A principal strategic goal of the RCMI Coordinating Center (RCMI-CC) is to improve the health of minority populations and to reduce ethnic and geographic disparities in health by coordinating the development and facilitating the implementation of clinical research across the RCMI Consortium. To more effectively spur inter-institutional collaborative research, the RCMI-CC supports a Clinical Research Pilot Projects Program for hypothesis-driven clinical research projects proposed by postdoctoral fellows, early-career faculty and/or early-stage investigators from two or more RCMI U54 Centers. The purpose of this brief report is to summarize the Science Speed Dating sessions to facilitate cross-site collaboration at the RCMI Investigator Development Core (IDC) Workshop, held in conjunction with the 2024 RCMI Consortium National Conference. RCMI investigators and IDC Directors from 20 RCMI U54 Centers participated in two rounds of highly interactive small-group presentations of research ideas and resource needs in search of new collaborative and mentoring partnerships. Workshop participants expressed a high level of satisfaction with the speed-networking format and strongly agreed that the workshop was beneficial to their professional-development goals.

Abstract Pancreatic cancer is a highly lethal disease with obesity as one of the risk factors. Oncogenic KRAS mutations are prevalent in pancreatic cancer and can rewire lipid metabolism by altering fatty acid (FA) uptake, FA oxidation (FAO), and lipogenesis. Identification of the underlying mechanisms could lead to improved therapeutic strategies for treating KRAS-mutant pancreatic cancer. Here, we observed that KRASG12D upregulated the expression of SLC25A1, a citrate transporter that is a key metabolic switch to mediate FAO, fatty acid synthesis, glycolysis, and gluconeogenesis. In genetically engineered mouse models and human pancreatic cancer cells, KRASG12D induced SLC25A1 upregulation via GLI1, which directly stimulated SLC25A1 transcription by binding its promoter. The enhanced expression of SLC25A1 increased levels of cytosolic citrate, FAs, and key enzymes in lipid metabolism. In addition, a high-fat diet (HFD) further stimulated the KRASG12D-GLI1-SLC25A1 axis and the associated increase in citrate and FAs. Pharmacologic inhibition of SLC25A1 and upstream GLI1 significantly suppressed pancreatic tumorigenesis in KrasG12D/+ mice on a HFD. These results reveal a KRASG12D-GLI1-SLC25A1 regulatory axis, with SLC25A1 as an important node that regulates lipid metabolism during pancreatic tumorigenesis, thus indicating an intervention strategy for oncogenic KRAS-driven pancreatic cancer. Significance: Upregulation of SLC25A1 induced by KRASG12D-GLI1 signaling rewires lipid metabolism and is exacerbated by HFD to drive the development of pancreatic cancer, representing a targetable metabolic axis to suppress pancreatic tumorigenesis.

Uncropped version of all Western blots

Hsp90 functionally interacts with a broad array of client proteins, but in every case examined Hsp90 is accompanied by one or more co-chaperones. One class of co-chaperone contains a tetratricopeptide repeat domain that targets the co-chaperone to the C-terminal region of Hsp90. Within this class are Hsp90-binding peptidylprolyl isomerases, most of which belong to the FK506-binding protein (FKBP) family. Despite the common association of FKBP co-chaperones with Hsp90, it is now clear that the client protein influences, and is influenced by, the particular FKBP bound to Hsp90. Examples include Xap2 in aryl hydrocarbon receptor complexes and FKBP52 in steroid receptor complexes. In this chapter, we discuss the known functional roles played by FKBP co-chaperones and, where possible, relate distinctive functions to structural differences between FKBP members.

Coordinated cochaperone interactions with Hsp90 and associated client proteins are crucial for a multitude of signaling pathways in normal physiology, as well as in disease settings. Research on the molecular mechanisms regulated by the Hsp90 multiprotein complexes has demonstrated increasingly diverse roles for cochaperones throughout Hsp90-regulated signaling pathways. Thus, the Hsp90-associated cochaperones have emerged as attractive therapeutic targets in a wide variety of disease settings. The tetratricopeptide repeat (TPR)-domain immunophilins FKBP51 and FKBP52 are of special interest among the Hsp90-associated cochaperones given their Hsp90 client protein specificity, ubiquitous expression across tissues, and their increasingly important roles in neuronal signaling, intracellular calcium release, peptide bond isomerization, viral replication, steroid hormone receptor function, and cell proliferation to name a few. This review summarizes the current knowledge of the structure and molecular functions of TPR-domain immunophilins FKBP51 and FKBP52, recent findings implicating these immunophilins in disease, and the therapeutic potential of targeting FKBP51 and FKBP52 for the treatment of disease.

The Second International Symposium on Cellular and Organismal Stress Responses took place virtually on September 8–9, 2022. This meeting was supported by the Cell Stress Society International (CSSI) and organized by Patricija Van Oosten-Hawle and Andrew Truman (University of North Carolina at Charlotte, USA) and Mehdi Mollapour (SUNY Upstate Medical University, USA). The goal of this symposium was to continue the theme from the initial meeting in 2020 by providing a platform for established researchers, new investigators, postdoctoral fellows, and students to present and exchange ideas on various topics on cellular stress and chaperones. We will summarize the highlights of the meeting here and recognize those that received recognition from the CSSI.

Background The lack of race/ethnic and gender diversity in grants funded by the National Institutes of Health (NIH) is a persistent challenge related to career advancement and the quality and relevance of health research. We describe pilot programs at nine institutions supported by the NIH-sponsored Building Infrastructure Leading to Diversity (BUILD) program aimed at increasing diversity in biomedical research. Methods We collected data from the 2016-2017 Higher Education Research Institute survey of faculty and NIH progress reports for the first four years of the program (2015-2018). We then conducted descriptive analyses of data from the nine BUILD institutions that had collected data and evaluated which activities were associated with research productivity. We used Poisson regression and rate ratios of the numbers of BUILD pilots funded, students included, abstracts, presentations, publications, and submitted and funded grant proposals. Results Teaching workshops were associated with more abstracts (RR 4.04, 95% CI 2.21-8.09). Workshops on grant writing were associated with more publications (RR 2.64, 95% CI 1.644.34) and marginally with marginally more presentations. Incentives to develop courses were associated with more abstracts published (RR 4.33, 95% CI 2.56-7.75). Workshops on research skills and other incentives were not associated with any positive effects. Conclusions Pilot interventions show promise in supporting diversity in NIH-level research. Longitudinal modeling that considers time lags in career development in moving from project development to grants submissions can provide more direction for future diversity pilot interventions.

Background: Prostate cancer (PCa) is the most common non-cutaneous cancer afflicting men, both in the United States and worldwide. Aberrant molecular signaling mechanisms mediated by the Androgen Receptor (AR) are attributed as the main culprits for the initiation and progression of PCa, and research efforts have focused on developing strategies to directly target the AR. In an alternative approach, molecular chaperones that are critical for the maturation and signaling of the AR have emerged as potential therapeutic targets. Our lab has demonstrated the potential of inhibitors that target AR cochaperones, FKBP51 and FKBP52, in PCa cells. Previously, FKBP51 and FKBP52 have been shown to play a role in cell proliferation and survival, steroid hormone receptor maturation, and other mechanisms highly implicated in prostate carcinogenesis. Therefore, it is imperative to understand the protein-protein interactions that regulate FKBP51 and FKBP52-mediated AR function in PCa. We hypothesize that unknown interactors cooperate with FKBP51 and FKBP52 to promote PCa progression via the AR signaling pathways. Here, we performed a series of experiments aimed to identify and characterize auxiliary proteins influencing FKBP51- and FKBP52-mediated AR activity. Ultimately, our goal is to define novel protein interactions involved in AR-mediated PCa progression for therapeutic intervention. Methods: We implemented tandem affinity purification followed by mass spectrometry analysis in 22RV1 PCa cells using FKBP51 and FKBP52 as bait. The spectral counts of proteins identified were then used to analyze protein-protein interactions through the IDEP workflow software. Gene Ontology (GO) term enrichment analysis was used to generate a STRING interaction network. Protein interactions of interest were then validated via reciprocal immunoprecipitations. Results: This interactome analysis resulted in 353 IDs for FKBP51 and FKBP52 interactors. Heatmaps representing top 100 interactors were generated for both bait proteins. Members of the peroxiredoxin family, implicated in development of PCa and resistance to treatment, are among the top candidates of interest resulting from this interactome. Conclusion: This analysis aims to provide important insights into the androgen signaling milieu, which can be applied in the development of novel treatments targeting AR interacting partners in PCa progression. This study contributes to the growing list of functionally diverse proteins that help modulate AR transcriptional activity, and supports the idea that AR signaling is dynamic with numerous molecules involved in the aberrant activation of AR in prostate-tumor cells Citation Format: Olga B. Soto, Abhijeet R. Patil, Sourav Roy, Marc B. Cox. Identification of androgen receptor co-chaperone interactors implicated in castration resistant prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr LB038.

The Research Centers in Minority Institutions (RCMI) Program was congressionally mandated in 1985 to build research capacity at institutions that currently and historically recruit, train, and award doctorate degrees in the health professions and health-related sciences, primarily to individuals from underrepresented and minority populations. RCMI grantees share similar infrastructure needs and institutional goals. Of particular importance is the professional development of multidisciplinary teams of academic and community scholars (the "workforce") and the harnessing of the heterogeneity of thought (the "thinkforce") to reduce health disparities. The purpose of this report is to summarize the presentations and discussion at the RCMI Investigator Development Core (IDC) Workshop, held in conjunction with the RCMI Program National Conference in Bethesda, Maryland, in December 2019. The RCMI IDC Directors provided information about their professional development activities and Pilot Projects Programs and discussed barriers identified by new and early-stage investigators that limit effective career development, as well as potential solutions to overcome such obstacles. This report also proposes potential alignments of professional development activities, targeted goals and common metrics to track productivity and success.

The core cytosolic Hsp90 chaperone/co-chaperone complex plays a critical role in proteostasis management of human health and disease. To identify novel compounds that alter the ability of the Hsp90 co-chaperone Aha1 to modulate the ATPase activity found in multiple folding diseases ranging from steroid hormone receptor (SHR) sensitive prostate cancer to tauopathies associated with neurodegenerative diseases, we employed a h igh t hroughput s creening (HTS) assay to monitor selectively A ha1- s timulated H sp90 (ASH) ATPase activity. The ASH assay identified SEW04784 (SEW), a small molecule that disrupts ASH activity without inhibiting the basal Hsp90 ATPase activity. NMR analysis reveals that SEW binds to the C-terminal domain of Aha1 to disrupt its asymmetric binding to Hsp90 leading to abrogation of its chaperoning activity of Hsp90. SEW exhibits therapeutic potential by blocking the transcriptional activity of prostate cancer (PCa) associated variants of the androgen receptor (AR) in a cell-based model of PCa. Additionally, SEW exhibits the ability to clear toxic, phosphorylated tau aggregated species associated with tauopathies. By not directly impacting the basal ATPase function of the abundant and ubiquitous Hsp90, SEW could provide a therapeutic approach for mitigation of client-specific proteostatic disease.

Background: GMC1 (2-(1H-benzimidazol-2-ylsulfanyl)-N-[(Z)-(4-methoxyphenyl) methylideneamino] acetamide) effectively inhibits androgen receptor function by binding directly to FKBP52. This is a novel mechanism for the treatment of castration resistant prostate cancer (CRPC). Methods: an LC-MS/MS method was developed and validated to quantify GMC1 in plasma and urine from pharmacokinetics studies in rats. An ultra-high-performance liquid chromatography (UHPLC) system equipped with a Waters XTerra MS C18 column was used for chromatographic separation by gradient elution with 0.1% (v/v) formic acid in water and methanol. A Sciex 4000 QTRAP® mass spectrometer was used for analysis by multiple reaction monitoring (MRM) in positive mode; the specific ions [M+H]+m/z 340.995 → m/z 191.000 and [M+H]+ m/z 266.013 → m/z 234.000 were monitored for GMC1 and internal standard (albendazole), respectively. Results: GMC1 and albendazole had retention times of 1.68 and 1.66 min, respectively. The calibration curves for the determination of GMC1 in rat plasma and urine were linear from 1–1000 ng/mL. The LC-MS/MS method was validated with intra- and inter-day accuracy and precision within the 15% acceptance limit. The extraction recovery values of GMC1 from rat plasma and urine were greater than 95.0 ± 2.1% and 97.6 ± 4.6%, respectively, with no significant interfering matrix effect. GMC1 is stable under expected sample handling, storage, preparation and LC-MS/MS analysis conditions. Conclusions: Pharmacokinetic evaluation of GMC1 revealed that the molecule has a biexponential disposition in rats, is distributed rapidly and extensively, has a long elimination half-life, and appears to be eliminated primarily by first order kinetics.

Previous studies demonstrated that the 52-kDa FK506-binding protein (FKBP52) proline-rich loop is functionally relevant in the regulation of steroid hormone receptor activity. While zebra fish (Danio rerio; Dr) FKBP52 contains all of the analogous domains and residues previously identified as critical for FKBP52 potentiation of receptor activity, it fails to potentiate activity. Thus, we used a cross-species comparative approach to assess the residues that are functionally critical for FKBP52 function. Random selection of gain-of-function DrFKBP52 mutants in Saccharomyces cerevisiae identified two critical residues, alanine 111 (A111) and threonine 157 (T157), for activation of receptor potentiation by DrFKBP52. In silico homology modeling suggests that alanine to valine substitution at position 111 in DrFKBP52 induces an open conformation of the proline-rich loop surface similar to that observed on human FKBP52, which may allow for sufficient surface area and increased hydrophobicity for interactions within the receptor-chaperone complex. A second mutation in the FKBP12-like domain 2 (FK2), threonine 157 to arginine (T157R), also enhanced potentiation, and the DrFKBP52-A111V/T157R double mutant potentiated receptor activity similar to human FKBP52. Collectively, these results confirm the functional importance of the FKBP52 proline-rich loop, suggest that an open conformation on the proline-rich loop surface is a predictor of activity, and highlight the importance of an additional residue within the FK2 domain.

In this chapter, we summarize the birth of the field of nuclear receptors. These receptors exhibit a multitude of roles in cell biology and hence have attracted a great deal of interest in the drug discovery field. It is not certain whether these receptors evolved independently or an ancestral protein acquired various functions upon binding to preexisting small molecules, ligands. Currently, members of this receptor superfamily are categorized in six groups, including "orphan receptors." Research in the area has resulted in several clinically used drugs and continues to reveal further previously unknown roles for these receptors paving the road toward more valuable discoveries in the future.

Molecular chaperones are a diverse group of highly conserved proteins that transiently interact with partially folded polypeptide chains during normal cellular processes such as protein translation, translocation, and disassembly of protein complexes. Prior to folding or after denaturation, hydrophobic residues that are normally sequestered within a folded protein are exposed to the aqueous environment and are prone to aggregation or misfolding. Multiple classes of molecular chaperones, such as Hsp70s and Hsp40s, recognize and transiently bind polypeptides with exposed hydrophobic stretches in order to prevent misfolding. Other types of chaperones, such as Hsp90, have more specialized functions in that they appear to interact with only a subset of cellular proteins. This chapter focuses on the role of Hsp90 and partner co-chaperones in promoting the folding and activation of a diverse group of proteins with critical roles in cellular signaling and function.

In this article we summarize the birth of the field of nuclear receptors, the discovery of untransformed and transformed isoforms of ligand-binding macromolecules, the discovery of the three-domain structure of the receptors, and the properties of the Hsp90-based heterocomplex responsible for the overall structure of the oligomeric receptor and many aspects of the biological effects.The discovery and properties of the subfamily of receptors called orphan receptors is also outlined.Novel molecular aspects of the mechanism of action of nuclear receptors and challenges to resolve in the near future are discussed.

BACKGROUND AND PURPOSE:With funding from the National Institutes of Health, BUILDing SCHOLARS was established at The University of Texas at El Paso with the goal of implementing, evaluating and sustaining a suite of institutional, faculty and student development interventions in order to train the next generation of biomedical researchers from the U.S. Southwest region, where the need is dire among underserved communities. The focus is on supporting the infrastructure necessary to train and mentor students so they persist on pathways across a range of biomedical research fields. The purpose of this article is to highlight the design and implementation of BUILDing SCHOLARS' key interventions, which offer a systemic student training model for the U.S. Southwest. In-depth reporting of evaluation results is reserved for other technical publications.PROGRAM AND KEY HIGHLIGHTS:BUILDing SCHOLARS uses a comprehensive regional approach to undergraduate training through a multi-institution consortium that includes 12 research partners and various pipeline partners across Texas, New Mexico, and Arizona. Through faculty collaborations and undergraduate research training, the program integrates social and behavioral sciences and biomedical engineering while emphasizing seven transdisciplinary nodes of biomedical research excellence that are common across partner institutions: addiction, cancer, degenerative and chronic diseases, environmental health, health disparities, infectious diseases, and translational biomedicine. Key interventions aim to: (1) improve institutional capacities by expanding undergraduate research training infrastructures; (2) develop an intra- and cross-institutional mentoring-driven "community of practice" to support undergraduate student researchers; (3) broaden the pool of student participants, improve retention, and increase matriculation into competitive graduate programs; and (4) support faculty and postdoctoral personnel by training them in research pedagogy and mentoring techniques and providing them with resources for increasing their research productivity. Student training activities focus on early interventions to maximize retention and on enabling students to overcome common barriers by addressing their educational endowments, science socialization, network development, family expectations, and material resources. Over the long term, BUILDing SCHOLARS will help increase the diversity of the biomedical research workforce in the U.S. by meeting the needs of students from the Southwest region and by serving as a model for other institutions.

Castration‐resistant prostate cancer (CRPC) is the emergence of prostate cancer cells that have adapted to the androgen‐depleted environment of the prostate. In recent years, targeting multiple chaperones and co‐chaperones (e.g., Hsp27, FKBP52) that promote androgen receptor (AR) signaling and/or novel AR regulatory mechanisms have emerged as promising alternative treatments for CRPC. We have shown that inactivation of inhibitor of differentiation 4 (ID4), a dominant‐negative helix loop helix protein, promotes de novo steroidogenesis and CRPC with a gene expression signature that resembles constitutive AR activity in castrated mice. In this study, we investigated the underlying mechanism through which loss of ID4 potentiates AR signaling. Proteomic analysis between prostate cancer cell line LNCaP (L+ns) and LNCaP lacking ID4 (L(−)ID4) revealed elevated levels of Hsp27 and FKBP52, suggesting a role for these AR‐associated co‐chaperones in promoting constitutively active AR signaling in L(−)ID4 cells. Interestingly, protein interaction studies demonstrated a direct interaction between ID4 and the 52‐kDa FK506‐binding protein (FKBP52) in vitro, but not with AR. An increase in FKBP52‐dependent AR transcriptional activity was observed in L(−)ID4 cells. Moreover, pharmacological inhibition of FKBP52‐AR signaling, by treatment with MJC13, attenuated the tumor growth, weight, and volume in L(−)ID4 xenografts. Together, our results demonstrate that ID4 selectively regulates AR activity through direct interaction with FKBP52, and its loss, promotes CRPC through FKBP52‐mediated AR signaling.

Polymorphisms in FKBP51 are associated with stress-related psychiatric disorders and influence the severity of pain symptoms experienced after trauma. We report that FKBP51-FK506 binding protein 51) is crucial for the full development and maintenance of long-term pain states. Indeed, FKBP51 knockout mice, as well as mice in which silencing of FKBP51 is restricted to the spinal cord, showed reduced hypersensitivity in several persistent pain models in rodents. FKBP51 deletion did not compromise the detection of acute painful stimuli, a critical protective mechanism. Moreover, the intrathecal administration of the specific FKBP51 inhibitor SAFit2 reduced the severity of an established pain state, confirming the crucial role of spinal FKBP51 in nociceptive processing. Finally, glucocorticoid signaling, which is known to modulate persistent pain states in rodents, was impaired in FKBP51 knockout mice. This finding suggested that FKBP51 regulates chronic pain by modulation of glucocorticoid signaling. Thus, FKBP51 is a central mediator of chronic pain, likely in humans as well as rodents, and is a new pharmacologically tractable target for the treatment of long-term pain states.