERL interface, FITZAP coevolves rapidly to maintain lysin binding. FITZAP-lysin interactions exhibit a similar species-specificity as lysin-VERL interactions. Thus, tethered molecular arms races driven by sexual selection can generally explain rapid sperm protein evolution.
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http://dx.doi.org/10.7554/eLife.52628DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6952181PMC
December 2019

RMSD analysis of structures of the bacterial protein FimH identifies five conformations of its lectin domain.

Proteins 2020 04 5;88(4):593-603. Epub 2019 Nov 5.

Department of Biochemistry, University of Washington, Seattle, WA.

FimH is a bacterial adhesin protein located at the tip of Escherichia coli fimbria that functions to adhere bacteria to host cells. Thus, FimH is a critical factor in bacterial infections such as urinary tract infections and is of interest in drug development. It is also involved in vaccine development and as a model for understanding shear-enhanced catch bond cell adhesion. To date, over 60 structures have been deposited in the Protein Data Bank showing interactions between FimH and mannose ligands, potential inhibitors, and other fimbrial proteins. In addition to providing insights about ligand recognition and fimbrial assembly, these structures provide insights into conformational changes in the two domains of FimH that are critical for its function. To gain further insights into these structural changes, we have superposed FimH's mannose binding lectin domain in all these structures and categorized the structures into five groups of lectin domain conformers using RMSD as a metric. Many structures also include the pilin domain, which anchors FimH to the fimbriae and regulates the conformation and function of the lectin domain. For these structures, we have also compared the relative orientations of the two domains. These structural analyses enhance our understanding of the conformational changes associated with FimH ligand binding and domain-domain interactions, including its catch bond behavior through allosteric action of force in bacterial adhesion.
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http://dx.doi.org/10.1002/prot.25840DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7058522PMC
April 2020

Interplay of disordered and ordered regions of a human small heat shock protein yields an ensemble of 'quasi-ordered' states.

Elife 2019 10 1;8. Epub 2019 Oct 1.

Department of Biochemistry, University of Washington, Seattle, United States.

Small heat shock proteins (sHSPs) are nature's 'first responders' to cellular stress, interacting with affected proteins to prevent their aggregation. Little is known about sHSP structure beyond its structured α-crystallin domain (ACD), which is flanked by disordered regions. In the human sHSP HSPB1, the disordered N-terminal region (NTR) represents nearly 50% of the sequence. Here, we present a hybrid approach involving NMR, hydrogen-deuterium exchange mass spectrometry, and modeling to provide the first residue-level characterization of the NTR. The results support a model in which multiple grooves on the ACD interact with specific NTR regions, creating an ensemble of 'quasi-ordered' NTR states that can give rise to the known heterogeneity and plasticity of HSPB1. Phosphorylation-dependent interactions inform a mechanism by which HSPB1 is activated under stress conditions. Additionally, we examine the effects of disease-associated NTR mutations on HSPB1 structure and dynamics, leveraging our emerging structural insights.
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http://dx.doi.org/10.7554/eLife.50259DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6791718PMC
October 2019

Cbl interacts with multiple E2s in vitro and in cells.

PLoS One 2019 23;14(5):e0216967. Epub 2019 May 23.

Women's Malignancy Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, Maryland, United States of America.

Many receptor tyrosine kinases (RTKs, such as EGFR, MET) are negatively regulated by ubiquitination and degradation mediated by Cbl proteins, a family of RING finger (RF) ubiquitin ligases (E3s). Loss of Cbl protein function is associated with malignant transformation driven by increased RTK activity. RF E3s, such as the Cbl proteins, interact with a ubiquitin-conjugating enzyme (E2) to confer specificity to the ubiquitination process and direct the transfer of ubiquitin from the E2 to one or more lysines on the target proteins. Using in vitro E3 assays and yeast two-hybrid screens, we found that Ube2d, Ube2e families, Ube2n/2v1, and Ube2w catalyze autoubiquitination of the Cbl protein and Ube2d2, Ube2e1, and Ube 2n/2v1 catalyze Cbl-mediated substrate ubiquitination of the EGFR and SYK. Phosphorylation of the Cbl protein by by Src resulted in increased E3 activity compared to unphosphorylated cbl or Cbl containing a phosphomimetic Y371E mutation. Ubiquitin chain formation depended on the E2 tested with Cbl with Ube2d2 forming both K48 and K63 linked chains, Ube2n/2v1 forming only K63 linked chains, and Ube2w inducing monoubiquitination. In cells, the Ube2d family, Ube2e family, and Ube2n/2v1 contributed to EGFR ubiquitination. Our data suggest that multiple E2s can interact with Cbl and modulate its E3 activity in vitro and in cells.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0216967PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6533038PMC
February 2020

Mechanisms of Small Heat Shock Proteins.

Cold Spring Harb Perspect Biol 2019 10 1;11(10). Epub 2019 Oct 1.

Department of Biochemistry, University of Washington, Seattle, Washington 98195.

Small heat shock proteins (sHSPs) are ATP-independent chaperones that delay formation of harmful protein aggregates. sHSPs' role in protein homeostasis has been appreciated for decades, but their mechanisms of action remain poorly understood. This gap in understanding is largely a consequence of sHSP properties that make them recalcitrant to detailed study. Multiple stress-associated conditions including pH acidosis, oxidation, and unusual availability of metal ions, as well as reversible stress-induced phosphorylation can modulate sHSP chaperone activity. Investigations of sHSPs reveal that sHSPs can engage in transient or long-lived interactions with client proteins depending on solution conditions and sHSP or client identity. Recent advances in the field highlight both the diversity of function within the sHSP family and the exquisite sensitivity of individual sHSPs to cellular and experimental conditions. Here, we will present and highlight current understanding, recent progress, and future challenges.
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http://dx.doi.org/10.1101/cshperspect.a034025DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6771367PMC
October 2019

Small heat shock proteins: multifaceted proteins with important implications for life.

Cell Stress Chaperones 2019 03 13;24(2):295-308. Epub 2019 Feb 13.

Laboratory of Cell and Developmental Genetics, IBIS, and Department of Molecular Biology, Medical Biochemistry and Pathology, Medical School, Université Laval, QC, Québec, G1V 0A6, Canada.

Small Heat Shock Proteins (sHSPs) evolved early in the history of life; they are present in archaea, bacteria, and eukaryota. sHSPs belong to the superfamily of molecular chaperones: they are components of the cellular protein quality control machinery and are thought to act as the first line of defense against conditions that endanger the cellular proteome. In plants, sHSPs protect cells against abiotic stresses, providing innovative targets for sustainable agricultural production. In humans, sHSPs (also known as HSPBs) are associated with the development of several neurological diseases. Thus, manipulation of sHSP expression may represent an attractive therapeutic strategy for disease treatment. Experimental evidence demonstrates that enhancing the chaperone function of sHSPs protects against age-related protein conformation diseases, which are characterized by protein aggregation. Moreover, sHSPs can promote longevity and healthy aging in vivo. In addition, sHSPs have been implicated in the prognosis of several types of cancer. Here, sHSP upregulation, by enhancing cellular health, could promote cancer development; on the other hand, their downregulation, by sensitizing cells to external stressors and chemotherapeutics, may have beneficial outcomes. The complexity and diversity of sHSP function and properties and the need to identify their specific clients, as well as their implication in human disease, have been discussed by many of the world's experts in the sHSP field during a dedicated workshop in Québec City, Canada, on 26-29 August 2018.
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http://dx.doi.org/10.1007/s12192-019-00979-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6439001PMC
March 2019

HSPB5 engages multiple states of a destabilized client to enhance chaperone activity in a stress-dependent manner.

J Biol Chem 2019 03 19;294(9):3261-3270. Epub 2018 Dec 19.

From the Department of Biochemistry, University of Washington, Seattle, Washington 98195-7350

Small heat shock proteins (sHSPs) delay protein aggregation in an ATP-independent manner by interacting with client proteins that are in states susceptible to aggregation, including destabilized states related to cellular stress. Up-regulation of sHSPs under stress conditions supports their critical role in cellular viability. Widespread distribution of sHSPs in most organisms implies conservation of function, but it remains unclear whether sHSPs implement common or distinct mechanisms to delay protein aggregation. Comparisons among various studies are confounded by the use of different model client proteins, different assays for both aggregation and sHSP/client interactions, and variable experimental conditions used to mimic cellular stress. To further define sHSP/client interactions and their relevance to sHSP chaperone function, we implemented multiple strategies to characterize sHSP interactions with α-lactalbumin, a model client whose aggregation pathway is well defined. We compared the chaperone activity of human αB-crystallin (HSPB5) with HSPB5 variants that mimic states that arise under conditions of cellular stress or disease. The results show that these closely related sHSPs vary not only in their activity under identical conditions but also in their interactions with clients. Importantly, under nonstress conditions, WT HSPB5 delays client aggregation solely through transient interactions early in the aggregation pathway, whereas HSPB5 mutants that mimic stress-activated conditions can also intervene at later stages of the aggregation pathway to further delay client protein aggregation.
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http://dx.doi.org/10.1074/jbc.RA118.003156DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6398148PMC
March 2019

Mechanistic insights revealed by a UBE2A mutation linked to intellectual disability.

Nat Chem Biol 2019 01 10;15(1):62-70. Epub 2018 Dec 10.

Brazilian Biosciences National Laboratory, Center for Research in Energy and Materials, Campinas, Brazil.

Ubiquitin-conjugating enzymes (E2) enable protein ubiquitination by conjugating ubiquitin to their catalytic cysteine for subsequent transfer to a target lysine side chain. Deprotonation of the incoming lysine enables its nucleophilicity, but determinants of lysine activation remain poorly understood. We report a novel pathogenic mutation in the E2 UBE2A, identified in two brothers with mild intellectual disability. The pathogenic Q93E mutation yields UBE2A with impaired aminolysis activity but no loss of the ability to be conjugated with ubiquitin. Importantly, the low intrinsic reactivity of UBE2A Q93E was not overcome by a cognate ubiquitin E3 ligase, RAD18, with the UBE2A target PCNA. However, UBE2A Q93E was reactive at high pH or with a low-pK amine as the nucleophile, thus providing the first evidence of reversion of a defective UBE2A mutation. We propose that Q93E substitution perturbs the UBE2A catalytic microenvironment essential for lysine deprotonation during ubiquitin transfer, thus generating an enzyme that is disabled but not dead.
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http://dx.doi.org/10.1038/s41589-018-0177-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6626659PMC
January 2019

The ubiquitin ligase SspH1 from uses a modular and dynamic E3 domain to catalyze substrate ubiquitylation.

J Biol Chem 2019 01 20;294(3):783-793. Epub 2018 Nov 20.

From the Departments of Biochemistry and

SspH/IpaH bacterial effector E3 ubiquitin (Ub) ligases, unrelated in sequence or structure to eukaryotic E3s, are utilized by a wide variety of Gram-negative bacteria during pathogenesis. These E3s function in a eukaryotic environment, utilize host cell E2 ubiquitin-conjugating enzymes of the Ube2D family, and target host proteins for ubiquitylation. Despite several crystal structures, details of Ube2D∼Ub binding and the mechanism of ubiquitin transfer are poorly understood. Here, we show that the catalytic E3 ligase domain of SspH1 can be divided into two subdomains: an N-terminal subdomain that harbors the active-site cysteine and a C-terminal subdomain containing the Ube2D∼Ub-binding site. SspH1 mutations designed to restrict subdomain motions show rapid formation of an E3∼Ub intermediate, but impaired Ub transfer to substrate. NMR experiments using paramagnetic spin labels reveal how SspH1 binds Ube2D∼Ub and targets the E2∼Ub active site. Unexpectedly, hydrogen/deuterium exchange MS shows that the E2∼Ub-binding region is dynamic but stabilized in the E3∼Ub intermediate. Our results support a model in which both subunits of an Ube2D∼Ub clamp onto a dynamic region of SspH1, promoting an E3 conformation poised for transthiolation. A conformational change is then required for Ub transfer from E3∼Ub to substrate.
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http://dx.doi.org/10.1074/jbc.RA118.004247DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6341402PMC
January 2019

A Bifunctional Role for the UHRF1 UBL Domain in the Control of Hemi-methylated DNA-Dependent Histone Ubiquitylation.

Mol Cell 2018 11 1;72(4):753-765.e6. Epub 2018 Nov 1.

Department of Biochemistry, University of Washington, Seattle, WA 98195, USA. Electronic address:

DNA methylation patterns regulate gene expression programs and are maintained through a highly coordinated process orchestrated by the RING E3 ubiquitin ligase UHRF1. UHRF1 controls DNA methylation inheritance by reading epigenetic modifications to histones and DNA to activate histone H3 ubiquitylation. Here, we find that all five domains of UHRF1, including the previously uncharacterized ubiquitin-like domain (UBL), cooperate for hemi-methylated DNA-dependent H3 ubiquitin ligation. Our structural and biochemical studies, including mutations found in cancer genomes, reveal a bifunctional requirement for the UBL in histone modification: (1) the UBL makes an essential interaction with the backside of the E2 and (2) the UBL coordinates with other UHRF1 domains that recognize epigenetic marks on DNA and histone H3 to direct ubiquitin to H3. Finally, we show UBLs from other E3s also have a conserved interaction with the E2, Ube2D, highlighting a potential prevalence of interactions between UBLs and E2s.
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http://dx.doi.org/10.1016/j.molcel.2018.09.029DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6239910PMC
November 2018

Characterization of RING-Between-RING E3 Ubiquitin Transfer Mechanisms.

Methods Mol Biol 2018 ;1844:3-17

Department of Biochemistry, University of Washington, Seattle, WA, USA.

Protein ubiquitination is an essential posttranslational modification that regulates nearly all cellular processes. E3 ligases catalyze the final transfer of ubiquitin (Ub) onto substrates and thus are important temporal regulators of ubiquitin modifications in the cell. E3s are classified by their distinct transfer mechanisms. RING E3s act as scaffolds to facilitate the transfer of Ub from E2-conjugating enzymes directly onto substrates, while HECT E3s form an E3~Ub thioester intermediate prior to Ub transfer. A third class, RING-Between-RING (RBR) E3s, are classified as RING/HECT hybrids based on their ability to engage the E2~Ub conjugate via a RING1 domain while subsequently forming an obligate E3~Ub intermediate prior to substrate modification. RBRs comprise the smallest class of E3s, consisting of only 14 family members in humans, yet their dysfunction has been associated with neurodegenerative diseases, susceptibility to infection, inflammation, and cancer. Additionally, their activity is suppressed by auto-inhibitory domains that block their catalytic activity, suggesting their regulation has important cellular consequences. Here, we identify technical hurdles faced in studying RBR E3s and provide protocols and guidelines to overcome these challenges.
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http://dx.doi.org/10.1007/978-1-4939-8706-1_1DOI Listing
May 2019

Mechanism of phosphoribosyl-ubiquitination mediated by a single Legionella effector.

Nature 2018 05 23;557(7707):729-733. Epub 2018 May 23.

Weill Institute for Cell and Molecular Biology and Department of Molecular Biology and Genetics, Cornell University, Ithaca, NY, USA.

Ubiquitination is a post-translational modification that regulates many cellular processes in eukaryotes. The conventional ubiquitination cascade culminates in a covalent linkage between the C terminus of ubiquitin (Ub) and a target protein, usually on a lysine side chain. Recent studies of the Legionella pneumophila SidE family of effector proteins revealed a ubiquitination method in which a phosphoribosyl ubiquitin (PR-Ub) is conjugated to a serine residue on substrates via a phosphodiester bond. Here we present the crystal structure of a fragment of the SidE family member SdeA that retains ubiquitination activity, and determine the mechanism of this unique post-translational modification. The structure reveals that the catalytic module contains two distinct functional units: a phosphodiesterase domain and a mono-ADP-ribosyltransferase domain. Biochemical analysis shows that the mono-ADP-ribosyltransferase domain-mediated conversion of Ub to ADP-ribosylated Ub (ADPR-Ub) and the phosphodiesterase domain-mediated ligation of PR-Ub to substrates are two independent activities of SdeA. Furthermore, we present two crystal structures of a homologous phosphodiesterase domain from the SidE family member SdeD in complexes with Ub and ADPR-Ub. The structures suggest a mechanism for how SdeA processes ADPR-Ub to PR-Ub and AMP, and conjugates PR-Ub to a serine residue in substrates. Our study establishes the molecular mechanism of phosphoribosyl-linked ubiquitination and will enable future studies of this unusual type of ubiquitination in eukaryotes.
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http://dx.doi.org/10.1038/s41586-018-0147-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5980775PMC
May 2018

Structural basis for tankyrase-RNF146 interaction reveals noncanonical tankyrase-binding motifs.

Protein Sci 2018 06 25;27(6):1057-1067. Epub 2018 Apr 25.

Department of Biological Structure, University of Washington, Seattle, Washington, 98195.

Poly(ADP-ribosyl)ation (PARylation) catalyzed by the tankyrase enzymes (Tankyrase-1 and -2; a.k.a. PARP-5a and -5b) is involved in mitosis, telomere length regulation, GLUT-4 vesicle transport, and cell growth and differentiation. Together with the E3 ubiquitin ligase RNF146 (a.k.a. Iduna), tankyrases regulate the cellular levels of several important proteins including Axin, 3BP2, and angiomotins, which are key regulators of Wnt, Src and Hippo signaling, respectively. These tankyrase substrates are first PARylated and then ubiquitylated by RNF146, which is allosterically activated by binding to PAR polymer. Each tankyrase substrate is recognized by a tankyrase-binding motif (TBM). Here we show that RNF146 binds directly to tankyrases via motifs in its C-terminal region. Four of these RNF146 motifs represent novel, extended TBMs, that have one or two additional amino acids between the most conserved Arg and Gly residues. The individual RNF146 motifs display weak binding, but together mediate a strong multivalent interaction with the substrate-binding region of TNKS, forming a robust one-to-one complex. A crystal structure of the first RNF146 noncanonical TBM in complex with the second ankyrin repeat domain of TNKS shows how an extended motif can be accommodated in a peptide-binding groove on tankyrases. Overall, our work demonstrates the existence of a new class of extended TBMs that exist in previously uncharacterized tankyrase-binding proteins including those of IF4A1 and NELFE.
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http://dx.doi.org/10.1002/pro.3413DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5980575PMC
June 2018

Gcn4-Mediator Specificity Is Mediated by a Large and Dynamic Fuzzy Protein-Protein Complex.

Cell Rep 2018 03;22(12):3251-3264

Department of Biochemistry, University of Washington, Seattle, WA 98195, USA. Electronic address:

Transcription activation domains (ADs) are inherently disordered proteins that often target multiple coactivator complexes, but the specificity of these interactions is not understood. Efficient transcription activation by yeast Gcn4 requires its tandem ADs and four activator-binding domains (ABDs) on its target, the Mediator subunit Med15. Multiple ABDs are a common feature of coactivator complexes. We find that the large Gcn4-Med15 complex is heterogeneous and contains nearly all possible AD-ABD interactions. Gcn4-Med15 forms via a dynamic fuzzy protein-protein interface, where ADs bind the ABDs in multiple orientations via hydrophobic regions that gain helicity. This combinatorial mechanism allows individual low-affinity and specificity interactions to generate a biologically functional, specific, and higher affinity complex despite lacking a defined protein-protein interface. This binding strategy is likely representative of many activators that target multiple coactivators, as it allows great flexibility in combinations of activators that can cooperate to regulate genes with variable coactivator requirements.
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http://dx.doi.org/10.1016/j.celrep.2018.02.097DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5908246PMC
March 2018

De novo mutation in with epigenetic effects on neurodevelopment.

Proc Natl Acad Sci U S A 2018 02 31;115(7):1558-1563. Epub 2018 Jan 31.

Department of Medicine, University of Washington, Seattle, WA 98195;

RING1 is an E3-ubiquitin ligase that is involved in epigenetic control of transcription during development. It is a component of the polycomb repressive complex 1, and its role in that complex is to ubiquitylate histone H2A. In a 13-year-old girl with syndromic neurodevelopmental disabilities, we identified a de novo mutation, RING1 p.R95Q, which alters a conserved arginine residue in the catalytic RING domain. In vitro assays demonstrated that the mutant RING1 retains capacity to catalyze ubiquitin chain formation, but is defective in its ability to ubiquitylate histone H2A in nucleosomes. Consistent with this in vitro effect, cells of the patient showed decreased monoubiquitylation of histone H2A. We modeled the mutant RING1 in by editing the comparable amino acid change into , the suggested ortholog. Animals with either the missense mutation or complete knockout of were defective in monoubiquitylation of histone H2A and had defects in neuronal migration and axon guidance. Relevant to our patient, animals heterozygous for either the missense or knockout allele also showed neuronal defects. Our results support three conclusions: mutation of is the likely cause of a human neurodevelopmental syndrome, mutation of can disrupt histone H2A ubiquitylation without disrupting RING1 catalytic activity, and the comparable mutation in both recapitulates the effects on histone H2A ubiquitylation and leads to neurodevelopmental abnormalities. This role for adds to our understanding of the importance of aberrant epigenetic effects as causes of human neurodevelopmental disorders.
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http://dx.doi.org/10.1073/pnas.1721290115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5816211PMC
February 2018

BARD1 is necessary for ubiquitylation of nucleosomal histone H2A and for transcriptional regulation of estrogen metabolism genes.

Proc Natl Acad Sci U S A 2018 02 24;115(6):1316-1321. Epub 2018 Jan 24.

Department of Biochemistry, University of Washington, Seattle, WA 98195;

Missense mutations that disrupt the RING domain of the tumor suppressor gene lead to increased risk of breast and ovarian cancer. The BRCA1 RING domain is a ubiquitin ligase, whose structure and function rely critically on forming a heterodimer with BARD1, which also harbors a RING domain. The function of the BARD1 RING domain is unknown. In families severely affected with breast cancer, we identified inherited BARD1 missense mutations Cys53Trp, Cys71Tyr, and Cys83Arg that alter three zinc-binding residues of the BARD1 RING domain. Each of these mutant BARD1 proteins retained the ability to form heterodimeric complexes with BRCA1 to make an active ubiquitin ligase, but the mutant BRCA1/BARD1 complexes were deficient in binding to nucleosomes and in ubiquitylating histone H2A. The BARD1 mutations also caused loss of transcriptional repression of BRCA1-regulated estrogen metabolism genes and ; breast epithelial cells edited to create heterozygous loss of showed significantly higher expression of and Reintroduction of wild-type into these cells restored and transcription to normal levels, but introduction of the cancer-predisposing RING mutants failed to do so. These results indicate that an intact BARD1 RING domain is critical to BRCA1/BARD1 binding to nucleosomes and hence to ubiquitylation of histone H2A and also critical to transcriptional repression of BRCA1-regulated genes active in estrogen metabolism.
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http://dx.doi.org/10.1073/pnas.1715467115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5819413PMC
February 2018

Solution structure of sperm lysin yields novel insights into molecular dynamics of rapid protein evolution.

Proc Natl Acad Sci U S A 2018 02 18;115(6):1310-1315. Epub 2018 Jan 18.

Department of Genome Sciences, University of Washington, Seattle, WA 98195.

Protein evolution is driven by the sum of different physiochemical and genetic processes that usually results in strong purifying selection to maintain biochemical functions. However, proteins that are part of systems under arms race dynamics often evolve at unparalleled rates that can produce atypical biochemical properties. In the marine mollusk abalone, lysin and vitelline envelope receptor for lysin (VERL) are a pair of rapidly coevolving proteins that are essential for species-specific interactions between sperm and egg. Despite extensive biochemical characterization of lysin-including crystal structures of multiple orthologs-it was unclear how sites under positive selection may facilitate recognition of VERL. Using a combination of targeted mutagenesis and multidimensional NMR, we present a high-definition solution structure of sperm lysin from red abalone (). Unapparent from the crystallography data, multiple NMR-based analyses conducted in solution reveal clustering of the N and C termini to form a nexus of 13 positively selected sites that constitute a VERL binding interface. Evolutionary rate was found to be a significant predictor of backbone flexibility, which may be critical for lysin bioactivity and/or accelerated evolution. Flexible, rapidly evolving segments that constitute the VERL binding interface were also the most distorted regions of the crystal structure relative to what was observed in solution. While lysin has been the subject of extensive biochemical and evolutionary analyses for more than 30 years, this study highlights the enhanced insights gained from applying NMR approaches to rapidly evolving proteins.
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http://dx.doi.org/10.1073/pnas.1709061115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5819389PMC
February 2018

HspB1 and Hsc70 chaperones engage distinct tau species and have different inhibitory effects on amyloid formation.

J Biol Chem 2018 02 3;293(8):2687-2700. Epub 2018 Jan 3.

Department of Medicinal Chemistry, University of Washington, Seattle, Washington 98195-7610. Electronic address:

The microtubule-associated protein tau forms insoluble, amyloid-type aggregates in various dementias, most notably Alzheimer's disease. Cellular chaperone proteins play important roles in maintaining protein solubility and preventing aggregation in the crowded cellular environment. Although tau is known to interact with numerous chaperones, it remains unclear how these chaperones function mechanistically to prevent tau aggregation and how chaperones from different classes compare in terms of mechanism. Here, we focused on the small heat shock protein HspB1 (also known as Hsp27) and the constitutive chaperone Hsc70 (also known as HspA8) and report how each chaperone interacts with tau to prevent its fibril formation. Using fluorescence and NMR spectroscopy, we show that the two chaperones inhibit tau fibril formation by distinct mechanisms. HspB1 delayed tau fibril formation by weakly interacting with early species in the aggregation process, whereas Hsc70 was highly efficient at preventing tau fibril elongation, possibly by capping the ends of tau fibrils. Both chaperones recognized aggregation-prone motifs within the microtubule-binding repeat region of tau. However, HspB1 binding remained transient in both aggregation-promoting and non-aggregating conditions, whereas Hsc70 binding was significantly tighter under aggregation-promoting conditions. These differences highlight the fact that chaperones from different families play distinct but complementary roles in the prevention of pathological protein aggregation.
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http://dx.doi.org/10.1074/jbc.M117.803411DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5827454PMC
February 2018

RING-Between-RING E3 Ligases: Emerging Themes amid the Variations.

J Mol Biol 2017 11 19;429(22):3363-3375. Epub 2017 Aug 19.

Department of Biochemistry, University of Washington, Seattle, WA, United States. Electronic address:

Covalent, reversible, post-translational modification of cellular proteins with the small modifier, ubiquitin (Ub), regulates virtually every known cellular process in eukaryotes. The process is carried out by a trio of enzymes: a Ub-activating (E1) enzyme, a Ub-conjugating (E2) enzyme, and a Ub ligase (E3) enzyme. RING-in-Between-RING (RBR) E3s constitute one of three classes of E3 ligases and are defined by a RING-HECT-hybrid mechanism that utilizes a E2-binding RING domain and a second domain (called RING2) that contains an active site Cys required for the formation of an obligatory E3~Ub intermediate. Albeit a small class, RBR E3s in humans regulate diverse cellular process. This review focuses on non-Parkin members such as HOIP/HOIL-1L (the only E3s known to generate linear Ub chains), HHARI and TRIAD1, both of which have been recently demonstrated to work together with Cullin RING E3 ligases. We provide a brief historical background and highlight, summarize, and discuss recent developments in the young field of RBR E3s. Insights reviewed here include new understandings of the RBR Ub-transfer mechanism, specifically the role of RING1 and various Ub-binding sites, brief structural comparisons among members, and different modes of auto-inhibition and activation.
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http://dx.doi.org/10.1016/j.jmb.2017.08.008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5675740PMC
November 2017

Two functionally distinct E2/E3 pairs coordinate sequential ubiquitination of a common substrate in development.

Proc Natl Acad Sci U S A 2017 08 24;114(32):E6576-E6584. Epub 2017 Jul 24.

Department of Biochemistry, University of Washington School of Medicine, Seattle, WA 98195

Ubiquitination, the crucial posttranslational modification that regulates the eukaryotic proteome, is carried out by a trio of enzymes, known as E1 [ubiquitin (Ub)-activating enzyme], E2 (Ub-conjugating enzyme), and E3 (Ub ligase). Although most E2s can work with any of the three mechanistically distinct classes of E3s, the E2 UBCH7 is unable to function with really interesting new gene (RING)-type E3s, thereby restricting it to homologous to E6AP C-terminus (HECT) and RING-in-between-RING (RBR) E3s. The UBCH7 homolog, UBC-18, plays a critical role in developmental processes through its cooperation with the RBR E3 ARI-1 (HHARI in humans). We discovered that another E2, , interacts genetically with in an unbiased genome-wide RNAi screen in These two E2s have nonoverlapping biochemical activities, and each is dedicated to distinct classes of E3s. UBC-3 is the ortholog of CDC34 that functions specifically with Cullin-RING E3 ligases, such as SCF (Skp1-Cullin-F-box). Our genetic and biochemical studies show that UBCH7 (UBC-18) and the RBR E3 HHARI (ARI-1) coordinate with CDC34 (UBC-3) and an SCF E3 complex to ubiquitinate a common substrate, a SKP1-related protein. We show that UBCH7/HHARI primes the substrate with a single Ub in the presence of CUL-1, and that CDC34 is required to build chains onto the Ub-primed substrate. Our study reveals that the association and coordination of two distinct E2/E3 pairs play essential roles in a developmental pathway and suggests that cooperative action among E3s is a conserved feature from worms to humans.
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http://dx.doi.org/10.1073/pnas.1705060114DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5559030PMC
August 2017

Structural Studies of HHARI/UbcH7∼Ub Reveal Unique E2∼Ub Conformational Restriction by RBR RING1.

Structure 2017 06 25;25(6):890-900.e5. Epub 2017 May 25.

Department of Biochemistry, University of Washington, 1705 Northeast Pacific Street, Seattle, WA 98195, USA. Electronic address:

RING-between-RING (RBR) E3s contain RING1 domains that are structurally similar yet mechanistically distinct from canonical RING domains. Both types of E3 bind E2∼ubiquitin (E2∼Ub) via their RINGs but canonical RING E3s promote closed E2∼Ub conformations required for direct Ub transfer from the E2 to substrate, while RBR RING1s promote open E2∼Ub to favor Ub transfer to the E3 active site. This different RING/E2∼Ub conformation determines its direct target, which for canonical RING E3s is typically a substrate or substrate-linked Ub, but is the E3 active-site cysteine in the case of RBR-type E3s. Here we show that a short extension of HHARI RING1, namely Zn-loop II, not present in any RING E3s, acts as a steric wedge to disrupt closed E2∼Ub, providing a structural explanation for the distinctive RING1-dependent conformational restriction mechanism utilized by RBR E3s.
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http://dx.doi.org/10.1016/j.str.2017.04.013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5462532PMC
June 2017

The growing world of small heat shock proteins: from structure to functions.

Cell Stress Chaperones 2017 07 31;22(4):601-611. Epub 2017 Mar 31.

Laboratory of Cell & Developmental Genetics, IBIS, and Department of Molecular Biology, Medical Biochemistry and Pathology, Medical School, Université Laval, Québec (Qc), G1V 0A6, Canada.

Small heat shock proteins (sHSPs) are present in all kingdoms of life and play fundamental roles in cell biology. sHSPs are key components of the cellular protein quality control system, acting as the first line of defense against conditions that affect protein homeostasis and proteome stability, from bacteria to plants to humans. sHSPs have the ability to bind to a large subset of substrates and to maintain them in a state competent for refolding or clearance with the assistance of the HSP70 machinery. sHSPs participate in a number of biological processes, from the cell cycle, to cell differentiation, from adaptation to stressful conditions, to apoptosis, and, even, to the transformation of a cell into a malignant state. As a consequence, sHSP malfunction has been implicated in abnormal placental development and preterm deliveries, in the prognosis of several types of cancer, and in the development of neurological diseases. Moreover, mutations in the genes encoding several mammalian sHSPs result in neurological, muscular, or cardiac age-related diseases in humans. Loss of protein homeostasis due to protein aggregation is typical of many age-related neurodegenerative and neuromuscular diseases. In light of the role of sHSPs in the clearance of un/misfolded aggregation-prone substrates, pharmacological modulation of sHSP expression or function and rescue of defective sHSPs represent possible routes to alleviate or cure protein conformation diseases. Here, we report the latest news and views on sHSPs discussed by many of the world's experts in the sHSP field during a dedicated workshop organized in Italy (Bertinoro, CEUB, October 12-15, 2016).
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http://dx.doi.org/10.1007/s12192-017-0787-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5465036PMC
July 2017