RESUMEN

In this issue of Molecular Cell, Xie et al.1 revealed that the proteasome is a constitutive component of plant stress granules (SGs), and that enhanced proteolytic activity is essential for efficient SG disassembly and plant survival during the stress response.

Asunto(s)

Gránulos Citoplasmáticos , Homeostasis , Complejo de la Endopetidasa Proteasomal , Estrés Fisiológico , Complejo de la Endopetidasa Proteasomal/metabolismo , Gránulos Citoplasmáticos/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , Arabidopsis/enzimología , Proteolisis

RESUMEN

The recA gene, encoding Recombinase A (RecA) is one of three Mycobacterium tuberculosis (Mtb) genes encoding an in-frame intervening protein sequence (intein) that must splice out of precursor host protein to produce functional protein. Ongoing debate about whether inteins function solely as selfish genetic elements or benefit their host cells requires understanding of interplay between inteins and their hosts. We measured environmental effects on native RecA intein splicing within Mtb using a combination of western blots and promoter reporter assays. RecA splicing was stimulated in bacteria exposed to DNA damaging agents or by treatment with copper in hypoxic, but not normoxic, conditions. Spliced RecA was processed by the Mtb proteasome, while free intein was degraded efficiently by other unknown mechanisms. Unspliced precursor protein was not observed within Mtb despite its accumulation during ectopic expression of Mtb recA within E. coli. Surprisingly, Mtb produced free N-extein in some conditions, and ectopic expression of Mtb N-extein activated LexA in E. coli. These results demonstrate that the bacterial environment greatly impacts RecA splicing in Mtb, underscoring the importance of studying intein splicing in native host environments and raising the exciting possibility of intein splicing as a novel regulatory mechanism in Mtb.

Asunto(s)

Proteínas Bacterianas , Escherichia coli , Inteínas , Mycobacterium tuberculosis , Empalme de Proteína , Rec A Recombinasas , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/metabolismo , Rec A Recombinasas/metabolismo , Rec A Recombinasas/genética , Inteínas/genética , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Exteínas/genética , Daño del ADN , Complejo de la Endopetidasa Proteasomal/metabolismo , Complejo de la Endopetidasa Proteasomal/genética , Regulación Bacteriana de la Expresión Génica , Regiones Promotoras Genéticas , Serina Endopeptidasas

RESUMEN

The ubiquitin-proteasome system is frequently employed to degrade viral proteins, thereby inhibiting viral replication and pathogenicity. Through an analysis of the degradation kinetics of all the SARS-CoV-2 proteins, our study revealed rapid degradation of several proteins, particularly NSP5. Additionally, we identified FBXO22, an E3 ubiquitin ligase, as the primary regulator of NSP5 ubiquitination. Moreover, we validated the interaction between FBXO22 and NSP5, demonstrating that FBXO22-mediated ubiquitination of NSP5 facilitated its recognition by the proteasome, leading to subsequent degradation. Specifically, FBXO22 catalyzed the formation of K48-linked polyubiquitin chains on NSP5 at lysine residues 5 and 90. Knockdown of FBXO22 resulted in decreased NSP5 ubiquitination levels, increased stability, and enhanced ability to evade the host innate immune response. Notably, the protein level of FBXO22 were negatively correlated with SARS-CoV-2 load, highlighting its importance in inhibiting viral replication. This study elucidates the molecular mechanism by which FBXO22 mediates the degradation of NSP5 and underscores its critical role in limiting viral replication. The identification of FBXO22 as a regulator of NSP5 stability provides new insights and potential avenues for targeting NSP5 in antiviral strategies.

Asunto(s)

Complejo de la Endopetidasa Proteasomal , SARS-CoV-2 , Ubiquitinación , Replicación Viral , Humanos , Complejo de la Endopetidasa Proteasomal/metabolismo , SARS-CoV-2/fisiología , SARS-CoV-2/metabolismo , COVID-19/virología , COVID-19/metabolismo , Proteínas F-Box/metabolismo , Proteínas F-Box/genética , Células HEK293 , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitina-Proteína Ligasas/genética , Proteolisis , Proteasas Similares a la Papaína de Coronavirus/metabolismo , Receptores Citoplasmáticos y Nucleares

RESUMEN

The yeast pre1-1(ß4-S142F) mutant accumulates late 20S proteasome core particle precursor complexes (late-PCs). We report a 2.1 Å cryo-EM structure of this intermediate with full-length Ump1 trapped inside, and Pba1-Pba2 attached to the α-ring surfaces. The structure discloses intimate interactions of Ump1 with ß2- and ß5-propeptides, which together fill most of the antechambers between the α- and ß-rings. The ß5-propeptide is unprocessed and separates Ump1 from ß6 and ß7. The ß2-propeptide is disconnected from the subunit by autocatalytic processing and localizes between Ump1 and ß3. A comparison of different proteasome maturation states reveals that maturation goes along with global conformational changes in the rings, initiated by structuring of the proteolytic sites and their autocatalytic activation. In the pre1-1 strain, ß2 is activated first enabling processing of ß1-, ß6-, and ß7-propeptides. Subsequent maturation of ß5 and ß1 precedes degradation of Ump1, tightening of the complex, and finally release of Pba1-Pba2.

Asunto(s)

Microscopía por Crioelectrón , Complejo de la Endopetidasa Proteasomal , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Complejo de la Endopetidasa Proteasomal/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Subunidades de Proteína/metabolismo , Subunidades de Proteína/química , Modelos Moleculares , Conformación Proteica , Péptidos/metabolismo , Péptidos/química , Unión Proteica , Chaperonas Moleculares

RESUMEN

The pathogen Agrobacterium tumefaciens is known for causing crown gall tumours in plants. However, it has also been harnessed as a valuable tool for plant genetic transformation. Apart from the T-DNA, Agrobacterium also delivers at least five virulence proteins into the host plant cells, which are required for an efficient infection. One of these virulence proteins is VirD5. F-box proteins, encoded in the host plant genome or the Ti plasmid, and the ubiquitin/26S proteasome system (UPS) also play an important role in facilitating Agrobacterium infection. Our study identified two Arabidopsis F-box proteins, D5BF1 and D5BF2, that bind VirD5 and facilitate its degradation via the UPS. Additionally, we found that Agrobacterium partially suppresses the expression of D5BF1 and D5BF2. Lastly, stable transformation and tumorigenesis efficiency assays revealed that D5BF1 and D5BF2 negatively regulate the Agrobacterium infection process, showing that the plant F-box proteins and UPS play a role in defending against Agrobacterium infection.

Asunto(s)

Agrobacterium tumefaciens , Proteínas de Arabidopsis , Arabidopsis , Proteínas F-Box , Transformación Genética , Arabidopsis/microbiología , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Agrobacterium tumefaciens/genética , Agrobacterium tumefaciens/patogenicidad , Proteínas F-Box/metabolismo , Proteínas F-Box/genética , Carcinogénesis/genética , Tumores de Planta/microbiología , Complejo de la Endopetidasa Proteasomal/metabolismo , Regulación de la Expresión Génica de las Plantas

RESUMEN

Immunoproteasome is a specialized form of proteasome which plays a crucial role in antigen processing and presentation, and enhances immune responses against malignant cells. This review explores the role of immunoproteasome in the anti-tumor immune responses, including immune surveillance and modulation of the tumor microenvironment, as well as its potential as a target for cancer immunotherapy. Furthermore, we have also discussed the therapeutic potential of immunoproteasome inhibitors, strategies to enhance antigen presentation and combination therapies. The ongoing trials and case studies in urology, melanoma, lung, colorectal, and breast cancers have also been summarized. Finally, the challenges facing clinical translation of immunoproteasome-targeted therapies, such as toxicity and resistance mechanisms, and the future research directions have been addressed. This review underscores the significance of targeting the immunoproteasome in combination with other immunotherapies for solid tumors and its potential broader applications in other diseases.

Asunto(s)

Inmunoterapia , Neoplasias , Complejo de la Endopetidasa Proteasomal , Microambiente Tumoral , Humanos , Neoplasias/inmunología , Neoplasias/terapia , Inmunoterapia/métodos , Complejo de la Endopetidasa Proteasomal/metabolismo , Complejo de la Endopetidasa Proteasomal/inmunología , Microambiente Tumoral/inmunología , Animales , Inhibidores de Proteasoma/uso terapéutico , Inhibidores de Proteasoma/farmacología , Carcinoma/inmunología , Carcinoma/terapia

RESUMEN

Proteasomes are essential molecular machines responsible for the degradation of proteins in eukaryotic cells. Altered proteasome activity has been linked to neurodegeneration, auto-immune disorders and cancer. Despite the relevance for human disease and drug development, no method currently exists to monitor proteasome composition and interactions in vivo in animal models. To fill this gap, we developed a strategy based on tagging of proteasomes with promiscuous biotin ligases and generated a new mouse model enabling the quantification of proteasome interactions by mass spectrometry. We show that biotin ligases can be incorporated in fully assembled proteasomes without negative impact on their activity. We demonstrate the utility of our method by identifying novel proteasome-interacting proteins, charting interactomes across mouse organs, and showing that proximity-labeling enables the identification of both endogenous and small-molecule-induced proteasome substrates.

Asunto(s)

Complejo de la Endopetidasa Proteasomal , Complejo de la Endopetidasa Proteasomal/metabolismo , Animales , Ratones , Humanos , Espectrometría de Masas/métodos , Mapeo de Interacción de Proteínas

RESUMEN

TRIM44, a tripartite motif (TRIM) family member, is pivotal in linking the ubiquitin-proteasome system (UPS) to autophagy in multiple myeloma (MM). However, its prognostic impact and therapeutic potential remain underexplored. Here, we report that TRIM44 overexpression is associated with poor prognosis in a Multiple Myeloma Research Foundation (MMRF) cohort of 858 patients, persisting across primary and recurrent MM cases. TRIM44 expression notably increases in advanced MM stages, indicating its potential role in disease progression. Single-cell RNA sequencing across MM stages showed significant TRIM44 upregulation in smoldering MM (SMM) and MM compared to normal bone marrow, especially in patients with t(4;14) cytogenetic abnormalities. This analysis further identified high TRIM44 expression as predictive of lower responsiveness to proteasome inhibitor (PI) treatments, underscoring its critical function in the unfolded protein response (UPR) in TRIM44-high MM cells. Our findings also demonstrate that TRIM44 facilitates SQSTM1 oligomerization under oxidative stress, essential for its phosphorylation and subsequent autophagic degradation. This process supports the survival of PI-resistant MM cells by activating the NRF2 pathway, which is crucial for oxidative stress response and, potentially, other chemotherapy-induced stressors. Additionally, TRIM44 counters the TRIM21-mediated suppression of the antioxidant response, enhancing MM cell survival under oxidative stress. Collectively, our discoveries highlight TRIM44's significant role in MM progression and resistance to therapy, suggesting its potential value as a therapeutic target.

Asunto(s)

Mieloma Múltiple , Complejo de la Endopetidasa Proteasomal , Proteínas de Motivos Tripartitos , Mieloma Múltiple/patología , Mieloma Múltiple/metabolismo , Mieloma Múltiple/genética , Humanos , Proteínas de Motivos Tripartitos/metabolismo , Proteínas de Motivos Tripartitos/genética , Pronóstico , Línea Celular Tumoral , Complejo de la Endopetidasa Proteasomal/metabolismo , Biomarcadores de Tumor/metabolismo , Biomarcadores de Tumor/genética , Autofagia/genética , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Péptidos y Proteínas de Señalización Intracelular/genética , Inhibidores de Proteasoma/farmacología , Resistencia a Antineoplásicos/genética , Proteína Sequestosoma-1/metabolismo , Proteína Sequestosoma-1/genética , Factor 2 Relacionado con NF-E2/metabolismo , Factor 2 Relacionado con NF-E2/genética , Regulación Neoplásica de la Expresión Génica

RESUMEN

Regulatory T (Treg) cells play a central role in immune homeostasis. Forkhead box P3 (Foxp3), a hallmark molecule in Treg cells, is a vital transcription factor for their development and function. Studies have shown that degradation of the Foxp3 could provide therapeutic benefits in achieving effective anti-tumor immunity. In this study, we designed three PROTAC molecules, P60-L1-VHL, P60-L2-VHL, and P60-L3-VHL, based on a 15-mer peptide inhibitor of Foxp3 (P60), and explored their potential in regulating Foxp3 expression and function. Our data show that, among these molecules, P60-L3-VHL can inhibit the expression and nuclear localization of Foxp3 in HEK 293 T and HeLa cells, respectively. Meanwhile, use of proteasome inhibitor in P60-L3-VHL treated cells revealed an increased Foxp3 expression, indicating that P60-L3-VHL mediates the inhibition of Foxp3 through its degradation in the proteasome pathway. We further substantiate that P60-L3-VHL reduces the differentiation and Foxp3 expression in the in-vitro activated Treg cells. Overall, our findings suggest that P60-L3-VHL inhibits the differentiation of Treg cells by degrading the Foxp3, which may have potential implications in cancer immunotherapy.

Asunto(s)

Factores de Transcripción Forkhead , Proteolisis , Humanos , Factores de Transcripción Forkhead/metabolismo , Proteolisis/efectos de los fármacos , Células HEK293 , Células HeLa , Linfocitos T Reguladores/efectos de los fármacos , Relación Estructura-Actividad , Estructura Molecular , Descubrimiento de Drogas , Relación Dosis-Respuesta a Droga , Complejo de la Endopetidasa Proteasomal/metabolismo , Quimera Dirigida a la Proteólisis

RESUMEN

Proteotoxic stress impairs cellular homeostasis and underlies the pathogenesis of many neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS). The proteasomal and autophagic degradation of proteins are two major pathways for protein quality control in the cell. Here, we report a genome-wide CRISPR screen uncovering a major regulator of cytotoxicity resulting from the inhibition of the proteasome. Dihydrolipoamide branched chain transacylase E2 (DBT) was found to be a robust suppressor, the loss of which protects against proteasome inhibition-associated cell death through promoting clearance of ubiquitinated proteins. Loss of DBT altered the metabolic and energetic status of the cell and resulted in activation of autophagy in an AMP-activated protein kinase (AMPK)-dependent mechanism in the presence of proteasomal inhibition. Loss of DBT protected against proteotoxicity induced by ALS-linked mutant TDP-43 in Drosophila and mammalian neurons. DBT is upregulated in the tissues of ALS patients. These results demonstrate that DBT is a master switch in the metabolic control of protein quality control with implications in neurodegenerative diseases.

Asunto(s)

Complejo de la Endopetidasa Proteasomal , Proteostasis , Animales , Complejo de la Endopetidasa Proteasomal/metabolismo , Humanos , Drosophila/metabolismo , Autofagia , Esclerosis Amiotrófica Lateral/metabolismo , Esclerosis Amiotrófica Lateral/genética , Neuronas/metabolismo , Drosophila melanogaster/metabolismo , Drosophila melanogaster/genética

RESUMEN

BACKGROUND: The antigen processing machinery (APM) plays a critical role in generating tumor-specific antigens that can be recognized and targeted by the immune system. Proper functioning of APM components is essential for presenting these antigens on the surface of tumor cells, enabling immune detection and destruction. In many cancers, defects in APM can lead to immune evasion, contributing to tumor progression and poor clinical outcomes. However, the status of the APM in sarcomas is not well characterized, limiting the development of effective immunotherapeutic strategies for these patients. METHODS: We investigated 126 patients with 8 types of bone and soft tissue sarcoma operated between 2001-2021. Tissue microarrays mapped 11 specific areas in each case. The presence/absence of APM protein was determined through immunohistochemistry. Bayesian networks were used. RESULTS: All investigated sarcomas had some defects in APM. The least damaged component was HLA Class I subunit ß2-microglobulin and HLA Class II. The proteasome LMP10 subunit was defective in leiomyosarcoma (LMS), myxoid liposarcoma (MLPS), and dedifferentiated liposarcoma (DDLPS), while MHC I transporting unit TAP2 was altered in undifferentiated pleomorphic sarcoma (UPS), gastrointestinal stromal tumor (GIST), and chordoma (CH). Among different neoplastic areas, high-grade areas showed different patterns of expression compared to high lymphocytic infiltrate areas. Heterogeneity at the patient level was also observed. Loss of any APM component was prognostic of distant metastasis (DM) for LMS and DDLPS and of overall survival (OS) for LMS. CONCLUSION: Sarcomas exhibit a high degree of defects in APM components, with differences among histotypes and tumoral areas. The most commonly altered APM components were HLA Class I subunit ß2-microglobulin, HLA Class I subunit α (HC10), and MHC I transporting unit TAP2. The loss of APM components was prognostic of DM and OS and clinically relevant for LMS and DDLPS. This study explores sarcoma molecular mechanisms, enriching personalized therapeutic approaches.

Asunto(s)

Presentación de Antígeno , Sarcoma , Humanos , Sarcoma/inmunología , Sarcoma/patología , Presentación de Antígeno/inmunología , Masculino , Femenino , Persona de Mediana Edad , Anciano , Adulto , Antígenos de Neoplasias/inmunología , Antígenos de Neoplasias/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Microglobulina beta-2/metabolismo , Pronóstico , Miembro 3 de la Subfamilia B de Transportadores de Casetes de Unión a ATP

RESUMEN

The WD repeat-containing protein 4 (WDR4) has repeatedly been associated with primary microcephaly, a condition of impaired brain and skull growth. Often, faulty centrosomes cause microcephaly, yet aberrant cilia may also be involved. Here, we show using a combination of approaches in human fibroblasts, zebrafish embryos and patient-derived cells that WDR4 facilitates cilium formation. Molecularly, we associated WDR4 loss-of-function with increased protein synthesis and concomitant upregulation of proteasomal activity, while ubiquitin precursor pools are reduced. Inhibition of proteasomal activity as well as supplementation with free ubiquitin restored normal ciliogenesis. Proteasome inhibition ameliorated microcephaly phenotypes. Thus, we propose that WDR4 loss-of-function impairs head growth and neurogenesis via aberrant cilia formation, initially caused by disturbed protein and ubiquitin homeostasis.

Asunto(s)

Cilios , Complejo de la Endopetidasa Proteasomal , Ubiquitina , Pez Cebra , Complejo de la Endopetidasa Proteasomal/metabolismo , Humanos , Cilios/metabolismo , Cilios/patología , Animales , Ubiquitina/metabolismo , Microcefalia/genética , Microcefalia/metabolismo , Microcefalia/patología , Fibroblastos/metabolismo , Neurogénesis

RESUMEN

Many cellular processes are regulated by proteasome-mediated protein degradation, including regulation of signaling pathways and gene expression. Among the pathways regulated by the ubiquitin-proteasome system is the Hedgehog pathway and its downstream effectors, the Gli transcription factors. Here we provide evidence that proteasomal activity is necessary for maintaining the activation of the Hedgehog pathway, and this crucial event takes place at the level of Gli proteins. We undertook extensive work to demonstrate the specificity of the observed phenomenon by ruling out the involvement of primary cilium, impaired nuclear import, failed dissociation from Sufu, microtubule stabilization, and stabilization of Gli repressor forms. Moreover, we showed that proteasomal-inhibition-mediated Hedgehog pathway downregulation is not restricted to the NIH-3T3 cell line. We demonstrated, using CRISPR/Ca9 mutagenesis, that neither Gli1, Gli2, nor Gli3 are solely responsible for the Hedgehog pathway downregulation upon proteasome inhibitor treatment, and that Cul3 KO renders the same phenotype. Finally, we report two novel E3 ubiquitin ligases, Btbd9 and Kctd3, known Cul3 interactors, as positive Hedgehog pathway regulators. Our data pave the way for a better understanding of the regulation of gene expression and the Hedgehog signaling pathway.

Asunto(s)

Proteínas Cullin , Proteínas Hedgehog , Complejo de la Endopetidasa Proteasomal , Transducción de Señal , Ubiquitinación , Animales , Complejo de la Endopetidasa Proteasomal/metabolismo , Proteínas Hedgehog/metabolismo , Proteínas Hedgehog/genética , Ratones , Células 3T3 NIH , Proteínas Cullin/metabolismo , Proteínas Cullin/genética , Proteína con Dedos de Zinc GLI1/metabolismo , Proteína con Dedos de Zinc GLI1/genética , Humanos , Regulación de la Expresión Génica

RESUMEN

(1): Atopic dermatitis and psoriasis vulgaris are chronic, inflammatory diseases. Clinical presentation usually leads to a proper diagnosis, but sometimes neither clinical examination nor histopathological evaluation can be conclusive. Therefore, we aimed to build up a novel diagnostic tool and check it for accuracy. The main objective of our work was to differentiate between healthy skin (C), atopic dermatitis (AD) and psoriasis vulgaris (PV) biopsies on the base of involucrin (IVL) and human ß-defensin-2 (hBD-2) concentrations and their mRNA, as well as mRNA for TPP2 and PSMB8. (2): ELISA for IVL and hBD-2 proteins and Real-time PCR for the relative expression of mRNA for: IVL (IVL mRNA), hBD-2 (hBD-2 mRNA), PSMB8 (PSMB8 mRNA) and TPP2 (TPP2 mRNA), isolated from skin biopsies taken from AD and PV patients and healthy volunteers were performed. (3): hBD-2 mRNA and PSMB8 mRNA correlated with some parameters of clinical assessment of inflammatory disease severity. hBD-2 mRNA expression, exclusively, was sufficient to distinguish inflammatory skin biopsies from the healthy ones. (4): hBD-2 mRNA and PSMB8 mRNA analysis were the most valuable parameters in differentiating AD and PV biopsies.

Asunto(s)

Dermatitis Atópica , Psoriasis , ARN Mensajero , Piel , beta-Defensinas , Humanos , Psoriasis/genética , Psoriasis/metabolismo , Psoriasis/patología , Psoriasis/diagnóstico , beta-Defensinas/genética , beta-Defensinas/metabolismo , Dermatitis Atópica/genética , Dermatitis Atópica/metabolismo , Dermatitis Atópica/patología , Dermatitis Atópica/diagnóstico , ARN Mensajero/genética , ARN Mensajero/metabolismo , Biopsia , Femenino , Masculino , Piel/metabolismo , Piel/patología , Adulto , Persona de Mediana Edad , Complejo de la Endopetidasa Proteasomal/genética , Complejo de la Endopetidasa Proteasomal/metabolismo , Diagnóstico Diferencial , Adulto Joven , Adolescente

RESUMEN

Capacitation is an essential post-testicular maturation event endowing spermatozoa with fertilizing capacity within the female reproductive tract, significant for fertility, reproductive health, and contraception. By using a human-relevant large animal model, the domestic boar, this study focuses on furthering our understanding of the involvement of the ubiquitin-proteasome system (UPS) in sperm capacitation. The UPS is a universal, evolutionarily conserved, cellular proteome-wide degradation and recycling machinery, that has been shown to play a significant role in reproduction during the past two decades. Herein, we have used a bottom-up proteomic approach to (i) monitor the capacitation-related changes in the sperm protein levels, and (ii) identify the targets of UPS regulation during sperm capacitation. Spermatozoa were capacitated under proteasomal activity-permissive and inhibiting conditions and extracted sperm proteins were subjected to high-resolution mass spectrometry. We report that 401 individual proteins differed at least two-fold in abundance (P < 0.05) after in vitro capacitation (IVC) and 13 proteins were found significantly different (P < 0.05) between capacitated spermatozoa with proteasomal inhibition compared to the vehicle control. These proteins were associated with biological processes including sperm capacitation, sperm motility, metabolism, binding to zona pellucida, and proteasome-mediated catabolism. Changes in RAB2A, CFAP161, and TTR during IVC were phenotyped by immunocytochemistry, image-based flow cytometry, and Western blotting. We conclude that (i) the sperm proteome is subjected to extensive remodeling during sperm capacitation, and (ii) the UPS has a narrow range of distinct protein substrates during capacitation. This knowledge highlights the importance of the UPS in sperm capacitation and offers opportunities to identify novel pharmacological targets to modulate sperm fertilizing ability for the benefit of human reproductive health, assisted reproductive therapy, and contraception, as well as reproductive management in food animal agriculture.

Asunto(s)

Complejo de la Endopetidasa Proteasomal , Proteómica , Capacitación Espermática , Espermatozoides , Ubiquitina , Capacitación Espermática/fisiología , Animales , Masculino , Complejo de la Endopetidasa Proteasomal/metabolismo , Ubiquitina/metabolismo , Porcinos , Espermatozoides/metabolismo , Espermatozoides/fisiología , Proteómica/métodos , Proteoma/metabolismo

RESUMEN

Spinal muscular atrophy (SMA) is one of the most frequent causes of death in childhood. The disease's molecular basis is deletion or mutations in the SMN1 gene, which produces reduced survival motor neuron protein (SMN) levels. As a result, there is spinal motor neuron degeneration and a large increase in muscle atrophy, in which the ubiquitin-proteasome system (UPS) plays a significant role. In humans, a paralogue of SMN1, SMN2 encodes the truncated protein SMNΔ7. Structural differences between SMN and SMNΔ7 affect the interaction of the proteins with UPS and decrease the stability of the truncated protein. SMN loss affects the general ubiquitination process by lowering the levels of UBA1, one of the main enzymes in the ubiquitination process. We discuss how SMN loss affects both SMN stability and the general ubiquitination process, and how the proteins involved in ubiquitination could be used as future targets for SMA treatment.

Asunto(s)

Atrofia Muscular Espinal , Proteína 1 para la Supervivencia de la Neurona Motora , Ubiquitinación , Humanos , Atrofia Muscular Espinal/metabolismo , Atrofia Muscular Espinal/terapia , Atrofia Muscular Espinal/genética , Atrofia Muscular Espinal/patología , Proteína 1 para la Supervivencia de la Neurona Motora/genética , Proteína 1 para la Supervivencia de la Neurona Motora/metabolismo , Animales , Complejo de la Endopetidasa Proteasomal/metabolismo , Ubiquitina/metabolismo , Proteína 2 para la Supervivencia de la Neurona Motora/genética , Proteína 2 para la Supervivencia de la Neurona Motora/metabolismo , Enzimas Activadoras de Ubiquitina

RESUMEN

Bone homeostasis relies on the delicate balance between osteoblast-mediated bone formation and osteoclast-mediated bone resorption. The casein kinase 2 interacting protein-1 (CKIP-1), a specific CK2α subunit-interacting protein, has been documented as one of the crucial negative regulators of bone formation. CKIP-1 siRNA therapy has constraints that limit its use in clinical applications. Therefore, it is necessary to explore effective targeting strategies for CKIP-1. In this study, we observed an upregulation of CKIP-1 protein expression in the microgravity environment, while its ubiquitination levels decreased. We further investigated the interaction between CKIP-1 and VHL and found that VHL enhanced CKIP-1 degradation through the ubiquitylation-proteasome system (UPS). Additionally, we discovered a small molecule ligand, named C77, through DNA-encoded library (DEL) screening, which binds to CKIP-1 both in vivo and in vitro, as confirmed by Surface Plasmon Resonance (SPR) and the Cellular Thermal shift assay (CETSA), respectively. Our findings demonstrated the potential of VHL and C77 as guiding factors in the development of CKIP-1-based Proteolysis-Targeting Chimeras (PROTACs), which could be future therapeutic interventions in disuse osteoporosis.

Asunto(s)

Osteoporosis , Proteína Supresora de Tumores del Síndrome de Von Hippel-Lindau , Humanos , Ligandos , Osteoporosis/metabolismo , Osteoporosis/tratamiento farmacológico , Osteoporosis/terapia , Proteína Supresora de Tumores del Síndrome de Von Hippel-Lindau/metabolismo , Proteína Supresora de Tumores del Síndrome de Von Hippel-Lindau/genética , Ubiquitinación , Proteínas Portadoras/metabolismo , Proteínas Portadoras/genética , Proteolisis , Animales , Complejo de la Endopetidasa Proteasomal/metabolismo , Unión Proteica , Ratones , Péptidos y Proteínas de Señalización Intracelular

RESUMEN

Proteasome inhibitors (PIs), bortezomib, carfilzomib, and ixazomib, are the first-line treatment for multiple myeloma (MM). They inhibit cytosolic protein degradation in cells, which leads to the accumulation of misfolded and malfunctioned proteins in the cytosol and endoplasmic reticulum, resulting in cell death. Despite being a breakthrough in MM therapy, malignant cells develop resistance to PIs via different mechanisms. Understanding these mechanisms drives research toward new anticancer agents to overcome PI resistance. In this review, we summarize the mechanism of action of PIs and how MM cells adapt to these drugs to develop resistance. Finally, we explore these mechanisms to present strategies to interfere with PI resistance. The strategies include new inhibitors of the ubiquitin-proteasome system, drug efflux inhibitors, autophagy disruption, targeting stress response mechanisms, affecting survival and cell cycle regulators, bone marrow microenvironment modulation, and immunotherapy. We list potential pharmacological targets examined in in vitro, in vivo, and clinical studies. Some of these strategies have already provided clinicians with new anti-MM medications, such as panobinostat and selinexor. We hope that further exploration of the subject will broaden the range of therapeutic options and improve patient outcomes.

Asunto(s)

Resistencia a Antineoplásicos , Mieloma Múltiple , Inhibidores de Proteasoma , Humanos , Inhibidores de Proteasoma/farmacología , Inhibidores de Proteasoma/uso terapéutico , Resistencia a Antineoplásicos/efectos de los fármacos , Mieloma Múltiple/tratamiento farmacológico , Mieloma Múltiple/metabolismo , Mieloma Múltiple/patología , Animales , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Complejo de la Endopetidasa Proteasomal/metabolismo , Autofagia/efectos de los fármacos

RESUMEN

CC chemokine receptor 2 (CCR2) has been linked to many inflammatory and immune diseases, making it a relevant drug target. Yet, all CCR2 antagonists developed so far have failed in clinical trials; thus, novel strategies are needed to target this receptor. Targeted protein degradation represents a novel approach to inhibit protein function by hijacking the cellular degradation machinery, such as the proteasome, to degrade the protein of interest. Here, we aimed to determine the amenability of CCR2 to chemically induced degradation by using a CCR2 fusion protein containing a HaloTag7 and HiBiT tag (CCR2-HaloTag-HiBiT). After characterization of the CCR2 construct, we used luminescence-based assays and immunofluorescence to quantify CCR2 levels, as well as a label-free, phenotypic assay to investigate the functional effect of CCR2 degradation. Treatment with HaloPROTAC3, which selectively degrades HaloTag fusion proteins, led to concentration- and time-dependent degradation of CCR2-HaloTag-HiBiT. HaloPROTAC3 induced degradation via the proteasome, as degradation was fully blocked with proteasomal inhibitors. Finally, functional assays showed that degradation of CCR2-HaloTag-HiBiT leads to a reduced functional response after agonist stimulation. Overall, our results indicate that CCR2 is amenable to targeted degradation, paving the way for the future development of CCR2 chemical degraders.

Asunto(s)

Complejo de la Endopetidasa Proteasomal , Proteolisis , Receptores CCR2 , Receptores CCR2/metabolismo , Humanos , Proteolisis/efectos de los fármacos , Complejo de la Endopetidasa Proteasomal/metabolismo , Células HEK293 , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Recombinantes de Fusión/genética

RESUMEN

Embryonic stem cells (ESCs) are remarkable for the high activity level of ubiquitin-proteasome system-the molecular machinery of protein degradation in the cell. Various forms of the proteasome complexes comprising different subunits and interacting regulators are responsible for the substrate selectivity and degradation. Immunoproteasomes are amongst these forms which play an important role in antigen presentation; however, a body of recent evidence suggests their functions in pluripotent stem cells. Previous studies have established three consecutive phases of pluripotency, featured by epiblast cells and their cultured counterparts: naïve, formative, and primed phase. In this work, we report that immunoproteasomes and their chaperone co-regulators are suppressed in the naïve state but are readily upregulated in the formative phase of the pluripotency continuum, featured by epiblast-like cells (EpiLCs). Our data lay ground for the further investigation of the biological functions of immunoproteasome in the regulation of proteostasis during early mammalian development.

Asunto(s)

Complejo de la Endopetidasa Proteasomal , Animales , Complejo de la Endopetidasa Proteasomal/metabolismo , Ratones , Células Madre Pluripotentes/metabolismo , Células Madre Pluripotentes/citología , Diferenciación Celular , Estratos Germinativos/metabolismo , Células Madre Embrionarias de Ratones/metabolismo