Publications by authors named "Jennifer N Rauch"

30 Publications

  • Page 1 of 1

Comparison of Severe Acute Respiratory Syndrome Coronavirus 2 Screening Using Reverse Transcriptase-Quantitative Polymerase Chain Reaction or CRISPR-Based Assays in Asymptomatic College Students.

JAMA Netw Open 2021 02 1;4(2):e2037129. Epub 2021 Feb 1.

Department of Molecular, Cellular, and Developmental Biology, University of California, Santa Barbara.

Importance: The reopening of colleges and universities in the US during the coronavirus disease 2019 (COVID-19) pandemic is a significant public health challenge. The development of accessible and practical approaches for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) detection in the college population is paramount for deploying recurrent surveillance testing as an essential strategy for virus detection, containment, and mitigation.

Objective: To determine the prevalence of SARS-CoV-2 in asymptomatic participants in a university community by using CREST (Cas13-based, rugged, equitable, scalable testing), a CRISPR-based test developed for accessible and large-scale viral screening.

Design, Setting, And Participants: For this cohort study, a total of 1808 asymptomatic participants were screened for SARS-CoV-2 using a CRISPR-based assay and a point-of-reference reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR) test. Viral prevalence in self-collected oropharyngeal swab samples collected from May 28 to June 11, 2020, and from June 23 to July 2, 2020, was evaluated.

Exposures: Testing for SARS-CoV-2.

Main Outcomes And Measures: SARS-CoV-2 status, viral load, and demographic information of the study participants were collected.

Results: Among the 1808 participants (mean [SD] age, 27.3 [11.0] years; 955 [52.8%] female), 732 underwent testing from May to early June (mean [SD] age, 28.4 [11.7] years; 392 [53.6%] female). All test results in this cohort were negative. In contrast, 1076 participants underwent testing from late June to early July (mean [SD] age, 26.6 [10.5] years; 563 [52.3%] female), with 9 positive results by RT-qPCR. Eight of these positive samples were detected by the CRISPR-based assay and confirmed by Clinical Laboratory Improvement Amendments-certified diagnostic testing. The mean (SD) age of the positive cases was 21.7 (3.3) years; all 8 individuals self-identified as students. These metrics showed that a CRISPR-based assay was effective at capturing positive SARS-CoV-2 cases in this student population. Notably, the viral loads detected in these asymptomatic cases resemble those seen in clinical samples, highlighting the potential of covert viral transmission. The shift in viral prevalence coincided with the relaxation of stay-at-home measures.

Conclusions And Relevance: These findings reveal a shift in SARS-CoV-2 prevalence in a young and asymptomatic population and uncover the leading edge of a local outbreak that coincided with rising case counts in the surrounding county and the state of California. The concordance between CRISPR-based and RT-qPCR testing suggests that CRISPR-based assays are reliable and offer alternative options for surveillance testing and detection of SARS-CoV-2 outbreaks, as is required to resume operations in higher-education institutions in the US and abroad.
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http://dx.doi.org/10.1001/jamanetworkopen.2020.37129DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7879237PMC
February 2021

Chemical validation of a druggable site on Hsp27/HSPB1 using in silico solvent mapping and biophysical methods.

Bioorg Med Chem 2021 Mar 24;34:115990. Epub 2021 Jan 24.

Department of Pharmaceutical Chemistry and the Institute for Neurodegenerative Disease, University of California at San Francisco, San Francisco, CA 94158, United States. Electronic address:

Destabilizing mutations in small heat shock proteins (sHsps) are linked to multiple diseases; however, sHsps are conformationally dynamic, lack enzymatic function and have no endogenous chemical ligands. These factors render sHsps as classically "undruggable" targets and make it particularly challenging to identify molecules that might bind and stabilize them. To explore potential solutions, we designed a multi-pronged screening workflow involving a combination of computational and biophysical ligand-discovery platforms. Using the core domain of the sHsp family member Hsp27/HSPB1 (Hsp27c) as a target, we applied mixed solvent molecular dynamics (MixMD) to predict three possible binding sites, which we confirmed using NMR-based solvent mapping. Using this knowledge, we then used NMR spectroscopy to carry out a fragment-based drug discovery (FBDD) screen, ultimately identifying two fragments that bind to one of these sites. A medicinal chemistry effort improved the affinity of one fragment by ~50-fold (16 µM), while maintaining good ligand efficiency (~0.32 kcal/mol/non-hydrogen atom). Finally, we found that binding to this site partially restored the stability of disease-associated Hsp27 variants, in a redox-dependent manner. Together, these experiments suggest a new and unexpected binding site on Hsp27, which might be exploited to build chemical probes.
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http://dx.doi.org/10.1016/j.bmc.2020.115990DOI Listing
March 2021

A Scalable, Easy-to-Deploy, Protocol for Cas13-Based Detection of SARS-CoV-2 Genetic Material.

J Clin Microbiol 2021 Jan 21. Epub 2021 Jan 21.

University of California Santa Barbara, Department of Molecular, Cellular, and Developmental Biology.

The COVID-19 pandemic has created massive demand for widespread, distributed tools for detecting SARS-CoV-2 genetic material. The hurdles to scalable testing include reagent and instrument accessibility, availability of highly trained personnel, and large upfront investment. Here we showcase an orthogonal pipeline we call CREST (Cas13-based, Rugged, Equitable, Scalable Testing) that addresses some of these hurdles. Specifically, CREST pairs commonplace and reliable biochemical methods (PCR) with low-cost instrumentation, without sacrificing detection sensitivity. By taking advantage of simple fluorescence visualizers, CREST allows for a binary interpretation of results. CREST may provide a point-of-care solution to increase the distribution of COVID-19 surveillance.
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http://dx.doi.org/10.1128/JCM.02402-20DOI Listing
January 2021

The proline-rich domain promotes Tau liquid-liquid phase separation in cells.

J Cell Biol 2020 Nov;219(11)

Department of Molecular, Cell and Developmental Biology, University of California, Santa Barbara, Santa Barbara, CA.

Tau protein in vitro can undergo liquid-liquid phase separation (LLPS); however, observations of this phase transition in living cells are limited. To investigate protein state transitions in living cells, we attached Cry2 to Tau and studied the contribution of each domain that drives the Tau cluster in living cells. Surprisingly, the proline-rich domain (PRD), not the microtubule binding domain (MTBD), drives LLPS and does so under the control of its phosphorylation state. Readily observable, PRD-derived cytoplasmic condensates underwent fusion and fluorescence recovery after photobleaching consistent with the PRD LLPS in vitro. Simulations demonstrated that the charge properties of the PRD predicted phase separation. Tau PRD formed heterotypic condensates with EB1, a regulator of plus-end microtubule dynamic instability. The specific domain properties of the MTBD and PRD serve distinct but mutually complementary roles that use LLPS in a cellular context to implement emergent functionalities that scale their relationship from binding α-beta tubulin heterodimers to the larger proportions of microtubules.
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http://dx.doi.org/10.1083/jcb.202006054DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7594490PMC
November 2020

LRP1 is a master regulator of tau uptake and spread.

Nature 2020 04 1;580(7803):381-385. Epub 2020 Apr 1.

Neuroscience Research Institute, Department of Molecular, Cellular, and Developmental Biology, University of California, Santa Barbara, CA, USA.

The spread of protein aggregates during disease progression is a common theme underlying many neurodegenerative diseases. The microtubule-associated protein tau has a central role in the pathogenesis of several forms of dementia known as tauopathies-including Alzheimer's disease, frontotemporal dementia and chronic traumatic encephalopathy. Progression of these diseases is characterized by the sequential spread and deposition of protein aggregates in a predictable pattern that correlates with clinical severity. This observation and complementary experimental studies have suggested that tau can spread in a prion-like manner, by passing to naive cells in which it templates misfolding and aggregation. However, although the propagation of tau has been extensively studied, the underlying cellular mechanisms remain poorly understood. Here we show that the low-density lipoprotein receptor-related protein 1 (LRP1) controls the endocytosis of tau and its subsequent spread. Knockdown of LRP1 significantly reduced tau uptake in H4 neuroglioma cells and in induced pluripotent stem cell-derived neurons. The interaction between tau and LRP1 is mediated by lysine residues in the microtubule-binding repeat region of tau. Furthermore, downregulation of LRP1 in an in vivo mouse model of tau spread was found to effectively reduce the propagation of tau between neurons. Our results identify LRP1 as a key regulator of tau spread in the brain, and therefore a potential target for the treatment of diseases that involve tau spread and aggregation.
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http://dx.doi.org/10.1038/s41586-020-2156-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7687380PMC
April 2020

Hsp70 and Hsp40 inhibit an inter-domain interaction necessary for transcriptional activity in the androgen receptor.

Nat Commun 2019 08 8;10(1):3562. Epub 2019 Aug 8.

Institute for Research in Biomedicine (IRB Barcelona), The Barcelona Institute of Science and Technology, Baldiri Reixac 10, 08028, Barcelona, Spain.

Molecular chaperones such as Hsp40 and Hsp70 hold the androgen receptor (AR) in an inactive conformation. They are released in the presence of androgens, enabling transactivation and causing the receptor to become aggregation-prone. Here we show that these molecular chaperones recognize a region of the AR N-terminal domain (NTD), including a FQNLF motif, that interacts with the AR ligand-binding domain (LBD) upon activation. This suggests that competition between molecular chaperones and the LBD for the FQNLF motif regulates AR activation. We also show that, while the free NTD oligomerizes, binding to Hsp70 increases its solubility. Stabilizing the NTD-Hsp70 interaction with small molecules reduces AR aggregation and promotes its degradation in cellular and mouse models of the neuromuscular disorder spinal bulbar muscular atrophy. These results help resolve the mechanisms by which molecular chaperones regulate the balance between AR aggregation, activation and quality control.
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http://dx.doi.org/10.1038/s41467-019-11594-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6687723PMC
August 2019

Narrow equilibrium window for complex coacervation of tau and RNA under cellular conditions.

Elife 2019 04 5;8. Epub 2019 Apr 5.

Department of Chemistry and Biochemistry, University of California Santa Barbara, Santa Barbara, United States.

The mechanism that leads to liquid-liquid phase separation (LLPS) of the tau protein, whose pathological aggregation is implicated in neurodegenerative disorders, is not well understood. Establishing a phase diagram that delineates the boundaries of phase co-existence is key to understanding whether LLPS is an equilibrium or intermediate state. We demonstrate that tau and RNA reversibly form complex coacervates. While the equilibrium phase diagram can be fit to an analytical theory, a more advanced model is investigated through field theoretic simulations (FTS) that provided direct insight into the thermodynamic driving forces of tau LLPS. Together, experiment and simulation reveal that tau-RNA LLPS is stable within a narrow equilibrium window near physiological conditions over experimentally tunable parameters including temperature, salt and tau concentrations, and is entropy-driven. Guided by our phase diagram, we show that tau can be driven toward LLPS under live cell coculturing conditions with rationally chosen experimental parameters.
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http://dx.doi.org/10.7554/eLife.42571DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6450672PMC
April 2019

A farnesyltransferase inhibitor activates lysosomes and reduces tau pathology in mice with tauopathy.

Sci Transl Med 2019 03;11(485)

Neuroscience Research Institute, University of California, Santa Barbara, Santa Barbara, CA 93106, USA.

Tau inclusions are a shared feature of many neurodegenerative diseases, among them frontotemporal dementia caused by tau mutations. Treatment approaches for these conditions include targeting posttranslational modifications of tau proteins, maintaining a steady-state amount of tau, and preventing its tendency to aggregate. We discovered a new regulatory pathway for tau degradation that operates through the farnesylated protein, Rhes, a GTPase in the Ras family. Here, we show that treatment with the farnesyltransferase inhibitor lonafarnib reduced Rhes and decreased brain atrophy, tau inclusions, tau sumoylation, and tau ubiquitination in the rTg4510 mouse model of tauopathy. In addition, lonafarnib treatment attenuated behavioral abnormalities in rTg4510 mice and reduced microgliosis in mouse brain. Direct reduction of Rhes in the rTg4510 mouse by siRNA reproduced the results observed with lonafarnib treatment. The mechanism of lonafarnib action mediated by Rhes to reduce tau pathology was shown to operate through activation of lysosomes. We finally showed in mouse brain and in human induced pluripotent stem cell-derived neurons a normal developmental increase in Rhes that was initially suppressed by tau mutations. The known safety of lonafarnib revealed in human clinical trials for cancer suggests that this drug could be repurposed for treating tauopathies.
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http://dx.doi.org/10.1126/scitranslmed.aat3005DOI Listing
March 2019

Myopathy associated BAG3 mutations lead to protein aggregation by stalling Hsp70 networks.

Nat Commun 2018 12 17;9(1):5342. Epub 2018 Dec 17.

University Medical Center Groningen, University of Groningen, Department of Biomedical Sciences of Cell & Systems, Groningen, AV, 9791, The Netherlands.

BAG3 is a multi-domain hub that connects two classes of chaperones, small heat shock proteins (sHSPs) via two isoleucine-proline-valine (IPV) motifs and Hsp70 via a BAG domain. Mutations in either the IPV or BAG domain of BAG3 cause a dominant form of myopathy, characterized by protein aggregation in both skeletal and cardiac muscle tissues. Surprisingly, for both disease mutants, impaired chaperone binding is not sufficient to explain disease phenotypes. Recombinant mutants are correctly folded, show unaffected Hsp70 binding but are impaired in stimulating Hsp70-dependent client processing. As a consequence, the mutant BAG3 proteins become the node for a dominant gain of function causing aggregation of itself, Hsp70, Hsp70 clients and tiered interactors within the BAG3 interactome. Importantly, genetic and pharmaceutical interference with Hsp70 binding completely reverses stress-induced protein aggregation for both BAG3 mutations. Thus, the gain of function effects of BAG3 mutants act as Achilles heel of the HSP70 machinery.
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http://dx.doi.org/10.1038/s41467-018-07718-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6297355PMC
December 2018

Cofactors are essential constituents of stable and seeding-active tau fibrils.

Proc Natl Acad Sci U S A 2018 12 11;115(52):13234-13239. Epub 2018 Dec 11.

Department of Chemistry and Biochemistry, University of California, Santa Barbara, CA 93106;

Amyloid fibrils are cross-β-rich aggregates that are exceptionally stable forms of protein assembly. Accumulation of tau amyloid fibrils is involved in many neurodegenerative diseases, including Alzheimer's disease (AD). Heparin-induced aggregates have been widely used and assumed to be a good tau amyloid fibril model for most biophysical studies. Here we show that mature fibrils made of 4R tau variants, prepared with heparin or RNA, spontaneously depolymerize and release monomers when their cofactors are removed. We demonstrate that the cross-β-sheet assembly formed in vitro with polyanion addition is unstable at room temperature. We furthermore demonstrate high seeding capacity with transgenic AD mouse brain-extracted tau fibrils in vitro that, however, is exhausted after one generation, while supplementation with RNA cofactors resulted in sustained seeding over multiple generations. We suggest that tau fibrils formed in brains are supported by unknown cofactors and inhere higher-quality packing, as reflected in a more distinct conformational arrangement in the mouse fibril-seeded, compared with heparin-induced, tau fibrils. Our study suggests that the role of cofactors in tauopathies is a worthy focus of future studies, as they may be viable targets for diagnosis and therapeutics.
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http://dx.doi.org/10.1073/pnas.1810058115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6310788PMC
December 2018

Mapping interactions with the chaperone network reveals factors that protect against tau aggregation.

Nat Struct Mol Biol 2018 05 30;25(5):384-393. Epub 2018 Apr 30.

Department of Neurology, University of California at San Francisco, San Francisco, CA, USA.

A network of molecular chaperones is known to bind proteins ('clients') and balance their folding, function and turnover. However, it is often unclear which chaperones are critical for selective recognition of individual clients. It is also not clear why these key chaperones might fail in protein-aggregation diseases. Here, we utilized human microtubule-associated protein tau (MAPT or tau) as a model client to survey interactions between ~30 purified chaperones and ~20 disease-associated tau variants (~600 combinations). From this large-scale analysis, we identified human DnaJA2 as an unexpected, but potent, inhibitor of tau aggregation. DnaJA2 levels were correlated with tau pathology in human brains, supporting the idea that it is an important regulator of tau homeostasis. Of note, we found that some disease-associated tau variants were relatively immune to interactions with chaperones, suggesting a model in which avoiding physical recognition by chaperone networks may contribute to disease.
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http://dx.doi.org/10.1038/s41594-018-0057-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5942583PMC
May 2018

Tau Internalization is Regulated by 6-O Sulfation on Heparan Sulfate Proteoglycans (HSPGs).

Sci Rep 2018 04 23;8(1):6382. Epub 2018 Apr 23.

Neuroscience Research Institute, Department of Molecular Cellular Developmental Biology, University of California, Santa Barbara, CA, 93106, USA.

The misfolding and accumulation of tau protein into intracellular aggregates known as neurofibrillary tangles is a pathological hallmark of neurodegenerative diseases such as Alzheimer's disease. However, while tau propagation is a known marker for disease progression, exactly how tau propagates from one cell to another and what mechanisms govern this spread are still unclear. Here, we report that cellular internalization of tau is regulated by quaternary structure and have developed a cellular assay to screen for genetic modulators of tau uptake. Using CRISPRi technology we have tested 3200 genes for their ability to regulate tau entry and identified enzymes in the heparan sulfate proteoglycan biosynthetic pathway as key regulators. We show that 6-O-sulfation is critical for tau-heparan sulfate interactions and that this modification regulates uptake in human central nervous system cell lines, iPS-derived neurons, and mouse brain slice culture. Together, these results suggest novel strategies to halt tau transmission.
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http://dx.doi.org/10.1038/s41598-018-24904-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5913225PMC
April 2018

High-throughput screen for inhibitors of protein-protein interactions in a reconstituted heat shock protein 70 (Hsp70) complex.

J Biol Chem 2018 03 2;293(11):4014-4025. Epub 2018 Feb 2.

From the Department of Pharmaceutical Chemistry, University of California, San Francisco, California 94158 and

Protein-protein interactions (PPIs) are an important category of putative drug targets. Improvements in high-throughput screening (HTS) have significantly accelerated the discovery of inhibitors for some categories of PPIs. However, methods suitable for screening multiprotein complexes ( those composed of three or more different components) have been slower to emerge. Here, we explored an approach that uses reconstituted multiprotein complexes (RMPCs). As a model system, we chose heat shock protein 70 (Hsp70), which is an ATP-dependent molecular chaperone that interacts with co-chaperones, including DnaJA2 and BAG2. The PPIs between Hsp70 and its co-chaperones stimulate nucleotide cycling. Thus, to re-create this ternary protein system, we combined purified human Hsp70 with DnaJA2 and BAG2 and then screened 100,000 diverse compounds for those that inhibited co-chaperone-stimulated ATPase activity. This HTS campaign yielded two compounds with promising inhibitory activity. Interestingly, one inhibited the PPI between Hsp70 and DnaJA2, whereas the other seemed to inhibit the Hsp70-BAG2 complex. Using secondary assays, we found that both compounds inhibited the PPIs through binding to allosteric sites on Hsp70, but neither affected Hsp70's intrinsic ATPase activity. Our RMPC approach expands the toolbox of biochemical HTS methods available for studying difficult-to-target PPIs in multiprotein complexes. The results may also provide a starting point for new chemical probes of the Hsp70 system.
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http://dx.doi.org/10.1074/jbc.RA117.001575DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5857975PMC
March 2018

RNA stores tau reversibly in complex coacervates.

PLoS Biol 2017 Jul 6;15(7):e2002183. Epub 2017 Jul 6.

Department of Chemistry and Biochemistry, University of California Santa Barbara, Santa Barbara, California, United States of America.

Nonmembrane-bound organelles that behave like liquid droplets are widespread among eukaryotic cells. Their dysregulation appears to be a critical step in several neurodegenerative conditions. Here, we report that tau protein, the primary constituent of Alzheimer neurofibrillary tangles, can form liquid droplets and therefore has the necessary biophysical properties to undergo liquid-liquid phase separation (LLPS) in cells. Consonant with the factors that induce LLPS, tau is an intrinsically disordered protein that complexes with RNA to form droplets. Uniquely, the pool of RNAs to which tau binds in living cells are tRNAs. This phase state of tau is held in an approximately 1:1 charge balance across the protein and the nucleic acid constituents, and can thus be maximal at different RNA:tau mass ratios, depending on the biopolymer constituents involved. This feature is characteristic of complex coacervation. We furthermore show that the LLPS process is directly and sensitively tuned by salt concentration and temperature, implying it is modulated by both electrostatic interactions between the involved protein and nucleic acid constituents, as well as net changes in entropy. Despite the high protein concentration within the complex coacervate phase, tau is locally freely tumbling and capable of diffusing through the droplet interior. In fact, tau in the condensed phase state does not reveal any immediate changes in local protein packing, local conformations and local protein dynamics from that of tau in the dilute solution state. In contrast, the population of aggregation-prone tau as induced by the complexation with heparin is accompanied by large changes in local tau conformations and irreversible aggregation. However, prolonged residency within the droplet state eventually results in the emergence of detectable β-sheet structures according to thioflavin-T assay. These findings suggest that the droplet state can incubate tau and predispose the protein toward the formation of insoluble fibrils.
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http://dx.doi.org/10.1371/journal.pbio.2002183DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5500003PMC
July 2017

Interactions between Microtubule-Associated Protein Tau (MAPT) and Small Molecules.

Cold Spring Harb Perspect Med 2017 Jul 5;7(7). Epub 2017 Jul 5.

Institute for Neurodegenerative Diseases, Weill Institute for Neurosciences, University of California, San Francisco, San Francisco, California 94158.

Tau aggregation is linked to multiple neurodegenerative disorders that are collectively termed tauopathies. Small molecules are powerful probes of the aggregation process, helping to reveal the key steps and serving as diagnostics and reporters. Moreover, some of these small molecules may have potential as therapeutics. This review details how small molecules and chemical biology have helped to elucidate the mechanisms of tau aggregation and how they are being used to detect and prevent tau aggregation. In addition, we comment on how new insights into tau prions are changing the approach to small molecule discovery.
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http://dx.doi.org/10.1101/cshperspect.a024034DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5495054PMC
July 2017

BAG3 Is a Modular, Scaffolding Protein that physically Links Heat Shock Protein 70 (Hsp70) to the Small Heat Shock Proteins.

J Mol Biol 2017 01 21;429(1):128-141. Epub 2016 Nov 21.

Department of Pharmaceutical Chemistry, Institute for Neurodegenerative Disease, University of California at San Francisco, San Francisco, CA 94158, USA. Electronic address:

Small heat shock proteins (sHsps) are a family of ATP-independent molecular chaperones that are important for binding and stabilizing unfolded proteins. In this task, the sHsps have been proposed to coordinate with ATP-dependent chaperones, including heat shock protein 70 (Hsp70). However, it is not yet clear how these two important components of the chaperone network are linked. We report that the Hsp70 co-chaperone, BAG3, is a modular, scaffolding factor to bring together sHsps and Hsp70s. Using domain deletions and point mutations, we found that BAG3 uses both of its IPV motifs to interact with sHsps, including Hsp27 (HspB1), αB-crystallin (HspB5), Hsp22 (HspB8), and Hsp20 (HspB6). BAG3 does not appear to be a passive scaffolding factor; rather, its binding promoted de-oligomerization of Hsp27, likely by competing for the self-interactions that normally stabilize large oligomers. BAG3 bound to Hsp70 at the same time as Hsp22, Hsp27, or αB-crystallin, suggesting that it might physically bring the chaperone families together into a complex. Indeed, addition of BAG3 coordinated the ability of Hsp22 and Hsp70 to refold denatured luciferase in vitro. Together, these results suggest that BAG3 physically and functionally links Hsp70 and sHsps.
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http://dx.doi.org/10.1016/j.jmb.2016.11.013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5186407PMC
January 2017

Stabilizing the Hsp70-Tau Complex Promotes Turnover in Models of Tauopathy.

Cell Chem Biol 2016 08 4;23(8):992-1001. Epub 2016 Aug 4.

Department of Pharmaceutical Chemistry, University of California at San Francisco, San Francisco, CA 94158, USA; Sandler Neuroscience Center, University of California at San Francisco, Room 311, 675 Nelson Rising Lane, San Francisco, CA 94158, USA. Electronic address:

Heat shock protein 70 (Hsp70) is a chaperone that normally scans the proteome and initiates the turnover of some proteins (termed clients) by linking them to the degradation pathways. This activity is critical to normal protein homeostasis, yet it appears to fail in diseases associated with abnormal protein accumulation. It is not clear why Hsp70 promotes client degradation under some conditions, while sparing that protein under others. Here, we used a combination of chemical biology and genetic strategies to systematically perturb the affinity of Hsp70 for the model client, tau. This approach revealed that tight complexes between Hsp70 and tau were associated with enhanced turnover while transient interactions favored tau retention. These results suggest that client affinity is one important parameter governing Hsp70-mediated quality control.
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http://dx.doi.org/10.1016/j.chembiol.2016.04.014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4992411PMC
August 2016

Non-canonical Interactions between Heat Shock Cognate Protein 70 (Hsc70) and Bcl2-associated Anthanogene (BAG) Co-Chaperones Are Important for Client Release.

J Biol Chem 2016 09 29;291(38):19848-57. Epub 2016 Jul 29.

From the Department of Biological Chemistry, University of Michigan, Ann Arbor, Michigan 48109 and Department of Pharmaceutical Chemistry, University of California, San Francisco, San Francisco, California 94158

Heat shock cognate protein 70 (Hsc70) regulates protein homeostasis through its reversible interactions with client proteins. Hsc70 has two major domains: a nucleotide-binding domain (NBD), that hydrolyzes ATP, and a substrate-binding domain (SBD), where clients are bound. Members of the BAG family of co-chaperones, including Bag1 and Bag3, are known to accelerate release of both ADP and client from Hsc70. The release of nucleotide is known to be mediated by interactions between the conserved BAG domain and the Hsc70 NBD. However, less is known about the regions required for client release, and it is often assumed that this activity also requires the BAG domain. It is important to better understand this step because it determines how long clients remain in the inactive, bound state. Here, we report the surprising observation that truncated versions of either human Bag1 or Bag3, comprised only the BAG domain, promoted rapid release of nucleotide, but not client, in vitro Rather, we found that a non-canonical interaction between Bag1/3 and the Hsc70 SBD is sufficient for accelerating this step. Moreover, client release did not seem to require the BAG domain or Hsc70 NBD. These results suggest that Bag1 and Bag3 control the stability of the Hsc70-client complex using at least two distinct protein-protein contacts, providing a previously under-appreciated layer of molecular regulation in the human Hsc70 system.
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http://dx.doi.org/10.1074/jbc.M116.742502DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5025674PMC
September 2016

Protein Cross-Linking Capillary Electrophoresis for Protein-Protein Interaction Analysis.

Anal Chem 2016 08 28;88(16):8272-8. Epub 2016 Jul 28.

Department of Chemistry, University of Michigan , 930 N. University Ave., Ann Arbor, Michigan 48109, United States.

Capillary electrophoresis (CE) has been identified as a useful platform for detecting, quantifying, and screening for modulators of protein-protein interactions (PPIs). In this method, one protein binding partner is labeled with a fluorophore, the protein binding partners are mixed, and then, the complex is separated from free protein to allow direct determination of bound to free ratios. Although it possesses many advantages for PPI studies, the method is limited by the need to have separation conditions that both prevent protein adsorption to capillary and maintain protein interactions during the separation. In this work, we use protein cross-linking capillary electrophoresis (PXCE) to overcome this limitation. In PXCE, the proteins are cross-linked under binding conditions and then separated. This approach eliminates the need to maintain noncovalent interactions during electrophoresis and facilitates method development. We report PXCE methods for an antibody-antigen interaction and heterodimer and homodimer heat shock protein complexes. Complexes are cross-linked by short treatments with formaldehyde after reaching binding equilibrium. Cross-linked complexes are separated by electrophoretic mobility using free solution CE or by size using sieving electrophoresis of SDS complexes. The method gives good quantitative results; e.g., a lysozyme-antibody interaction was found to have Kd = 24 ± 3 nM by PXCE and Kd = 17 ± 2 nM using isothermal calorimetry (ITC). Heat shock protein 70 (Hsp70) in complex with bcl2 associated athanogene 3 (Bag3) was found to have Kd = 25 ± 5 nM by PXCE which agrees with Kd values reported without cross-linking. Hsp70-Bag3 binding site mutants and small molecule inhibitors of Hsp70-Bag3 were characterized by PXCE with good agreement to inhibitory constants and IC50 values obtained by a bead-based flow cytometry protein interaction assay (FCPIA). PXCE allows rapid method development for quantitative analysis of PPIs.
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http://dx.doi.org/10.1021/acs.analchem.6b02126DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5111828PMC
August 2016

Structural and Biological Interaction of hsc-70 Protein with Phosphatidylserine in Endosomal Microautophagy.

J Biol Chem 2016 08 12;291(35):18096-106. Epub 2016 Jul 12.

From the Departments of Pathology and

hsc-70 (HSPA8) is a cytosolic molecular chaperone, which plays a central role in cellular proteostasis, including quality control during protein refolding and regulation of protein degradation. hsc-70 is pivotal to the process of macroautophagy, chaperone-mediated autophagy, and endosomal microautophagy. The latter requires hsc-70 interaction with negatively charged phosphatidylserine (PS) at the endosomal limiting membrane. Herein, by combining plasmon resonance, NMR spectroscopy, and amino acid mutagenesis, we mapped the C terminus of the hsc-70 LID domain as the structural interface interacting with endosomal PS, and we estimated an hsc-70/PS equilibrium dissociation constant of 4.7 ± 0.1 μm. This interaction is specific and involves a total of 4-5 lysine residues. Plasmon resonance and NMR results were further experimentally validated by hsc-70 endosomal binding experiments and endosomal microautophagy assays. The discovery of this previously unknown contact surface for hsc-70 in this work elucidates the mechanism of hsc-70 PS/membrane interaction for cytosolic cargo internalization into endosomes.
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http://dx.doi.org/10.1074/jbc.M116.736744DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5000059PMC
August 2016

Defining Hsp70 Subnetworks in Dengue Virus Replication Reveals Key Vulnerability in Flavivirus Infection.

Cell 2015 Nov 12;163(5):1108-1123. Epub 2015 Nov 12.

Department of Biology and Genetics, Stanford University, Stanford, CA 94305, USA. Electronic address:

Viral protein homeostasis depends entirely on the machinery of the infected cell. Accordingly, viruses can illuminate the interplay between cellular proteostasis components and their distinct substrates. Here, we define how the Hsp70 chaperone network mediates the dengue virus life cycle. Cytosolic Hsp70 isoforms are required at distinct steps of the viral cycle, including entry, RNA replication, and virion biogenesis. Hsp70 function at each step is specified by nine distinct DNAJ cofactors. Of these, DnaJB11 relocalizes to virus-induced replication complexes to promote RNA synthesis, while DnaJB6 associates with capsid protein and facilitates virion biogenesis. Importantly, an allosteric Hsp70 inhibitor, JG40, potently blocks infection of different dengue serotypes in human primary blood cells without eliciting viral resistance or exerting toxicity to the host cells. JG40 also blocks replication of other medically-important flaviviruses including yellow fever, West Nile and Japanese encephalitis viruses. Thus, targeting host Hsp70 subnetworks provides a path for broad-spectrum antivirals.
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http://dx.doi.org/10.1016/j.cell.2015.10.046DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4869517PMC
November 2015

Isoform-selective Genetic Inhibition of Constitutive Cytosolic Hsp70 Activity Promotes Client Tau Degradation Using an Altered Co-chaperone Complement.

J Biol Chem 2015 May 11;290(21):13115-27. Epub 2015 Apr 11.

From the Department of Molecular Medicine, College of Medicine, USF Health Byrd Alzheimer's Institute, University of South Florida, Tampa, Florida 33613, James A. Haley Veteran's Hospital, Tampa, Florida 33612,

The constitutively expressed heat shock protein 70 kDa (Hsc70) is a major chaperone protein responsible for maintaining proteostasis, yet how its structure translates into functional decisions regarding client fate is still unclear. We previously showed that Hsc70 preserved aberrant Tau, but it remained unknown if selective inhibition of the activity of this Hsp70 isoform could facilitate Tau clearance. Using single point mutations in the nucleotide binding domain, we assessed the effect of several mutations on the functions of human Hsc70. Biochemical characterization revealed that one mutation abolished both Hsc70 ATPase and refolding activities. This variant resembled the ADP-bound conformer at all times yet remained able to interact with cofactors, nucleotides, and substrates appropriately, resembling a dominant negative Hsc70 (DN-Hsc70). We then assessed the effects of this DN-Hsc70 on its client Tau. DN-Hsc70 potently facilitated Tau clearance via the proteasome in cells and brain tissue, in contrast to wild type Hsc70 that stabilized Tau. Thus, DN-Hsc70 mimics the action of small molecule pan Hsp70 inhibitors with regard to Tau metabolism. This shift in Hsc70 function by a single point mutation was the result of a change in the chaperome associated with Hsc70 such that DN-Hsc70 associated more with Hsp90 and DnaJ proteins, whereas wild type Hsc70 was more associated with other Hsp70 isoforms. Thus, isoform-selective targeting of Hsc70 could be a viable therapeutic strategy for tauopathies and possibly lead to new insights in chaperone complex biology.
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http://dx.doi.org/10.1074/jbc.M115.637595DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4505567PMC
May 2015

Validation of the Hsp70-Bag3 protein-protein interaction as a potential therapeutic target in cancer.

Mol Cancer Ther 2015 Mar 6;14(3):642-8. Epub 2015 Jan 6.

Department of Pharmaceutical Chemistry and the Institute for Neurodegenerative Disease, University of California at San Francisco, San Francisco, California.

Hsp70 is a stress-inducible molecular chaperone that is required for cancer development at several steps. Targeting the active site of Hsp70 has proven relatively challenging, driving interest in alternative approaches. Hsp70 collaborates with the Bcl2-associated athanogene 3 (Bag3) to promote cell survival through multiple pathways, including FoxM1. Therefore, inhibitors of the Hsp70-Bag3 protein-protein interaction (PPI) may provide a noncanonical way to target this chaperone. We report that JG-98, an allosteric inhibitor of this PPI, indeed has antiproliferative activity (EC50 values between 0.3 and 4 μmol/L) across cancer cell lines from multiple origins. JG-98 destabilized FoxM1 and relieved suppression of downstream effectors, including p21 and p27. On the basis of these findings, JG-98 was evaluated in mice for pharmacokinetics, tolerability, and activity in two xenograft models. The results suggested that the Hsp70-Bag3 interaction may be a promising, new target for anticancer therapy.
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http://dx.doi.org/10.1158/1535-7163.MCT-14-0650DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4456214PMC
March 2015

Rehabilitating mutant GCase.

Chem Biol 2014 Aug;21(8):919-20

Department of Pharmaceutical Chemistry, Institute for Neurodegenerative Disease, University of California at San Francisco, 675 Nelson Rising Lane, San Francisco, CA 94158, USA. Electronic address:

Gaucher's disease is a hereditary deficiency of the enzyme β-glucocerebrosidase (GCase) that is most commonly treated by enzyme replacement therapy. In this issue of Chemistry & Biology, Tan and colleagues search for alternative ways to rehabilitate mutant GCase by understanding how it interacts with the proteostasis network.
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http://dx.doi.org/10.1016/j.chembiol.2014.07.010DOI Listing
August 2014

Hsp70-Bag3 interactions regulate cancer-related signaling networks.

Cancer Res 2014 Sep 3;74(17):4731-40. Epub 2014 Jul 3.

Department of Biochemistry, Boston University School of Medicine, Boston, Massachusetts.

Bag3, a nucleotide exchange factor of the heat shock protein Hsp70, has been implicated in cell signaling. Here, we report that Bag3 interacts with the SH3 domain of Src, thereby mediating the effects of Hsp70 on Src signaling. Using several complementary approaches, we established that the Hsp70-Bag3 module is a broad-acting regulator of cancer cell signaling by modulating the activity of the transcription factors NF-κB, FoxM1, Hif1α, the translation regulator HuR, and the cell-cycle regulators p21 and survivin. We also identified a small-molecule inhibitor, YM-1, that disrupts the Hsp70-Bag3 interaction. YM-1 mirrored the effects of Hsp70 depletion on these signaling pathways, and in vivo administration of this drug was sufficient to suppress tumor growth in mice. Overall, our results defined Bag3 as a critical factor in Hsp70-modulated signaling and offered a preclinical proof-of-concept that the Hsp70-Bag3 complex may offer an appealing anticancer target.
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http://dx.doi.org/10.1158/0008-5472.CAN-14-0747DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4174322PMC
September 2014

Binding of human nucleotide exchange factors to heat shock protein 70 (Hsp70) generates functionally distinct complexes in vitro.

J Biol Chem 2014 Jan 5;289(3):1402-14. Epub 2013 Dec 5.

From the Departments of Biochemistry and Pathology and the Life Sciences Institute, University of Michigan, Ann Arbor, Michigan 48109 and.

Proteins with Bcl2-associated anthanogene (BAG) domains act as nucleotide exchange factors (NEFs) for the molecular chaperone heat shock protein 70 (Hsp70). There are six BAG family NEFs in humans, and each is thought to link Hsp70 to a distinct cellular pathway. However, little is known about how the NEFs compete for binding to Hsp70 or how they might differentially shape its biochemical activities. Toward these questions, we measured the binding of human Hsp72 (HSPA1A) to BAG1, BAG2, BAG3, and the unrelated NEF Hsp105. These studies revealed a clear hierarchy of affinities: BAG3 > BAG1 > Hsp105 ≫ BAG2. All of the NEFs competed for binding to Hsp70, and their relative affinity values predicted their potency in nucleotide and peptide release assays. Finally, we combined the Hsp70-NEF pairs with cochaperones of the J protein family (DnaJA1, DnaJA2, DnaJB1, and DnaJB4) to generate 16 permutations. The activity of the combinations in ATPase and luciferase refolding assays were dependent on the identity and stoichiometry of both the J protein and NEF so that some combinations were potent chaperones, whereas others were inactive. Given the number and diversity of cochaperones in mammals, it is likely that combinatorial assembly could generate a large number of distinct permutations.
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http://dx.doi.org/10.1074/jbc.M113.521997DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3894324PMC
January 2014

Development of a capillary electrophoresis platform for identifying inhibitors of protein-protein interactions.

Anal Chem 2013 Oct 23;85(20):9824-31. Epub 2013 Sep 23.

Department of Biological Chemistry, ‡Department of Chemistry, and the§ Life Sciences Institute, University of Michigan , Ann Arbor, Michigan 48109, United States.

Methods for identifying chemical inhibitors of protein-protein interactions (PPIs) are often prone to discovery of false positives, particularly those caused by molecules that induce protein aggregation. Thus, there is interest in developing new platforms that might allow earlier identification of these problematic compounds. Capillary electrophoresis (CE) has been evaluated as a method to screen for PPI inhibitors using the challenging system of Hsp70 interacting with its co-chaperone Bag3. In the method, Hsp70 is labeled with a fluorophore, mixed with Bag3, and the resulting bound and free Hsp70 are separated and detected by CE with laser-induced fluorescence detection. The method used a chemically modified CE capillary to prevent protein adsorption. Inhibitors of the Hsp70-Bag3 interaction were detected by observing a reduction in the bound-to-free ratio. The method was used to screen a library of 3443 compounds, and the results were compared to those from a flow cytometry protein interaction assay. CE was found to produce a lower hit rate with more compounds that were reconfirmed in subsequent testing, suggesting greater specificity. This finding was attributed to the use of electropherograms to detect artifacts such as aggregators and to differences in protein modifications required to perform the different assays. Increases in throughput are required to make the CE method suitable for primary screens, but at the current stage of development it is attractive as a secondary screen to test hits found by higher-throughput methods.
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http://dx.doi.org/10.1021/ac4023082DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3845662PMC
October 2013

Cysteine reactivity distinguishes redox sensing by the heat-inducible and constitutive forms of heat shock protein 70.

Chem Biol 2012 Nov;19(11):1391-9

Department of Pathology, Department of Biological Chemistry, and the Life Sciences Institute, University of Michigan, Ann Arbor, MI 48109-2216, USA.

The heat shock protein 70 (Hsp70) family of molecular chaperones has important functions in maintaining proteostasis under stress conditions. Several Hsp70 isoforms, especially Hsp72 (HSPA1A), are dramatically upregulated in response to stress; however, it is unclear whether these family members have biochemical properties that are specifically adapted to these scenarios. The redox-active compound, methylene blue (MB), has been shown to inhibit the ATPase activity of Hsp72 in vitro, and it promotes degradation of the Hsp72 substrate, tau, in cellular and animal models. Here, we report that MB irreversibly inactivates Hsp72 but not the nearly identical, constitutively expressed isoform, heat shock cognate 70 (Hsc70; HSPA8). Mass spectrometry results show that MB oxidizes Cys306, which is not conserved in Hsc70. Molecular models suggested that oxidation of Cys306 exposes Cys267 to modification and that both events contribute to loss of ATP binding in response to MB. Consistent with this model, mutating Cys267 and Cys306 to serine made Hsp72 largely resistant to MB in vitro, and overexpression of the C306S mutant blocked MB-mediated loss of tau in a cellular model. Furthermore, mutating Cys267 and Cys306 to the pseudo-oxidation mimic, aspartic acid, mirrored MB treatment: the C267D and C306D mutants had reduced ATPase activity in vitro, and overexpression of the C267/306D double mutant significantly reduced tau levels in cells. Together, these results suggest that redox sensing by specific cysteine residues in Hsp72, but not Hsc70, may be an important component of the chaperone response to oxidative stress.
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http://dx.doi.org/10.1016/j.chembiol.2012.07.026DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3508472PMC
November 2012

Hsp70 protein complexes as drug targets.

Curr Pharm Des 2013 ;19(3):404-17

Department of Pathology, Life Sciences Institute, University of Michigan, 210 Washtenaw Ave., Ann Arbor, MI 48109-2216, USA.

Heat shock protein 70 (Hsp70) plays critical roles in proteostasis and is an emerging target for multiple diseases. However, competitive inhibition of the enzymatic activity of Hsp70 has proven challenging and, in some cases, may not be the most productive way to redirect Hsp70 function. Another approach is to inhibit Hsp70's interactions with important co-chaperones, such as J proteins, nucleotide exchange factors (NEFs) and tetratricopeptide repeat (TPR) domain-containing proteins. These co-chaperones normally bind Hsp70 and guide its many diverse cellular activities. Complexes between Hsp70 and co-chaperones have been shown to have specific functions, including roles in pro-folding, pro-degradation and pro-trafficking pathways. Thus, a promising strategy may be to block protein- protein interactions between Hsp70 and its co-chaperones or to target allosteric sites that disrupt these contacts. Such an approach might shift the balance of Hsp70 complexes and re-shape the proteome and it has the potential to restore healthy proteostasis. In this review, we discuss specific challenges and opportunities related to these goals. By pursuing Hsp70 complexes as drug targets, we might not only develop new leads for therapeutic development, but also discover new chemical probes for use in understanding Hsp70 biology.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3593251PMC
http://dx.doi.org/10.2174/138161213804143699DOI Listing
October 2013