RESUMEN
In the face of a challenging climate STEM (Science, Technology, Engineering and Mathematics) higher education that is resistant to Diversity, Equity, and Inclusion efforts aimed to increase and retain students from historically excluded groups (HEGs), there is a critical need for a support structure to ensure students from HEGs continue to be recruited retained. The Biology Undergraduate and Master's Mentorship Program (BUMMP) embodies this commitment to fostering scientific identity, efficacy, and a sense of belonging for first-generation and historically underserved undergraduate and master's students at UC San Diego. The mission of BUMMP is to cultivate a sense of belonging, instill confidence, and nurture a strong scientific identity amongst all its participants. At its core, the three pillars of BUMMP are (1) mentorship, (2) professional development, and (3) research. Quality mentorship is provided where students receive personal guidance from faculty, graduate students, postdocs, and industry leaders in navigating their career pathways. Complementing mentorship, BUMMP provides paid research opportunities and prioritizes professional development by offering workshops designed to enhance students' professional skills. These three pillars form the backbone of BUMMP, empowering students from all backgrounds and ensuring their retention and persistence in STEM. So far, we've served over 1350 mentees, collaborated with 809 mentors, and had over 180 mentees actively engaged in BUMMP-sponsored research activities. The primary focus of this paper is to provide a programmatic guideline for the three pillars of BUMMP: mentorship, professional development, and research. This will offer a blueprint for other institutions to establish similar mentorship programs. Additionally, the paper highlights the impact of the BUMMP program and surveyed mentees who have participated in the mentorship and research component of BUMMP. We showed that mentorship and research experience enhance students' sense of belonging, science identity, and science efficacy, which are key predictors of retention and persistence in pursuing a STEM career. Overall, BUMMP's expansive efforts have made a tremendous impact at UC San Diego and will continue to foster a community of future leaders who will be prepared to make meaningful contributions to the scientific community and beyond.
Asunto(s)
Ingeniería , Mentores , Estudiantes , Humanos , Estudiantes/psicología , Ingeniería/educación , Universidades , Tecnología/educación , Ciencia/educación , Empoderamiento , Matemática/educación , Tutoría/métodosRESUMEN
We are 52 Black scientists. Here, we establish the context of Juneteenth in STEMM and discuss the barriers Black scientists face, the struggles they endure, and the lack of recognition they receive. We review racism's history in science and provide institutional-level solutions to reduce the burdens on Black scientists.
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Población Negra , HumanosRESUMEN
Protein aggregates are a common feature of diseased and aged cells. Membrane proteins comprise a quarter of the proteome, and yet, it is not well understood how aggregation of membrane proteins is regulated and what effects these aggregates can have on cellular health. We have determined in yeast that the derlin Dfm1 has a chaperone-like activity that influences misfolded membrane protein aggregation. We establish that this function of Dfm1 does not require recruitment of the ATPase Cdc48 and it is distinct from Dfm1's previously identified function in dislocating misfolded membrane proteins from the endoplasmic reticulum (ER) to the cytosol for degradation. Additionally, we assess the cellular impacts of misfolded membrane proteins in the absence of Dfm1 and determine that misfolded membrane proteins are toxic to cells in the absence of Dfm1 and cause disruptions to proteasomal and ubiquitin homeostasis.
Asunto(s)
Proteínas de la Membrana , Pliegue de Proteína , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Proteínas de la Membrana/metabolismo , Chaperonas Moleculares/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
The endoplasmic reticulum (ER) is an essential organelle in eukaryotic cells and is a major site for protein folding, modification, and lipid synthesis. Perturbations within the ER, such as protein misfolding and high demand for protein folding, lead to dysregulation of the ER protein quality control network and ER stress. Recently, the rhomboid superfamily has emerged as a critical player in ER protein quality control because it has diverse cellular functions, including ER-associated degradation (ERAD), endosome Golgi-associated degradation (EGAD), and ER preemptive quality control (ERpQC). This breadth of function both illustrates the importance of the rhomboid superfamily in health and diseases and emphasizes the necessity of understanding their mechanisms of action. Because dysregulation of rhomboid proteins has been implicated in various diseases, such as neurological disorders and cancers, they represent promising potential therapeutic drug targets. This review provides a comprehensive account of the various roles of rhomboid proteins in the context of ER protein quality control and discusses their significance in health and disease.
Asunto(s)
Degradación Asociada con el Retículo Endoplásmico , Proteínas , Proteínas/metabolismo , Retículo Endoplásmico/metabolismo , Estrés del Retículo Endoplásmico , Pliegue de ProteínaRESUMEN
Nearly one-third of nascent proteins are initially targeted to the endoplasmic reticulum (ER), where they are correctly folded and assembled before being delivered to their final cellular destinations. To prevent the accumulation of misfolded membrane proteins, ER-associated degradation (ERAD) removes these client proteins from the ER membrane to the cytosol in a process known as retrotranslocation. Our previous work demonstrated that rhomboid pseudoprotease Dfm1 is involved in the retrotranslocation of ubiquitinated membrane integral ERAD substrates. Herein, we found that Dfm1 associates with the SPOTS complex, which is composed of serine palmitoyltransferase (SPT) enzymes and accessory components that are critical for catalyzing the first rate-limiting step of the sphingolipid biosynthesis pathway. Furthermore, Dfm1 employs an ERAD-independent role for facilitating the ER export and endosome- and Golgi-associated degradation (EGAD) of Orm2, which is a major antagonist of SPT activity. Given that the accumulation of human Orm2 homologs, ORMDLs, is associated with various pathologies, our study serves as a molecular foothold for understanding how dysregulation of sphingolipid metabolism leads to various diseases.
Asunto(s)
Degradación Asociada con el Retículo Endoplásmico , Esfingolípidos , Humanos , Esfingolípidos/metabolismo , Ubiquitina/metabolismo , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , HomeostasisRESUMEN
Nearly one-third of proteins are initially targeted to the endoplasmic reticulum (ER) membrane, where they are correctly folded and then delivered to their final cellular destinations. To prevent the accumulation of misfolded membrane proteins, ER-associated degradation (ERAD) moves these clients from the ER membrane to the cytosol, a process known as retrotranslocation. Our recent work in Saccharomyces cerevisiae reveals a derlin rhomboid pseudoprotease, Dfm1, is involved in the retrotranslocation of ubiquitinated ERAD membrane substrates. In this study, we identify conserved residues of Dfm1 that are critical for retrotranslocation. We find several retrotranslocation-deficient Loop 1 mutants that display impaired binding to membrane substrates. Furthermore, Dfm1 possesses lipid thinning function to facilitate in the removal of ER membrane substrates, and this feature is conserved in its human homolog, Derlin-1, further implicating that derlin-mediated retrotranslocation is a well-conserved process.
Asunto(s)
Degradación Asociada con el Retículo Endoplásmico , Metabolismo de los Lípidos , Proteínas de la Membrana/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de la Membrana/genética , Mutación , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Transporte de Proteínas , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteína que Contiene Valosina/genética , Proteína que Contiene Valosina/metabolismoRESUMEN
In S. cerevisiae, we identified rhomboid pseudoprotease Dfm1 as the major mediator for removing or retrotranslocating misfolded membrane substrates from the ER (endoplasmic reticulum). Long-standing challenges with rapid suppression of dfm1-null cells have limited the biochemical study of Dfm1's role in ER protein quality control. Here, we provide a protocol for the generation and handling of dfm1-null cells and procedures for studying normal vs. suppressive alternative retrotranslocation pathways. Our methods can be utilized to study other components involved in retrotranslocation. For complete information on the generation and use of this protocol, please refer to Neal et al. (2017, 2018); Neal et al. (2019); Neal et al. (2020).
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Técnicas de Cultivo de Célula/métodos , Degradación Asociada con el Retículo Endoplásmico , Técnicas de Inactivación de Genes/métodos , Proteínas de la Membrana , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Degradación Asociada con el Retículo Endoplásmico/genética , Degradación Asociada con el Retículo Endoplásmico/fisiología , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Proteínas de la Membrana/fisiología , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/fisiologíaRESUMEN
Phosphatidylethanolamine (PE) made in mitochondria has long been recognized as an important precursor for phosphatidylcholine production that occurs in the endoplasmic reticulum (ER). Recently, the strict mitochondrial localization of the enzyme that makes PE in the mitochondrion, phosphatidylserine decarboxylase 1 (Psd1), was questioned. Since a dual localization of Psd1 to the ER would have far-reaching implications, we initiated our study to independently re-assess the subcellular distribution of Psd1. Our results support the unavoidable conclusion that the vast majority, if not all, of functional Psd1 resides in the mitochondrion. Through our efforts, we discovered that mutant forms of Psd1 that impair a self-processing step needed for it to become functional are dually localized to the ER when expressed in a PE-limiting environment. We conclude that severely impaired cellular PE metabolism provokes an ER-assisted adaptive response that is capable of identifying and resolving nonfunctional mitochondrial precursors.
RESUMEN
Cells are equipped with protein quality control pathways in order to maintain a healthy proteome; a process known as protein homeostasis. Dysfunction in protein homeostasis leads to the development of many diseases that are associated with proteinopathies. Recently, the rhomboid superfamily has attracted much attention concerning their involvement in protein homeostasis. While their functional role has become much clearer in the last few years, their systemic significance in mammals remains elusive. Here we delineate the current knowledge of rhomboids in protein quality control and how these functions are integrated at the organismal level.
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Proteínas de la Membrana/metabolismo , Familia de Multigenes , Proteostasis , Animales , Enfermedad , Humanos , Péptido Hidrolasas/metabolismoRESUMEN
Prokaryotes have aerobic and anaerobic electron acceptors for oxidative folding of periplasmic proteins. The mitochondrial intermembrane space has an analogous pathway with the oxidoreductase Mia40 and sulfhydryl oxidase Erv1, termed the mitochondrial intermembrane space assembly (MIA) pathway. The aerobic electron acceptors include oxygen and cytochrome c, but an acceptor that can function under anaerobic conditions has not been identified. Here we show that the fumarate reductase Osm1, which facilitates electron transfer from fumarate to succinate, fills this gap as a new electron acceptor. In addition to microsomes, Osm1 localizes to the mitochondrial intermembrane space and assembles with Erv1 in a complex. In reconstitution studies with reduced Tim13, Mia40, and Erv1, the addition of Osm1 and fumarate completes the disulfide exchange pathway that results in Tim13 oxidation. From in vitro import assays, mitochondria lacking Osm1 display decreased import of MIA substrates, Cmc1 and Tim10. Comparative reconstitution assays support that the Osm1/fumarate couple accepts electrons with similar efficiency to cytochrome c and that the cell has strategies to coordinate expression of the terminal electron acceptors. Thus Osm1/fumarate is a new electron acceptor couple in the mitochondrial intermembrane space that seems to function in both aerobic and anaerobic conditions.
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Mitocondrias/metabolismo , Proteínas Mitocondriales/metabolismo , Oxidorreductasas actuantes sobre Donantes de Grupos Sulfuro/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Succinato Deshidrogenasa/metabolismo , Citocromos c/metabolismo , Disulfuros/metabolismo , Transporte de Electrón , Electrones , Fumaratos/metabolismo , Membranas Intracelulares/enzimología , Membranas Intracelulares/metabolismo , Microsomas/enzimología , Microsomas/metabolismo , Mitocondrias/enzimología , Proteínas de Transporte de Membrana Mitocondrial/metabolismo , Proteínas Mitocondriales/genética , Oxidación-Reducción , Oxidorreductasas/metabolismo , Oxidorreductasas actuantes sobre Donantes de Grupos Sulfuro/genética , Pliegue de Proteína , Transporte de Proteínas , Saccharomyces cerevisiae/enzimología , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Succinato Deshidrogenasa/genéticaRESUMEN
The HRD (HMG-CoA reductase degradation) pathway is a conserved route of endoplasmic reticulum-associated degradation (ERAD), by which misfolded ER proteins are ubiquitinated and degraded. ERAD substrates are ubiquitinated by the action of the Hrd1 RING-H2 E3 ligase. Hrd1 is always present in a stoichiometric complex with the ER membrane protein Hrd3, which is also required for HRD-dependent degradation. Despite its conserved presence, unequivocal study of Hrd3 function has been precluded by its central role in Hrd1 stability. Loss of Hrd3 causes unrestricted self-degradation of Hrd1, resulting in significant loss of the core ligase. Accordingly, the degree to which Hrd3 functions independently of Hrd1 stabilization has remained unresolved. By capitalizing on our studies of Usa1 in Hrd1 degradation, we have devised a new approach to evaluate Hrd3 functions in ERAD. We now show that Hrd3 has a direct and critical role in ERAD in addition to Hrd1 stabilization. This direct component of Hrd3 is phenotypically as important as Hrd1 in the native HRD complex. Hrd3 was required the E3 activity of Hrd1, rather than substrate or E2 recruitment to Hrd1. Although Hrd1 can function in some circumstances independent of Hrd3, these studies show an indispensable role for Hrd3 in living cells.
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Degradación Asociada con el Retículo Endoplásmico/fisiología , Retículo Endoplásmico/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Retículo Endoplásmico/genética , Estabilidad de Enzimas/fisiología , Glicoproteínas de Membrana/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Ubiquitina-Proteína Ligasas/genéticaRESUMEN
A redox-regulated import pathway consisting of Mia40 and Erv1 mediates the import of cysteine-rich proteins into the mitochondrial intermembrane space. Mia40 is the oxidoreductase that inserts two disulfide bonds into the substrate simultaneously. However, Mia40 has one redox-active cysteine pair, resulting in ambiguity about how Mia40 accepts numerous electrons during substrate oxidation. In this study, we have addressed the oxidation of Tim13 in vitro and in organello. Reductants such as glutathione and ascorbate inhibited both the oxidation of the substrate Tim13 in vitro and the import of Tim13 and Cmc1 into isolated mitochondria. In addition, a ternary complex consisting of Erv1, Mia40, and substrate, linked by disulfide bonds, was not detected in vitro. Instead, Mia40 accepted six electrons from substrates, and this fully reduced Mia40 was sensitive to protease, indicative of conformational changes in the structure. Mia40 in mitochondria from the erv1-101 mutant was also trapped in a completely reduced state, demonstrating that Mia40 can accept up to six electrons as substrates are imported. Therefore, these studies support that Mia40 functions as an electron sink to facilitate the insertion of two disulfide bonds into substrates.
Asunto(s)
Electrones , Regulación Fúngica de la Expresión Génica , Mitocondrias/metabolismo , Proteínas de Transporte de Membrana Mitocondrial/metabolismo , Proteínas Mitocondriales/metabolismo , Oxidorreductasas actuantes sobre Donantes de Grupos Sulfuro/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Ácido Ascórbico/farmacología , Disulfuros/química , Disulfuros/metabolismo , Glutatión/farmacología , Metalochaperonas/genética , Metalochaperonas/metabolismo , Mitocondrias/efectos de los fármacos , Proteínas de Transporte de Membrana Mitocondrial/genética , Proteínas del Complejo de Importación de Proteínas Precursoras Mitocondriales , Proteínas Mitocondriales/genética , Mutación , Oxidación-Reducción , Oxidorreductasas actuantes sobre Donantes de Grupos Sulfuro/genética , Plásmidos/química , Plásmidos/metabolismo , Transporte de Proteínas , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Sustancias Reductoras/farmacología , Saccharomyces cerevisiae/efectos de los fármacos , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Transducción de SeñalRESUMEN
Mia40 and Erv1 execute a disulfide relay to import the small Tim proteins into the mitochondrial intermembrane space. Here, we have reconstituted the oxidative folding pathway in vitro with Tim13 as a substrate and determined the midpoint potentials of Mia40 and Tim13. Specifically, Mia40 served as a direct oxidant of Tim13, and Erv1 was required to reoxidize Mia40. During oxidation, four electrons were transferred from Tim13 with the insertion of two disulfide bonds in succession. The extent of Tim13 oxidation was directly dependent on Mia40 concentration and independent of Erv1 concentration. Characterization of the midpoint potentials showed that electrons flowed from Tim13 with a more negative midpoint potential of -310 mV via Mia40 with an intermediate midpoint potential of -290 mV to the C130-C133 pair of Erv1 with a positive midpoint potential of -150 mV. Intermediary complexes between Tim13-Mia40 and Mia40-Erv1 were trapped. Last, mutating C133 of the catalytic C130-C133 pair or C30 of the shuttle C30-C33 pair in Erv1 abolished oxidation of Tim13, whereas mutating the cysteines in the redox-active CPC motif, but not the structural disulfide linkages of the CX(9)C motif of Mia40, prevented Tim13 oxidation. Thus, we demonstrate that Mia40, Erv1, and oxygen are the minimal machinery for Tim13 oxidation.