Jeffrey Brodsky - US grants

The endoplasmic reticulum (ER) receives, folds, and sorts proteins that are secreted from the eukaryotic cell or that are targeted to other intracellular organelles. To facilitate the import, or translocation, of these proteins into the ER, a myriad of cytoplasmic and lumenal factors associated with the ER are housed in multi-protein machines. Included in these machines are molecular chaperones, factors that play vital roles during protein folding and degradation in the cell and that have been shown to regulate multi-protein complexes in vitro. Compromising the activities of molecular chaperones arrests the import of some proteins into the ER. The mechanism by which these factors facilitate protein translocation is poorly understood, in part because pleiotrophic defects in many cellular processes occur when the activity of a single chaperone is compromised in vivo. To circumvent this problem in order to understand the specific effects of chaperones on ER import, reconstituted in vitro systems have been developed. The subsequent isolation of novel chaperone mutants from the model yeast, Saccharomyces cerevisiae, and their analysis in such in vitro assays, permits a molecular detailing of which chaperone activities are required to support protein translocation.

Chaperones required for protein translocation in yeast include the hsp70 ATPases BiP and Ssa1p in the ER lumen and cytoplasm, respectively, and the DnaJ homologues Sec63p and Ydj1p in the lumen and cytoplasm, respectively. Hsp70s and DnaJ proteins are known to cooperate and facilitate a number of cellular processes in all species in which they have been identified.

A recent analysis of new dominant lethal BiP mutants indicates that an ATP-dependent conformational change is imperative to support the interaction between BiP and Sec63p and is necessary to drive protein import into the ER. This project includes biochemical studies to determine why dominance arises in these mutants. The results of these experiments will indicate which essential role(s) BiP plays during protein import, and may either validate or refute existing hypotheses describing the action of molecular chaperones during protein translocation.

The mechanism by which the cytosolic chaperones Ssap1 and Ydjp1 facilitate translocation and the requirement for ATP hydrolysis by Ssa1p during import remains mysterious. To unravel this mystery, novel ATPase-defective ssa1 mutants were constructed and the resulting proteins purified during the preceding funding period. In this renewal project, the mutant and wild type proteins will be subjected to a battery of established biochemical tests to elucidate how Ssa1p ATPase activity is coupled to protein translocation.

Ydj1p plays ill-defined roles during both translocation and protein translation on ribosomes; therefore multicopy suppressors of a ydj1p temperature sensitive mutants were obtained, one of which was a gene encoding an uncharacterized hsp110 chaperon, Sse1p. The contribution of this chaperone to the translocation and translation reactions, both in vivo and in vitro, will be determined.

Because of their vital, diverse roles in cellular processes, studies of molecular chaperone function have yielded clues regarding the fundamental mechanisms underlying DNA replication, protein degradation and phosphorylation, and cell cycle control. The continued analysis of chaperone functions using novel biochemical, cell biological and genetic tools will undoubtedly continue to contribute to the understanding of these and other cellular functions.

DESCRIPTION (provided by applicant): The majority of wild type and nearly all of the disease-causing AF508 variant of CFTR fold inefficiently in the ER, and are ubiquitinated and destroyed by the cytoplasmic proteasome; however, the molecular mechanism by which CFTR is targeted for degradation is not completely clear. To begin to define which factors are specifically required for CFTR proteolysis, the protein was expressed in the yeast S. cerevisiae and found to be degraded by the ubiquitin-proteasome pathway, but was stabilized in yeast mutated for the cytoplasmic Hsc70, a molecular chaperone that associates with immature CFTR in the mammalian ER. In yeast, CFTR localizes to a unique site in the ER in the proteasome mutant, whereas it is dispersed throughout the ER in wild type or Hsc70 mutant cells. These combined results indicate that Hsc70 facilitates CFTR degradation and acts up-stream of the proteasome. To more precisely define the degradation pathway, CFTR-associated proteins will be identified by co-opting established yeast biochemical and proteomic methods. Analyzing the stability of CFTR in cells mutated or depleted for the corresponding genes will indicate which MR-interacting factors are required for proteolysis. Because CFTR turn-over can be abrogated at two distinct steps (catalyzed by Hsc70 and the proteasome, respectively), components acting at each point in the degradation pathway will be identified. As a complementary approach, it is hypothesized that genes required for the removal of CFTR will be induced in the CFTR-expressing Hsc70 and proteasome mutant strains. Candidate genes will be identified by screening microarrays using cDNA probes prepared from the appropriate strains. As above, CFTR degradation will be assayed in yeast mutated or depleted for the candidate genes. In sum, these studies serve as a prelude for examining whether mammalian homologues of the yeast factors play a similar role during CFTR degradation in human cells, long-term studies that will be catalyzed by continued collaborations with the local CF research community.

When cells make proteins for export (secretory proteins), it is critically important that the proteins are as they should be. If not, there is a quality control mechanism, termed Endoplasmic Reticulum-Associated Degradation (ERAD) that detects aberrant proteins and destroys them. The importance of cleansing the secretory pathway of aberrant proteins is underscored by the fact that if mis-folded proteins accumulate in the endoplasmic reticulum (ER), they induce the "unfolded protein response" (UPR), a cellular response that can lead in extreme cases to programmed cell death.

Drs. McCracken and Brodsky originally discovered that ERAD involves the selection of aberrant proteins (ERAD substrates), transport of the substrate proteins back across the ER membrane into the cytoplasm, and subsequent proteolytic degradation of the substrate proteins via the proteasome. This pathway has since been shown by several laboratories to be involved in the degradation of at least 20 different substrate proteins and to be conserved across eukaryotic species from yeast to humans. Subsequent work demonstrated that at least two ER-lumenal chaperones, BiP (KAR2) and calnexin, are required for ERAD export of soluble protein substrates. One of these, BiP, is also required for protein import into the ER. Brodsky and McCracken have recently identified mutations in BiP that are specific for ERAD, and as part of this project they will biochemically characterize these mutations (plus others that they plan to identify or create via site-directed mutagenesis) in order to determine what aspects of BiP structure and activity are specifically required for ERAD.

McCracken and Brodsky have also demonstrated that the ERAD pathway for an integral membrane protein, CFTR, is substantially different from that for soluble substrate proteins and involves a different set of chaperones. Neither BiP nor calnexin are required for CFTR degradation, but a cytosolic Hsp70 chaperone, Ssa1p, is; conversely, Ssa1p is not required for ERAD of soluble substrate proteins. As part of this project, the molecular basis for this distinction will be explored. Specifically, two hypotheses will be examined using genetic and biochemical techniques: (1) Ssa1p is required for CFTR ubiquitination; and (2) Ssa1p is required to maintain an aggregation-prone cytoplasmic domain of CFTR in solution.

A tabulation of the factors necessary and dispensable for the degradation of multiple ERAD
substrates indicates that the requirements for the degradation of ERAD substrates may or may not
utilize common factors. Thus, the continued identification of genes required for the turnover of a
given substrate is essential. To this end, Brodsky and McCracken have isolated mutations in which the degradation of the Z variant of Alpha1-Protease Inhibitor (A1PiZ) is compromised in yeast. In addition, because the presence of mis-folded proteins in the ER activate both ERAD and the UPR, known UPR-target genes that are required for the degradation of A1PiZ have been identified. As part of this project, McCracken and Brodsky will carry out a functional characterization of both classes of genes necessary for the proteolysis of A1PiZ; results from this study are expected to provide a better mechanistic understanding of the ERAD selection and targeting process.

In sum, these studies represent a combination of genetic and biochemical methods aimed
toward understanding a recently discovered cellular pathway in cell biology. The project will employ multiple approaches and will benefit from the synergistic expertise of the two collaborating scientists, Drs. Ardythe McCracken and Jeffrey Brodsky, who initially discovered the ERAD pathway. The project will also continue to contribute to both classroom and laboratory research instruction of undergraduate and graduate students.

[unreadable] DESCRIPTION (provided by applicant)Members of the oncogenic Polyomavirus family, such as Simian Virus 40 (SV40) and the human BK and JC viruses, require only one virally-encoded protein to transform mammalian cells: T antigen. T antigen is also required for viral replication, and the opportunistic growth of ICV and BKV in humans leads to disease. In addition, SV40 has been identified in several human cancers, induces brain tumors in rodents, and causes chromosomal re-arrangements in human fibroblasts. Three domains within T antigen are essential for these viruses to replicate and transform cells, two of which mediate binding to members of the p53 and Rb tumor suppressor families. The function of the third domain was mysterious until the discovery that this domain functioned in vitro as a DnaJ/Hsp40 molecular chaperone; members of the DnaJ/Hsp40 family activate Hsp70 chaperones and with Hsp70 can function as molecular "machines" that catalyze protein transport, folding, degradation, and activation in vivo and in vitro. The existing in vitro assay for T antigen chaperone activity measures the protein's ability to enhance the chaperone properties of a yeast Hsp70 chaperone. Notably, mutations that compromised T antigen chaperone activity also inhibited viral replication and tumorigenesis, a result that provided the first direct link between chaperone function and viral-mediated tumor formation. [unreadable] [unreadable] Because of this link, it was surmised that small molecule inhibitors of the interaction between DnaJ/Hsp40 and Hsp70 might block T antigen function. As a first step toward this goal, novel chaperone modulators have been identified through computational methods and the in vitro chaperone assay describe above. To identify specific, high affinity chaperone modulators that can ultimately be developed for therapeutic intervention, the Specific Aims of this grant application are to:1. Better define the structural motifs in a group of related organic compounds that compromise Hsp70 function in order to obtain more potent chaperone modulators.2. Assay existing and newly obtained Hsp70-modulators for their ability to inhibit T antigen activation of Hsp70 activity in vitro, to prevent the chaperone-dependent release of a polypeptide substrate from Hsp70, and to inhibit the chaperone-mediated transport of a precursor protein into yeast ER-derived microsomal vesicles.

[unreadable] DESCRIPTION (provided by applicant): Cystic fibrosis (CF) is the most common, inherited lethal disease in Caucasians in North America, and arises from mutations in the cystic fibrosis transmembrane conductance regulator (CFTR). The majority of disease-causing mutations block the maturation of this secreted protein, such that CFTR becomes trapped in the endoplasmic reticulum (ER) and is degraded by the proteasome. This process is referred to as ER associated degradation (ERAD), and >30 ERAD substrates from yeast to man have been identified, many of which are linked to specific diseases. ERAD substrate selection and targeting are catalyzed by molecular chaperones, but to date it has been difficult to define how and specifically at which step the chaperones impact CFTR degradation. Moreover, it has been challenging to define how a membrane protein, like CFTR, is delivered to the proteasome, and to identify uncharacterized genes required for maximal ERAD efficiency. To surmount existing technical barriers, the PI's laboratory established a yeast CFTR expression system and showed that unique chaperones play distinct roles during ERAD. To identify novel factors that catalyze ERAD, a micro-array "screen" was performed and a chaperone class with no previous connection to ERAD was found to facilitate CFTR degradation in yeast. In parallel with these studies, an in vitro system was established that recapitulates the polyubiquitination of CFTR and a CFTR homologue in yeast membranes. Based on this new data and tools, the goals of this grant application are to determine at which step in the CFTR degradation pathway known and newly identified chaperones function. And, for the first time, the requirements for substrate de-ubiquitination and proteasome targeting during ERAD will be investigated in a defined system. Importantly, data obtained from the in vitro assay will be complemented through in vivo studies in wild type and mutant yeast strains. This project reflects the PI's long-term interest in defining the molecular machines responsible for protein biogenesis in the ER, and this grant application constitutes the primary focus of ongoing research in the PI's laboratory. Finally, the results obtained from the experiments described in this application will direct future efforts to delineate the CFTR maturation pathway in mammalian cells, an effort that is vital as ongoing chaperone-based therapies to treat CF and other protein conformational diseases are entering clinical trials. [unreadable] [unreadable] [unreadable]

[unreadable] DESCRIPTION (provided by applicant): Cystic fibrosis (CF) is the most common, inherited lethal disease in Caucasians in North America, and arises from mutations in the cystic fibrosis transmembrane conductance regulator (CFTR). The majority of disease-causing mutations block the maturation of this secreted protein, such that CFTR becomes trapped in the endoplasmic reticulum (ER) and is degraded by the proteasome. This process is referred to as ER associated degradation (ERAD), and >30 ERAD substrates from yeast to man have been identified, many of which are linked to specific diseases. ERAD substrate selection and targeting are catalyzed by molecular chaperones, but to date it has been difficult to define how and specifically at which step the chaperones impact CFTR degradation. Moreover, it has been challenging to define how a membrane protein, like CFTR, is delivered to the proteasome, and to identify uncharacterized genes required for maximal ERAD efficiency. To surmount existing technical barriers, the PI's laboratory established a yeast CFTR expression system and showed that unique chaperones play distinct roles during ERAD. To identify novel factors that catalyze ERAD, a micro-array "screen" was performed and a chaperone class with no previous connection to ERAD was found to facilitate CFTR degradation in yeast. In parallel with these studies, an in vitro system was established that recapitulates the polyubiquitination of CFTR and a CFTR homologue in yeast membranes. Based on these new data and tools, the goals of this grant application are to determine at which step in the CFTR degradation pathway known and newly identified chaperones function. And, for the first time, the requirements for substrate de-ubiquitination and proteasome targeting during ERAD will be investigated in a defined system. Importantly, data obtained from the in vitro assay will be complemented through in vivo studies in wild type and mutant yeast strains. This project reflects the PI's long-term interest in defining the molecular machines responsible for protein biogenesis in the ER, and this grant application constitutes the primary focus of ongoing research in the PI's laboratory. Finally, the results obtained from the experiments described in this application will direct future efforts to delineate the CFTR maturation pathway in mammalian cells, an effort that is vital as ongoing chaperone-based therapies to treat CF and other protein conformational diseases are entering clinical trials. [unreadable] [unreadable] [unreadable]

[unreadable] DESCRIPTION (provided by applicant): The Polyomaviruses (Py) normally form latent infections in the kidney but upon re-activation can trigger the onset of incurable diseases. For example, re-activation of JC virus (JCV) results in Progressive Multifocal Leukoencephalopathy, which kills ~5% of AIDS patients, and re-activation of BK virus (BKV) leads to BKV Associated Nephropathy, which is the leading cause of rejection in kidney transplant recipients. Another Py is SV40, whose natural hosts are monkeys, and ~10% of the human population is infected with SV40 because the first polio vaccines were inadvertently contaminated with the virus; the long-term effects of SV40 infection in humans are unclear, although SV40 has been linked to several cancers. Each member of the Py family encodes the large tumor antigen (TAg), which is essential for viral replication and tumorigenesis. Previous work in the PI's laboratory contributed to the discovery that TAg possesses a chaperone-like domain, and that the interaction between this domain and the cellular Hsp70 chaperone is essential to orchestrate viral replication and cellular transformation. Furthermore, small molecule modulators that compromise TAg-Hsp70 interaction have been identified. One modulator (MAL2-11B) inhibits TAg's endogenous ATPase activity, an activity that is also essential for viral replication and cellular transformation. Indeed, MAL2-11B inhibits SV40 DNA synthesis in vitro and viral replication in tissue culture cells. Through these efforts, a significant battery of in vitro methods have been developed that have been re-configured for a high throughput screen to identify new, more potent TAg and Py inhibitors. Specifically, TAg's ATPase activity has recently been assessed in a 96-well format. Thus, the hypothesis underlying these studies is that chemical modulators of TAg ATPase activity might represent a new avenue to combat the catastrophic effects of Polyomavirus-associated diseases. The Specific Aims of this application are: (1) To perform high throughput screens to isolate TAg inhibitors using small molecule libraries at the resident University of Pittsburgh MLSCN, and (2) To utilize established assays as secondary and counter screens to assess the effects of "hits" from Aim 1. In both aims, MAL2-11B will serve as a positive control. Further tests will be conducted with the MLSCN and resident medicinal chemists to obtain more refined chemical probes. The Relevance of this study is underscored by the lack of approved and efficacious methods to treat diseases that arise from Polyomavirus infection. The described efforts employ a novel target, a positive control, and local collaborators, which will aid the completion of the stated goals. [unreadable] [unreadable] [unreadable]

This award is funded under the American Recovery and Reinvestment Act of 2009 (Public Law 111-5).

Proteins that reside in the cell membrane play important structural roles, but they are also critical to the cell's ability to sense and respond to environmental stimuli. For instance, ion channels open in response to changes in membrane voltage or membrane tension to permit the flow of molecules into and out of the cell. Knowledge of how proteins interact with the membrane is essential to our understanding of these functions. The research goal of this project is to build and use computational tools for elucidating the nature of membrane proteins' interactions with the membrane. Such tools are needed to understand the folding and trafficking of proteins from the endoplasmic reticulum (ER) to the plasma membrane and to understand the functional properties of proteins that undergo conformational rearrangements in the membrane. Currently, molecular simulations of charged helical peptides predict membrane insertion energies that are much larger than those derived from experiment. Therefore, new computational approaches are needed to pinpoint the source of these differences and help to resolve them. This CAREER project will (a) allow for the development of a fast, continuum based method for calculating the insertion energies and relative stabilities of membrane proteins, (b) use this computational model to predict the energy and kinetics of protein extraction from the membrane and compare these rates with in vivo extraction experiments from the ER, and (c) study how voltage changes manipulate voltage-sensor proteins to control their behavior. The membrane insertion model will be packaged into a freely available, cross-platform JAVA program with an intuitive graphical interface for carrying out membrane protein energetic calculations.

Broader Impacts. The educational component of this CAREER project involves continued development of a new mathematical biology course that introduces upper division undergraduate students to mathematical concepts that have greatly impacted biology. This project will enable the writing of an undergraduate textbook by the investigator and his colleagues. The undergraduate course and textbook will train future biologists in the mathematics needed to understand cutting-edge technologies in the biological sciences. Additionally, this research program includes the development of a summer course in basic mathematics for high school students who are members of minorities under-represented in science and who are enrolled in the School-to-Career Teen Program (Pittsburgh, PA). It will permit continued math and science education for these high school students and encourage them to attend and succeed at post-secondary educational institutions.

PROJECT SUMMARY (See instructions): The Model Organisms Core will employ two genetically-tractable systems, the yeast S. cerevisiae and the zebrafish D. rerio, each with distinct advantages. These experimental systems will help dissect fundamental aspects of kidney development and protein structure and function. Experiments associated with these model systems will be complemented by the use of small molecule modulators that have emerged from Core associated activities over the past four years. Hypotheses arising from the unique attributes of the yeast and zebrafish models and from the use of chemical modulators will continue to be tested in higher cell types and organisms via the other Cores. In turn, experiments using yeast and zebrafish provide rapid assessments of predictions from more complex systems. The goals of the Yeast Core are to develop and continue to utilize established expression systems for wild type and disease-causing proteins that transit the secretory pathway in kidney cells. Genomic and proteomic attacks will identify factors that impact their biogenesis, and the mechanism of action of these factors will be established. Toward these goals, the Yeast Core has created over a dozen expression systems and offers collaborators the expertise and tools to co-opt this model organism. Specific assays developed in the Core include methods to assess how chaperones, the ubiquitin proteasome pathway, autophagy, and chemical chaperones impact secretory protein biogenesis. The Zebrafish Core will utilize established transgenic kidney reporter lines and to utilize established automated screening technologies in small molecule screens to identify chemical probes for kidney development and disease. The Zebrafish Core has a number of transgenic lines and identified small molecules that impact kidney development, and has pinpointed when specific factors act during kidney development. Collaborators will be able to establish and analyze results from newly created zebrafish lines and perform small molecule screens. Overall, the knowledge gained from the use of these complementary model organisms will be expanded via collaborations with the other Cores, and in turn the hypotheses that arise from more complex systems can be rapidly and in some cases more thoroughly tested in yeast and zebrafish. The Core will co-

? DESCRIPTION (provided by applicant): A significant fraction of newly synthesized proteins translocate into the endoplasmic reticulum (ER). Once associated with this compartment, nascent polypeptides are post- translationally processed, acquire their native confirmations, and are sorted for delivery to other organelles or to the extracellular milieu. However, disease-causing mutations may compromise protein folding and maturation, which in turn generate aggregation-prone species. To off-set the catastrophic effects that accompany the accumulation of defective polypeptides, these substrates can be selected, delivered back to the cytoplasm, and then degraded via a process defined in the Brodsky laboratory and termed ER associated degradation, or ERAD. The long- term goal of research in the laboratory is to understand how disease-associated ERAD substrates are identified and destroyed so that specific factors or steps in this pathway can be modulated to prevent disease onset. As a first step toward this goal, yeast expression systems for ERAD substrates linked to specific maladies have been generated. Model substrates have also been constructed to explore how different classes of aberrant proteins are selected and routed for degradation. To complement these approaches, an in vitro assay that recapitulates the ubiquitination of membrane-embedded substrates was developed. Using these tools, the proposed studies will first examine whether there is a direct link between the aggregation propensity of a membrane protein and its selection for ERAD. Next, the membrane-associated components that maintain substrate solubility during ERAD will be identified and characterized. Secreted proteins that traffic beyond the ER-and even some ERAD escapees-can be captured and destroyed in the vacuole/lysosome. The factors involved in deciding whether a protein in the late secretory pathway should be sorted to its final destinations or should be delivered to the vacuole/lysosome are poorly defined. To better characterize this pathway, a genetic platform was developed. Recent data demonstrate that members of the a-arrestin family play a key role during post-ER degradation. A molecular dissection of how these proteins function and with which partners they operate will be undertaken using biochemical methods. A new cell surface fluorescence assay will then be used to generate complementary, quantitative data on plasma membrane residence when a-arrestin activity is altered. The outcome of these efforts will test new hypotheses, lead to deeper mechanistic insights into the etiology of protein conformational diseases, and characterize factors that may serve as novel therapeutic targets.

Approximately one-third of all newly synthesized proteins in eukaryotes enter the endoplasmic reticulum (ER). Once associated with this compartment, these nascent polypeptides are post- translationally processed, acquire their native confirmations, oligomerize, and are sorted for extracellular secretion or delivery to other organelles. However, many disease-causing mutations compromise protein folding and maturation, which in turn can generate aggregation- prone species. To off-set the catastrophic effects that accompany the accumulation of protein aggregates, misfolded protein substrates are: (i) selected by molecular chaperones associated with the ER, (ii) modified with ubiquitin, (iii) delivered to the cytoplasm via a process known as retrotranslocation, and (iv) degraded by the 26S proteasome. Brodsky and colleagues named this pathway ER associated degradation (ERAD), and over the past 21 years many of the molecular mechanisms underlying this sequence of events were defined in the Brodsky lab. To date, ~80 human diseases are linked to the ERAD pathway and >1,200 publications have been authored on various aspects of this pathway. Ongoing efforts are defining the pathophysiological foundation of several ERAD-related disorders. In parallel, members of the Brodsky lab have revealed how key components orchestrate each step during ERAD. In the past 5 years, the lab has published 64 papers, and tools and technologies were developed that provide an unprecedented view of the mechanisms that lead to the selection, ubiquitination, retrotranslocation, and degradation of diverse substrates. Nevertheless, recent discoveries dictate that more challenging research directions are pursued: By necessity, these next efforts will require additional method development and a pursuit of longer-term goals. Specific questions that the research program will address include: What biochemical features define an ERAD substrate? Which factors are sufficient to drive the retrotranslocation of ERAD substrates, some of which are aggregation-prone? Do ER-associated proteases function in tandem with the 26S proteasome to destroy substrates that are stably integrated into the ER membrane, and thus might be retrotranslocation resistant? And, how are retrotranslocated membrane proteins? which can reside in the cytosol after being liberated from the ER?retained in a soluble state? Answers to these questions, which lie at the core of research in the field, will significantly advance an understanding of how cellular health is maintained in the face of proteotoxic stress as well as how ERAD-associated diseases arise and might be rectified.

Summary/Abstract Funds from NIGMS are requested for the purchase of an Amersham Typhoon IP phosphorimager along with 8 imaging plates and cassettes, an imaging tray to accommodate the plates, and software licenses for data analysis on remote computers. In turn, the University of Pittsburgh and Department of Biological Sciences have committed funds to directly off-set the cost of the imager ($6,000), to purchase the computer (~$2,500), and to provide a long-term maintenance contract. The imager rapidly scans gels and plates up to 34 x 46 cm, produces high resolution figures, yields quantitative data over a long dynamic range, and has state-of-the-art software to accommodate diverse applications. In contrast, results of radioisotope-based experiments in the Brodsky and Berman labs currently rely on an 8-year old GE Typhoon FLA 7000 phosphorimager. This imager uses an older software package that is unlinked from the scanning software and not user-friendly, suffers from mechanical and software failures, and produces lower quality images that restrict data analysis and quantitation. The imager also only accommodates gels that are smaller than 20 x 40 cm. The purchase of the Amersham Typhoon IP phosphorimager is linked to grant R01 GM075061 to Prof. Jeffrey Brodsky and grant R01 GM116889 to Prof. Andrea Berman. The most critical experiments outlined in grant R01 GM075061 to Prof. Brodsky require the use of radioisotopes, which allows for quantitative assessment of the selection, ubiquitination, and degradation of misfolded proteins in the endoplasmic reticulum. Thus, an optimally functioning phosphorimager is essential to achieve each of the project?s goals. Furthermore, an application for a MIRA (R35 GM131732) to Prof. Brodsky was approved for funding with a requested start date of May 1, 2019. The experiments proposed in this recently approved grant application also require the use of radioisotopes and thus a phosphorimager to achieve each of the project?s goals. Together, the procurement of the Amersham Typhoon IP phosphorimager is critical for both the immediate and longer-term goals of Prof. Brodsky?s research program.

The graduate-level training program ?Interinstitutional Program in Cell & Molecular Biology? addresses the demands of changing job prospects, an urgent need to increase diversity in the scientific community, and the importance of enhancing ethics and rigor in research. The program unites three internationally recognized research and educational entities, the University of Pittsburgh, the University of Pittsburgh School of Medicine, and Carnegie-Mellon University, all in close proximity within one of America?s most livable cities. The merger of these distinct entities capitalizes on pre-existing connections between these groups, such as the Pittsburgh- area membrane trafficking group, which is now in its 18th year of organizing annual meetings and courses. The merger of the three groups also broadens training potential, particularly with respect to available expertise and infrastructure, and emphasizes the importance of scientific collaboration to the trainees. Indeed, to capitalize on these strengths, each trainee will have a co-mentor from one of the other institutions. The administrations at each institution are fully committed to the research plan, and Faculty in the program were chosen based on past training outcomes or (for younger Faculty) future potential. The Faculty encompass diverse areas of expertise in basic cell and molecular biology, professional ranks and backgrounds, and will participate in robust mentor training, which strengthens their ability to develop personalized training programs and to mentor trainees both daily and at twice-yearly thesis meetings. The result will be a personalized and supportive training plan that is rigorously monitored using objective assessment tools. Faculty labs within the program attract outstanding and diverse trainees with career goals that span a wide spectrum. Dual mentorship brings a breadth of scientific approaches and an increased base of professional contacts. Thanks to culling from the best of the participating departments, trainees will attend outstanding courses and seminars that collectively hone critical thinking, enable broad knowledge, introduce cutting-edge technologies, emphasize scientific ethics and rigor and reproducibility, and highlight career and professional development opportunities. Additionally, instruction and practice in communication skills, including participation in outreach and classroom teaching, will be an integral feature of the program. In sum, the program will recruit and retain a diverse trainee cohort that will be provided with a tailored and collaboration-focused pathway, preparing them for entry into a range of career paths open to PhD recipients in cell and molecular biology.

The folding of cellular proteins is inefficient, and the protein folding problem is even more problematic as cells age and encounter stressful conditions. The concentration of misfolded proteins rises even further in non-replicative cells, such as neurons. Therefore, it is not surprising that the most common ?protein conformational diseases? are linked to neurodegeneration, one of which is Alzheimer?s Disease (AD). AD is exemplified by the accumulation of amyloid plaques and neurofibrillary tangles, which are respectively due to Ab and tau. Although the amyloid hypothesis and the cellular effects of aggregated tau are controversial, protein homeostasis?or ?proteostasis??is clearly compromised in AD neurons. Therefore, if the toxicity associated with the accumulation of Ab and tau could be lessened, the tragic consequences and financial burden of AD could be ameliorated. Fortunately, cells surmount a stress response that can rectify toxicity arising from the accumulation of misfolded proteins. One protective factor that is induced under stress is the Hsp70 chaperone. Hsp70 binds and maintains the solubility of misfolded, aggregation-prone proteins. In addition, higher levels of Hsp70 can protect cells from compromised proteostasis. Unfortunately, while higher levels of Hsp70 are temporarily protective, a sustained increase in Hsp70 levels is toxic or requires genetic manipulations that cannot currently be administered to humans. In contrast, if a compound could directly increase Hsp70 chaperone activity without changing its levels, then improved survival of AD neurons might be evident. In 2008, a first-in-class compound that selectively activates Hsp70 function was identified. NMR analysis established the specificity and mechanism of action of the compound. Moreover, this Hsp70 activator reduces toxic levels of a- synuclein, which gives rise to Parkinson?s Disease, as well as an aggregation-prone polypeptide associated with Huntington?s disease. These efforts emerged from a collaboration between the PI and an expert in drug discovery and medicinal chemistry and were independent of the PI?s parent grant from GMS, which focuses instead on a specific proteostatic pathway. The goal of the current project is to: (1) synthesize and characterize more drug-like derivatives of existing Hsp70 chaperone activators, and (2) screen and then establish the mechanism of action of lead compounds in cellular assays that recapitulate the proteotoxic effects associated with AD. To catalyze progress, local AD experts will act as project consultants, and ultimately preclinical lead compounds will be identified for subsequent in vivo studies.