RESUMEN
The Hsp90 chaperone is known to interact with a diverse array of client proteins. However, in every case examined, Hsp90 is also accompanied by a single or several co-chaperone proteins. One class of co-chaperone contains a tetratricopeptide repeat (TPR) 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 abundantly clear that the client protein influences, and is often 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.
Asunto(s)
Proteínas HSP90 de Choque Térmico , Proteínas de Unión a Tacrolimus , Humanos , Proteínas HSP90 de Choque Térmico/química , Proteínas HSP90 de Choque Térmico/genética , Proteínas HSP90 de Choque Térmico/metabolismo , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Isomerasa de Peptidilprolil/metabolismo , Unión Proteica , Proteínas de Unión a Tacrolimus/genética , Proteínas de Unión a Tacrolimus/química , Proteínas de Unión a Tacrolimus/metabolismo , Inmunofilinas/genética , Inmunofilinas/metabolismoRESUMEN
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.
RESUMEN
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.
Asunto(s)
Proteínas de Unión a Tacrolimus/química , Proteínas de Pez Cebra/química , Animales , Fibroblastos/efectos de los fármacos , Fibroblastos/enzimología , Mutación con Ganancia de Función , Proteínas HSP90 de Choque Térmico/metabolismo , Humanos , Ratones , Ratones Noqueados , Simulación de Dinámica Molecular , Dominios Proteicos Ricos en Prolina/genética , Receptores Androgénicos/efectos de los fármacos , Receptores Androgénicos/metabolismo , Receptores de Glucocorticoides/metabolismo , Saccharomyces cerevisiae/enzimología , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Transducción de Señal , Proteínas de Unión a Tacrolimus/genética , Proteínas de Unión a Tacrolimus/metabolismo , Proteínas de Pez Cebra/genética , Proteínas de Pez Cebra/metabolismoRESUMEN
Sti1/Hop is a modular protein required for the transfer of client proteins from the Hsp70 to the Hsp90 chaperone system in eukaryotes. It binds Hsp70 and Hsp90 simultaneously via TPR (tetratricopeptide repeat) domains. Sti1/Hop contains three TPR domains (TPR1, TPR2A and TPR2B) and two domains of unknown structure (DP1 and DP2). We show that TPR2A is the high affinity Hsp90-binding site and TPR1 and TPR2B bind Hsp70 with moderate affinity. The DP domains exhibit highly homologous α-helical folds as determined by NMR. These, and especially DP2, are important for client activation in vivo. The core module of Sti1 for Hsp90 inhibition is the TPR2A-TPR2B segment. In the crystal structure, the two TPR domains are connected via a rigid linker orienting their peptide-binding sites in opposite directions and allowing the simultaneous binding of TPR2A to the Hsp90 C-terminal domain and of TPR2B to Hsp70. Both domains also interact with the Hsp90 middle domain. The accessory TPR1-DP1 module may serve as an Hsp70-client delivery system for the TPR2A-TPR2B-DP2 segment, which is required for client activation in vivo.
Asunto(s)
Proteínas HSP90 de Choque Térmico/química , Proteínas HSP90 de Choque Térmico/metabolismo , Proteínas de Choque Térmico/química , Proteínas de Choque Térmico/metabolismo , Chaperonas Moleculares/química , Chaperonas Moleculares/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Adenosina Trifosfatasas/metabolismo , Sitios de Unión , Proteínas HSP70 de Choque Térmico/química , Proteínas HSP70 de Choque Térmico/metabolismo , Modelos Moleculares , Proteína Oncogénica pp60(v-src)/metabolismo , Unión Proteica , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Receptores de Glucocorticoides/metabolismo , Saccharomyces cerevisiae/metabolismoRESUMEN
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.
Asunto(s)
Proteínas HSP90 de Choque Térmico/metabolismo , Proteínas de Unión a Tacrolimus/metabolismo , Animales , Sitios de Unión , Proteínas HSP90 de Choque Térmico/química , Humanos , Modelos Moleculares , Unión Proteica , Conformación Proteica , Dominios y Motivos de Interacción de Proteínas , Transducción de Señal , Relación Estructura-Actividad , Proteínas de Unión a Tacrolimus/químicaRESUMEN
MJC13, a novel FKBP52 targeting agent, has potential use for the treatment of castration-resistant prostate cancer. The purpose of this work was to develop a solution formulation of MJC13, and obtain its efficacy profile in a human prostate cancer xenograft mouse model. Preformulation studies were conducted to evaluate the physicochemical properties. Co-solvent systems were evaluated for aqueous solubility and tolerance. A human prostate cancer xenograft mouse model was established by growing 22Rv1 prostate cancer cells in C.B-17 SCID mice. The optimal formulation was used to study the efficacy of MJC13 in this preclinical model of castrate-resistant prostate cancer. We found that MJC13 was stable (at least for 1 month), highly lipophilic (logP = 6.49), poorly soluble in water (0.28 µg/mL), and highly plasma protein bound (>98%). The optimal formulation consisting of PEG 400 and Tween 80 (1:1, v/v) allowed us to achieve a MJC13 concentration of 7.5 mg/mL, and tolerated an aqueous environment. After twice weekly intratumoral injection with 10 mg/kg MJC13 in this formulation for four consecutive weeks, tumor volumes were significantly reduced compared to vehicle-treated controls.
Asunto(s)
Anilidas/síntesis química , Antineoplásicos/síntesis química , Química Farmacéutica/métodos , Ciclohexanos/síntesis química , Modelos Animales de Enfermedad , Neoplasias de la Próstata Resistentes a la Castración/tratamiento farmacológico , Anilidas/uso terapéutico , Animales , Antineoplásicos/uso terapéutico , Línea Celular Tumoral , Ciclohexanos/uso terapéutico , Humanos , Inyecciones Intralesiones , Masculino , Ratones , Ratones SCID , Soluciones Farmacéuticas/síntesis química , Soluciones Farmacéuticas/uso terapéutico , Ratas , Resultado del Tratamiento , Ensayos Antitumor por Modelo de Xenoinjerto/métodosRESUMEN
Steroid hormone receptors are ligand-dependent transcription factors that require the ordered assembly of multichaperone complexes for transcriptional activity. Although heat shock protein (Hsp) 90 and Hsp70 are key players in this process, multiple Hsp70- and Hsp90-associated cochaperones associate with receptor-chaperone complexes to regulate receptor folding and activation. Small glutamine-rich tetratricopeptide repeat-containing protein alpha (SGTA) was recently characterized as an Hsp70 and Hsp90-associated cochaperone that specifically regulates androgen receptor activity. However, the specificity of SGTA for additional members of the steroid hormone receptor superfamily and the mechanism by which SGTA regulates receptor activity remain unclear. Here we report that SGTA associates with and specifically regulates the androgen, glucocorticoid, and progesterone receptors and has no effect on the mineralocorticoid and estrogen receptors in both yeast and mammalian cell-based reporter assays. In both systems, SGTA knockdown/deletion enhances receptor activity, whereas SGTA overexpression suppresses receptor activity. We demonstrate that SGTA binds directly to Hsp70 and Hsp90 in vitro with similar affinities yet predominately precipitates with Hsp70 from cell lysates, suggesting a role for SGTA in early, Hsp70-mediated folding. Furthermore, SGTA expression completely abrogates the regulation of receptor function by FKBP52 (52-kDa FK506-binding protein), which acts at a later stage of the chaperone cycle. Taken together, our data suggest a role for SGTA at distinct steps in the chaperone-dependent modulation of androgen, glucocorticoid, and progesterone receptor activity.
Asunto(s)
Proteínas Portadoras/metabolismo , Receptores Androgénicos/metabolismo , Receptores de Glucocorticoides/metabolismo , Receptores de Progesterona/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Secuencia de Aminoácidos , Proteínas Portadoras/genética , Técnicas de Silenciamiento del Gen , Proteínas HSP70 de Choque Térmico/genética , Proteínas HSP70 de Choque Térmico/metabolismo , Proteínas HSP90 de Choque Térmico/genética , Proteínas HSP90 de Choque Térmico/metabolismo , Células HeLa , Humanos , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Datos de Secuencia Molecular , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Unión a Tacrolimus/metabolismo , Técnicas del Sistema de Dos HíbridosRESUMEN
The androgen receptor (AR) is a ligand-activated transcription factor that is essential for prostate cancer development. It is activated by androgens through its ligand-binding domain (LBD), which consists predominantly of 11 α-helices. Upon ligand binding, the last helix is reorganized to an agonist conformation termed activator function-2 (AF-2) for coactivator binding. Several coactivators bind to the AF-2 pocket through conserved LXXLL or FXXLF sequences to enhance the activity of the receptor. Recently, a small compound-binding surface adjacent to AF-2 has been identified as an allosteric modulator of the AF-2 activity and is termed binding function-3 (BF-3). However, the role of BF-3 in vivo is currently unknown, and little is understood about what proteins can bind to it. Here we demonstrate that a duplicated GARRPR motif at the N terminus of the cochaperone Bag-1L functions through the BF-3 pocket. These findings are supported by the fact that a selective BF-3 inhibitor or mutations within the BF-3 pocket abolish the interaction between the GARRPR motif(s) and the BF-3. Conversely, amino acid exchanges in the two GARRPR motifs of Bag-1L can impair the interaction between Bag-1L and AR without altering the ability of Bag-1L to bind to chromatin. Furthermore, the mutant Bag-1L increases androgen-dependent activation of a subset of AR targets in a genome-wide transcriptome analysis, demonstrating a repressive function of the GARRPR/BF-3 interaction. We have therefore identified GARRPR as a novel BF-3 regulatory sequence important for fine-tuning the activity of the AR.
Asunto(s)
Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/metabolismo , Receptores Androgénicos/metabolismo , Factores de Transcripción/química , Factores de Transcripción/metabolismo , Regulación Alostérica , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Línea Celular , Proteínas de Unión al ADN/genética , Humanos , Mutación , Oligopéptidos/química , Oligopéptidos/metabolismo , Unión Proteica , Estructura Terciaria de Proteína , Receptores Androgénicos/química , Receptores Androgénicos/genética , Secuencias Repetitivas de Aminoácido , Factores de Transcripción/genética , Activación TranscripcionalRESUMEN
Hsp90 binding immunophilins FKBP51 and FKBP52 modulate steroid receptor trafficking and hormone-dependent biological responses. With the purpose to expand this model to other nuclear factors that are also subject to nuclear-cytoplasmic shuttling, we analyzed whether these immunophilins modulate NF-κB signaling. It is demonstrated that FKBP51 impairs both the nuclear translocation rate of NF-κB and its transcriptional activity. The inhibitory action of FKBP51 requires neither the peptidylprolyl-isomerase activity of the immunophilin nor its association with Hsp90. The TPR domain of FKBP51 is essential. On the other hand, FKBP52 favors the nuclear retention time of RelA, its association to a DNA consensus binding sequence, and NF-κB transcriptional activity, the latter effect being strongly dependent on the peptidylprolyl-isomerase activity and also on the TPR domain of FKBP52, but its interaction with Hsp90 is not required. In unstimulated cells, FKBP51 forms endogenous complexes with cytoplasmic RelA. Upon cell stimulation with phorbol ester, the NF-κB soluble complex exchanges FKBP51 for FKBP52, and the NF-κB biological effect is triggered. Importantly, FKBP52 is functionally recruited to the promoter region of NF-κB target genes, whereas FKBP51 is released. Competition assays demonstrated that both immunophilins antagonize one another, and binding assays with purified proteins suggest that the association of RelA and immunophilins could be direct. These observations suggest that the biological action of NF-κB in different cell types could be positively regulated by a high FKBP52/FKBP51 expression ratio by favoring NF-κB nuclear retention, recruitment to the promoter regions of target genes, and transcriptional activity.
Asunto(s)
Proteínas de Unión a Tacrolimus/fisiología , Factor de Transcripción ReIA/metabolismo , Transporte Activo de Núcleo Celular , Animales , Núcleo Celular/metabolismo , Células HEK293 , Humanos , Regiones Promotoras Genéticas , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Ratas , Receptores de Glucocorticoides/metabolismo , Transcripción Genética , Activación TranscripcionalRESUMEN
Drugs that target novel surfaces on the androgen receptor (AR) and/or novel AR regulatory mechanisms are promising alternatives for the treatment of castrate-resistant prostate cancer. The 52 kDa FK506 binding protein (FKBP52) is an important positive regulator of AR in cellular and whole animal models and represents an attractive target for the treatment of prostate cancer. We used a modified receptor-mediated reporter assay in yeast to screen a diversified natural compound library for inhibitors of FKBP52-enhanced AR function. The lead compound, termed MJC13, inhibits AR function by preventing hormone-dependent dissociation of the Hsp90-FKBP52-AR complex, which results in less hormone-bound receptor in the nucleus. Assays in early and late stage human prostate cancer cells demonstrated that MJC13 inhibits AR-dependent gene expression and androgen-stimulated prostate cancer cell proliferation.
Asunto(s)
Regulación de la Expresión Génica/efectos de los fármacos , Proteínas HSP90 de Choque Térmico/metabolismo , Modelos Moleculares , Complejos Multiproteicos/metabolismo , Neoplasias de la Próstata/metabolismo , Receptores Androgénicos/metabolismo , Proteínas de Unión a Tacrolimus/antagonistas & inhibidores , Animales , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Descubrimiento de Drogas , Ensayo de Inmunoadsorción Enzimática , Fluorescencia , Humanos , Immunoblotting , Inmunoprecipitación , Masculino , Ratones , Simulación de Dinámica Molecular , Estructura Molecular , Receptores Androgénicos/química , Proteínas de Unión a Tacrolimus/metabolismo , Levaduras , beta-GalactosidasaRESUMEN
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.
Asunto(s)
Metabolismo de los Lípidos , Neoplasias Pancreáticas , Animales , Humanos , Ratones , Carcinogénesis/genética , Transformación Celular Neoplásica/genética , Transformación Celular Neoplásica/metabolismo , Citratos , Ácidos Grasos , Metabolismo de los Lípidos/genética , Ratones Transgénicos , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Proteínas Proto-Oncogénicas p21(ras)/genética , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Proteína con Dedos de Zinc GLI1/metabolismoRESUMEN
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.
Asunto(s)
Chaperonas Moleculares , Estrés Fisiológico , Humanos , Proteínas HSP70 de Choque Térmico , Chaperonas Moleculares/fisiología , Estrés Fisiológico/fisiologíaRESUMEN
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 1.64-4.34) 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.
Asunto(s)
Investigación Biomédica , Organización de la Financiación , Academias e Institutos , Humanos , National Institutes of Health (U.S.) , Estados Unidos , EscrituraRESUMEN
Imbalanced protein load within cells is a critical aspect for most diseases of aging. In particular, the accumulation of proteins into neurotoxic aggregates is a common thread for a host of neurodegenerative diseases. Our previous work demonstrated that age-related changes to the cellular chaperone repertoire contributes to abnormal buildup of the microtubule-associated protein tau that accumulates in a group of diseases termed tauopathies, the most common being Alzheimer's disease. Here, we show that the Hsp90 cochaperone, FK506-binding protein 51 (FKBP51), which possesses both an Hsp90-interacting tetratricopeptide domain and a peptidyl-prolyl cis-trans isomerase (PPIase) domain, prevents tau clearance and regulates its phosphorylation status. Regulation of the latter is dependent on the PPIase activity of FKBP51. FKB51 enhances the association of tau with Hsp90, but the FKBP51/tau interaction is not dependent on Hsp90. In vitro FKBP51 stabilizes microtubules with tau in a reaction depending on the PPIase activity of FKBP51. Based on these new findings, we propose that FKBP51 can use the Hsp90 complex to isomerize tau, altering its phosphorylation pattern and stabilizing microtubules.
Asunto(s)
Encéfalo/metabolismo , Proteínas HSP90 de Choque Térmico/metabolismo , Microtúbulos/metabolismo , Proteínas de Unión a Tacrolimus/metabolismo , Proteínas tau/metabolismo , Animales , Benzoquinonas/farmacología , Línea Celular Transformada , Quimotripsina/farmacología , Femenino , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/fisiología , Proteínas HSP90 de Choque Térmico/antagonistas & inhibidores , Humanos , Lactamas Macrocíclicas/farmacología , Masculino , Ratones , Ratones Endogámicos C57BL , Microtúbulos/efectos de los fármacos , Mutación/genética , Oocitos , Isomerasa de Peptidilprolil/metabolismo , ARN Interferente Pequeño/farmacología , Proteínas de Unión a Tacrolimus/genética , Transfección/métodos , XenopusRESUMEN
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.
Asunto(s)
Investigación Biomédica , Grupos Minoritarios , Humanos , Maryland , Investigadores , Recursos HumanosRESUMEN
BACKGROUND: Male infertility is a common cause of reproductive failure in humans. In mice, targeted deletions of the genes coding for FKBP6 or FKBP52, members of the FK506 binding protein family, can result in male infertility. In the case of FKBP52, this reflects an important role in potentiating Androgen Receptor (AR) signalling in the prostate and accessory glands, but not the testis. In infertile men, no mutations of FKBP52 or FKBP6 have been found so far, but the gene for FKBP-like (FKBPL) maps to chromosome 6p21.3, an area linked to azoospermia in a group of Japanese patients. METHODS: To determine whether mutations in FKBPL could contribute to the azoospermic phenotype, we examined expression in mouse and human tissues by RNA array blot, RT-PCR and immunohistochemistry and sequenced the complete gene from two azoospermic patient cohorts and matching control groups. FKBPL-AR interaction was assayed using reporter constructs in vitro. RESULTS: FKBPL is strongly expressed in mouse testis, with expression upregulated at puberty. The protein is expressed in human testis in a pattern similar to FKBP52 and also enhanced AR transcriptional activity in reporter assays. We examined sixty patients from the Japanese patient group and found one inactivating mutation and one coding change, as well as a number of non-coding changes, all absent in fifty-six controls. A second, Irish patient cohort of thirty showed another two coding changes not present in thirty proven fertile controls. CONCLUSIONS: Our results describe the first alterations in the gene for FKBPL in azoospermic patients and indicate a potential role in AR-mediated signalling in the testis.
Asunto(s)
Inmunofilinas/genética , Infertilidad Masculina/genética , Secuencia de Aminoácidos , Animales , Estudios de Casos y Controles , Estudios de Cohortes , Ligamiento Genético , Predisposición Genética a la Enfermedad , Humanos , Masculino , Ratones , Datos de Secuencia Molecular , Mutación/fisiología , Homología de Secuencia de Aminoácido , Proteínas de Unión a Tacrolimus , Análisis de Matrices TisularesRESUMEN
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 â m/z 191.000 and [M+H]+ m/z 266.013 â 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.
RESUMEN
Hsp90 plays an important role in health and is a therapeutic target for managing misfolding disease. Compounds that disrupt co-chaperone delivery of clients to Hsp90 target a subset of Hsp90 activities, thereby minimizing the toxicity of pan-Hsp90 inhibitors. Here, we have identified SEW04784 as a first-in-class inhibitor of the Aha1-stimulated Hsp90 ATPase activity without inhibiting basal Hsp90 ATPase. Nuclear magnetic resonance analysis reveals that SEW84 binds to the C-terminal domain of Aha1 to weaken its asymmetric binding to Hsp90. Consistent with this observation, SEW84 blocks Aha1-dependent Hsp90 chaperoning activities, including the in vitro and in vivo refolding of firefly luciferase, and the transcriptional activity of the androgen receptor in cell-based models of prostate cancer and promotes the clearance of phosphorylated tau in cellular and tissue models of neurodegenerative tauopathy. We propose that SEW84 provides a novel lead scaffold for developing therapeutic approaches to treat proteostatic disease.
Asunto(s)
Proteínas HSP90 de Choque Térmico/antagonistas & inhibidores , Chaperonas Moleculares/antagonistas & inhibidores , Bibliotecas de Moléculas Pequeñas/farmacología , Células HEK293 , Proteínas HSP90 de Choque Térmico/metabolismo , Humanos , Chaperonas Moleculares/metabolismo , Estructura Molecular , Pliegue de Proteína/efectos de los fármacos , Bibliotecas de Moléculas Pequeñas/químicaRESUMEN
Steroid hormone receptor-mediated reporter assays in the budding yeast Saccharomyces cerevisiae have been an invaluable tool for the identification and functional characterization of steroid hormone receptor-associated chaperones and cochaperones. This chapter describes a hormone-inducible androgen receptor-mediated beta-galactosidase reporter assay in yeast. In addition, the immunophilin FKBP52 is used as a specific example of a receptor-associated cochaperone that acts as a positive regulator of receptor function. With the right combination of receptor and cochaperone expression plasmids, reporter plasmid, and ligand, the assay protocol described here could be used to functionally characterize a wide variety of nuclear receptor-cochaperone interactions. In addition to the functional characterization of receptor regulatory proteins, a modified version of this assay is currently being used to screen compound libraries for selective FKBP52 inhibitors that represent attractive therapeutic candidates for the treatment of steroid hormone receptor-associated diseases.
Asunto(s)
Bioensayo/métodos , Genes Reporteros , Chaperonas Moleculares/metabolismo , Receptores de Esteroides/metabolismo , Saccharomyces cerevisiae/metabolismo , Animales , Bioensayo/instrumentación , Chaperonas Moleculares/genética , Receptores Androgénicos/genética , Receptores Androgénicos/metabolismo , Receptores de Esteroides/genética , Saccharomyces cerevisiae/genética , Proteínas de Unión a Tacrolimus/genética , Proteínas de Unión a Tacrolimus/metabolismo , beta-Galactosidasa/genética , beta-Galactosidasa/metabolismoRESUMEN
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.