Regulation of Human Hsp70 by its Nucleotide Exchange Factors (NEFs).

Heat shock protein 70 (Hsp70) is an abundant and ubiquitous molecular chaperone that is responsible for maintenance of the human proteome. Accordingly, Hsp70 has become an attractive drug target for neurodegenerative and hyperproliferative disorders; however it is difficult to imagine strategies for inhibiting its pathobiology without impacting its essential roles. Fortunately, Hsp70 does not work alone, and instead employs a large network of co-chaperone proteins, which can tune Hsp70 activity and influence disease state. These co-chaperone proteins provide potential handles for targeting Hsp70 without disrupting overall proteostasis. One such class of co-chaperones proteins known as the Nucleotide Exchange Factors (NEFs), are a particular appealing target. NEFs bind Hsp70 and help to facilitate the exchange of ADP for ATP. The biochemistry of the NEF family of co-chaperones has classically been investigated using the prokaryotic NEF, GrpE, as a model. However, the eukaryotic cytosol does not contain a GrpE homolog. Rather, there are three main sub-classes of human NEFs: Hsp110, HspBP1, and the BAG proteins, all of which are structurally distinct with little sequence homology. Consistent with their diverse structures, they also differ in their mode of binding to Hsp70 and their roles in guiding Hsp70 biology. For example, BAG2 is associated with proteasomal degradation of the Hsp70 substrate, tau, while BAG1-Hsp70 is linked to increased tau stability. These observations suggest that the formation of specific NEF-Hsp70 complexes may help decide the fate of Hsp70-bound substrates. Additionally, these findings illustrate that differential disruption of specific Hsp70-NEF contacts might be beneficial in disease. In this thesis work I have systematically characterized the human Hsp70 NEFs, including how they interact with Hsp70, how the influence Hsp70 biochemistry and how they can bridge Hsp70 with other classes of chaperone proteins. I have used high throughput screening methods to search for chemical matter that can modulate Hsp70-NEF interactions, and we have shown that inhibitors of Hsp70-NEF interactions can be beneficial for treating disease. This thesis work has significantly advanced our knowledge of human Hsp70 regulation, and has provided groundwork for future studies on other Hsp70 co-chaperones and proteostasis components.PhDBiological ChemistryUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/111611/1/rauchjn_1.pd

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

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

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

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

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

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

Genome-Wide DNA Methylation Maps in Follicular Lymphoma Cells Determined by Methylation-Enriched Bisulfite Sequencing

BACKGROUND: Follicular lymphoma (FL) is a form of non-Hodgkin's lymphoma (NHL) that arises from germinal center (GC) B-cells. Despite the significant advances in immunotherapy, FL is still not curable. Beyond transcriptional profiling and genomics datasets, there currently is no epigenome-scale dataset or integrative biology approach that can adequately model this disease and therefore identify novel mechanisms and targets for successful prevention and treatment of FL. METHODOLOGY/PRINCIPAL FINDINGS: We performed methylation-enriched genome-wide bisulfite sequencing of FL cells and normal CD19(+) B-cells using 454 sequencing technology. The methylated DNA fragments were enriched with methyl-binding proteins, treated with bisulfite, and sequenced using the Roche-454 GS FLX sequencer. The total number of bases covered in the human genome was 18.2 and 49.3 million including 726,003 and 1.3 million CpGs in FL and CD19(+) B-cells, respectively. 11,971 and 7,882 methylated regions of interest (MRIs) were identified respectively. The genome-wide distribution of these MRIs displayed significant differences between FL and normal B-cells. A reverse trend in the distribution of MRIs between the promoter and the gene body was observed in FL and CD19(+) B-cells. The MRIs identified in FL cells also correlated well with transcriptomic data and ChIP-on-Chip analyses of genome-wide histone modifications such as tri-methyl-H3K27, and tri-methyl-H3K4, indicating a concerted epigenetic alteration in FL cells. CONCLUSIONS/SIGNIFICANCE: This study is the first to provide a large scale and comprehensive analysis of the DNA methylation sequence composition and distribution in the FL epigenome. These integrated approaches have led to the discovery of novel and frequent targets of aberrant epigenetic alterations. The genome-wide bisulfite sequencing approach developed here can be a useful tool for profiling DNA methylation in clinical samples

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

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.\ua0Mutations 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

Use of cancer-specific yeast-secreted in vivo biotinylated recombinant antibodies for serum biomarker discovery

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