RESUMO
Through a network of progressively maturing vesicles, the endosomal system connects the cell's interior with extracellular space. Intriguingly, this network exhibits a bilateral architecture, comprised of a relatively immobile perinuclear vesicle "cloud" and a highly dynamic peripheral contingent. How this spatiotemporal organization is achieved and what function(s) it curates is unclear. Here, we reveal the endoplasmic reticulum (ER)-located ubiquitin ligase Ring finger protein 26 (RNF26) as the global architect of the entire endosomal system, including the trans-Golgi network (TGN). To specify perinuclear vesicle coordinates, catalytically competent RNF26 recruits and ubiquitinates the scaffold p62/sequestosome 1 (p62/SQSTM1), in turn attracting ubiquitin-binding domains (UBDs) of various vesicle adaptors. Consequently, RNF26 restrains fast transport of diverse vesicles through a common molecular mechanism operating at the ER membrane, until the deubiquitinating enzyme USP15 opposes RNF26 activity to allow vesicle release into the cell's periphery. By drawing the endosomal system's architecture, RNF26 orchestrates endosomal maturation and trafficking of cargoes, including signaling receptors, in space and time.
Assuntos
Retículo Endoplasmático/metabolismo , Endossomos/metabolismo , Membranas Intracelulares/metabolismo , Proteínas de Neoplasias/metabolismo , Linhagem Celular Tumoral , Células Dendríticas/citologia , Células Dendríticas/metabolismo , Humanos , Macrófagos/citologia , Macrófagos/metabolismo , Proteína Sequestossoma-1/metabolismo , Vesículas Transportadoras/metabolismo , Proteases Específicas de Ubiquitina/metabolismoRESUMO
MHC class I (MHC-I) molecules are critical for CD8+ T cell responses to viral infections and malignant cells, and tumors can downregulate MHC-I expression to promote immune evasion. In this study, using a genome-wide CRISPR screen on a human melanoma cell line, we identified the polycomb repressive complex 1 (PRC1) subunit PCGF1 and the deubiquitinating enzyme BAP1 as opposite regulators of MHC-I transcription. PCGF1 facilitates deposition of ubiquitin at H2AK119 at the MHC-I promoters to silence MHC-I, whereas BAP1 removes this modification to restore MHC-I expression. PCGF1 is widely expressed in tumors and its depletion increased MHC-I expression in multiple tumor lines, including MHC-Ilow tumors. In cells characterized by poor MHC-I expression, PRC1 and PRC2 act in parallel to impinge low transcription. However, PCGF1 depletion was sufficient to increase MHC-I expression and restore T cell-mediated killing of the tumor cells. Taken together, our data provide an additional layer of regulation of MHC-I expression in tumors: epigenetic silencing by PRC1 subunit PCGF1.
Assuntos
Histonas , Ubiquitina , Humanos , Histonas/metabolismo , Ubiquitina/metabolismo , Epigênese Genética , Complexo Repressor Polycomb 1/metabolismo , Linhagem Celular , Proteínas Supressoras de Tumor/metabolismo , Ubiquitina Tiolesterase/genética , Ubiquitina Tiolesterase/metabolismoRESUMO
The endolysosomal system fulfils a myriad of cellular functions predicated on regulated membrane identity progressions, collectively termed maturation. Mature or "late" endosomes are designated by small membrane-bound GTPases Rab7 and Arl8b, which can either operate independently or collaborate to form a joint compartment. Whether, and how, Rab7 and Arl8b resolve this hybrid identity compartment to regain functional autonomy is unknown. Here, we report that Arl8b employs its effector SKIP to instigate inactivation and removal of Rab7 from select membranes. We find that SKIP interacts with Rab7 and functions as its negative effector, delivering the cognate GAP, TBC1D15. Recruitment of TBC1D15 to SKIP occurs via the HOPS complex, whose assembly is facilitated by contacts between Rab7 and the KMI motif of SKIP. Consequently, SKIP mediates reinstatement of single identity Arl8b sub-compartment through an ordered Rab7-to-Arl8b handover, and, together with Rab7's positive effector RILP, enforces spatial, temporal and morphological compartmentalization of endolysosomal organelles.
Assuntos
Fatores de Ribosilação do ADP/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Ativadoras de GTPase/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Fatores de Ribosilação do ADP/genética , Proteínas Adaptadoras de Transdução de Sinal/genética , Compartimento Celular , Endossomos/metabolismo , Proteínas Ativadoras de GTPase/genética , Células HEK293 , Humanos , Lisossomos/metabolismo , Ligação Proteica , Transporte Proteico , Proteínas rab de Ligação ao GTP/genética , proteínas de unión al GTP Rab7RESUMO
The PD-L1/2-PD-1 immune checkpoint is essential for the proper induction of peripheral tolerance and limits autoimmunity, whereas tumor cells exploit their expression to promote immune evasion. Many different cell types express PD-L1/2, either constitutively or upon stimulation, but the factors driving this expression are often poorly defined. In this study, using genome-wide CRISPR activation screening, we identified three factors that upregulate PD-L1 expression: GATA2, MBD6, and transcription cofactor vestigial-like protein 3 (VGLL3). VGLL3 acts as a transcriptional regulator, and its expression induced PD-L1 in many different cell types. Conversely, loss of VGLL3 impaired IFN-γ-induced PD-L1/2 expression in human keratinocytes. Mechanistically, by performing a second screen to identify proteins acting in concert with VGLL3, we found that VGLL3 forms a complex with TEAD1 and RUNX1/3 to drive expression of PD-L1/2. Collectively, our work identified a new transcriptional complex controlling PD-L1/2 expression and suggests that VGLL3, in addition to its known role in the expression of proinflammatory genes, can balance inflammation by upregulating the anti-inflammatory factors PD-L1 and PD-L2.
Assuntos
Antígeno B7-H1 , Receptor de Morte Celular Programada 1 , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Subunidade alfa 2 de Fator de Ligação ao Core/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Humanos , Evasão da Resposta Imune , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Receptor de Morte Celular Programada 1/genética , Fatores de Transcrição de Domínio TEA , Fatores de Transcrição/genéticaRESUMO
There is growing interest in HLA-E-restricted T-cell responses as a possible novel, highly conserved, vaccination targets in the context of infectious and malignant diseases. The developing field of HLA multimers for the detection and study of peptide-specific T cells has allowed the in-depth study of TCR repertoires and molecular requirements for efficient antigen presentation and T-cell activation. In this study, we developed a method for efficient peptide thermal exchange on HLA-E monomers and multimers allowing the high-throughput production of HLA-E multimers. We optimized the thermal-mediated peptide exchange, and flow cytometry staining conditions for the detection of TCR and NKG2A/CD94 receptors, showing that this novel approach can be used for high-throughput identification and analysis of HLA-E-binding peptides which could be involved in T-cell and NK cell-mediated immune responses. Importantly, our analysis of NKG2A/CD94 interaction in the presence of modified peptides led to new molecular insights governing the interaction of HLA-E with this receptor. In particular, our results reveal that interactions of HLA-E with NKG2A/CD94 and the TCR involve different residues. Altogether, we present a novel HLA-E multimer technology based on thermal-mediated peptide exchange allowing us to investigate the molecular requirements for HLA-E/peptide interaction with its receptors.
Assuntos
Antígenos de Histocompatibilidade Classe I , Células Matadoras Naturais , Ligação Proteica , Antígenos de Histocompatibilidade Classe I/metabolismo , Peptídeos , Receptores de Antígenos de Linfócitos T , Subfamília D de Receptores Semelhantes a Lectina de Células NK/química , Subfamília D de Receptores Semelhantes a Lectina de Células NK/metabolismo , Subfamília C de Receptores Semelhantes a Lectina de Células NK , Antígenos HLA-ERESUMO
The endoplasmic reticulum (ER) is the largest organelle contacting virtually every other organelle for information exchange and control of processes such as transport, fusion, and fission. Here, we studied the role of the other organelles on ER network architecture in the cell periphery. We show that the co-migration of the ER with other organelles, called ER hitchhiking facilitated by late endosomes and lysosomes is a major mechanism controlling ER network architecture. When hitchhiking occurs, emerging ER structures may fuse with the existing ER tubules to alter the local ER architecture. This couples late endosomal/lysosomal positioning and mobility to ER network architecture. Conditions restricting late endosomal movement-including cell starvation-or the depletion of tether proteins that link the ER to late endosomes reduce ER dynamics and limit the complexity of the peripheral ER network architecture. This indicates that among many factors, the ER is controlled by late endosomal movement resulting in an alteration of the ER network architecture.
Assuntos
Retículo Endoplasmático , Endossomos , Transporte Biológico , Retículo Endoplasmático/metabolismo , Endossomos/metabolismo , Lisossomos/metabolismoRESUMO
The cytokine interferon-γ (IFNγ) can induce expression of MHC class II (MHCII) on many different cell types, leading to antigen presentation to CD4+ T cells and immune activation. This has also been linked to anti-tumour immunity and graft-versus-host disease. The extent of MHCII upregulation by IFNγ is cell type-dependent and under extensive control of epigenetic regulators and signalling pathways. Here, we identify novel genetic and chemical factors that control this form of MHCII expression. Loss of the oxidative stress sensor Keap1, autophagy adaptor p62/SQSTM1, ubiquitin E3-ligase Cullin-3 and chromatin remodeller BPTF impair IFNγ-mediated MHCII expression. A similar phenotype is observed for arsenite, an oxidative stressor. Effects of the latter can be reversed by the inhibition of HDAC1/2, linking oxidative stress conditions to epigenetic control of MHCII expression. Furthermore, dimethyl fumarate, an antioxidant used for the treatment of several autoimmune diseases, impairs the IFNγ response by manipulating transcriptional control of MHCII We describe novel pathways and drugs related to oxidative conditions in cells impacting on IFNγ-mediated MHCII expression, which provide a molecular basis for the understanding of MHCII-associated diseases.
Assuntos
Arsenitos/farmacologia , Antígenos de Histocompatibilidade Classe II/metabolismo , Interferon gama/metabolismo , Estresse Oxidativo , Imunidade Adaptativa , Apresentação de Antígeno , Antígenos Nucleares/metabolismo , Antioxidantes/farmacologia , Proteínas Culina/metabolismo , Fumarato de Dimetilo/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Células HeLa , Antígenos de Histocompatibilidade Classe II/genética , Antígenos de Histocompatibilidade Classe II/imunologia , Humanos , Interferon gama/imunologia , Interferon gama/farmacologia , Proteína 1 Associada a ECH Semelhante a Kelch/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Proteína Sequestossoma-1/metabolismo , Transdução de Sinais/efeitos dos fármacos , Fatores de Transcrição/metabolismo , Regulação para Cima/efeitos dos fármacosRESUMO
Infected or transformed cells must present peptides derived from endogenous proteins on MHC class I molecules to be recognized and targeted for elimination by Ag-specific cytotoxic T cells. In the first step of peptide generation, proteins are degraded by the proteasome. In this study, we investigated the role of the ubiquitin-specific protease 14 (Usp14), a proteasome-associated deubiquitinase, in direct Ag presentation using a ligand-stabilized model protein expressed as a self-antigen. Chemical inhibition of Usp14 diminished direct presentation of the model antigenic peptide, and the effect was especially pronounced when presentation was restricted to the defective ribosomal product (DRiP) form of the protein. Additionally, presentation specifically from DRiP Ags was diminished by expression of a catalytically inactive form of Usp14. Usp14 inhibition did not appreciably alter protein synthesis and only partially delayed protein degradation as measured by a slight increase in the half-life of the model protein when its degradation was induced. Taken together, these data indicate that functional Usp14 enhances direct Ag presentation, preferentially of DRiP-derived peptides, suggesting that the processing of DRiPs is in some ways different from other forms of Ag.
Assuntos
Apresentação de Antígeno/imunologia , Antígenos de Histocompatibilidade Classe I/imunologia , Peptídeos/imunologia , Linfócitos T Citotóxicos/imunologia , Ubiquitina Tiolesterase/antagonistas & inibidores , Animais , Apresentação de Antígeno/efeitos dos fármacos , Linhagem Celular Tumoral , Camundongos , Biossíntese de Proteínas , Proteólise , Pirróis/farmacologia , Pirrolidinas/farmacologiaRESUMO
The interpretation of high-dimensional structure-activity data sets in drug discovery to predict ligand-protein interaction landscapes is a challenging task. Here we present Drug Discovery Maps (DDM), a machine learning model that maps the activity profile of compounds across an entire protein family, as illustrated here for the kinase family. DDM is based on the t-distributed stochastic neighbor embedding (t-SNE) algorithm to generate a visualization of molecular and biological similarity. DDM maps chemical and target space and predicts the activities of novel kinase inhibitors across the kinome. The model was validated using independent data sets and in a prospective experimental setting, where DDM predicted new inhibitors for FMS-like tyrosine kinase 3 (FLT3), a therapeutic target for the treatment of acute myeloid leukemia. Compounds were resynthesized, yielding highly potent, cellularly active FLT3 inhibitors. Biochemical assays confirmed most of the predicted off-targets. DDM is further unique in that it is completely open-source and available as a ready-to-use executable to facilitate broad and easy adoption.
Assuntos
Descoberta de Drogas/métodos , Inibidores de Proteínas Quinases/metabolismo , Proteínas Quinases/metabolismo , Aprendizado de Máquina , Modelos Moleculares , Ligação Proteica , Conformação Proteica , Proteínas Quinases/química , Tirosina Quinase 3 Semelhante a fms/antagonistas & inibidores , Tirosina Quinase 3 Semelhante a fms/química , Tirosina Quinase 3 Semelhante a fms/metabolismoRESUMO
Acute myeloid leukemia (AML) is characterized by fast progression and low survival rates, in which Fms-like tyrosine kinase 3 (FLT3) receptor mutations have been identified as a driver mutation in cancer progression in a subgroup of AML patients. Clinical trials have shown emergence of drug resistant mutants, emphasizing the ongoing need for new chemical matter to enable the treatment of this disease. Here, we present the discovery and topological structure-activity relationship (SAR) study of analogs of isoquinolinesulfonamide H-89, a well-known PKA inhibitor, as FLT3 inhibitors. Surprisingly, we found that the SAR was not consistent with the observed binding mode of H-89 in PKA. Matched molecular pair analysis resulted in the identification of highly active sub-nanomolar azaindoles as novel FLT3-inhibitors. Structure based modelling using the FLT3 crystal structure suggested an alternative, flipped binding orientation of the new inhibitors.
Assuntos
Compostos Aza/química , Indóis/química , Inibidores de Proteínas Quinases/química , Tirosina Quinase 3 Semelhante a fms/antagonistas & inibidores , Compostos Aza/síntese química , Compostos Aza/metabolismo , Sítios de Ligação , Humanos , Indóis/síntese química , Indóis/metabolismo , Simulação de Acoplamento Molecular , Estrutura Molecular , Ligação Proteica , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/metabolismo , Relação Estrutura-Atividade , Tirosina Quinase 3 Semelhante a fms/química , Tirosina Quinase 3 Semelhante a fms/metabolismoRESUMO
Endosomes shuttle select cargoes between cellular compartments and, in doing so, maintain intracellular homeostasis and enable interactions with the extracellular space. Directionality of endosomal transport critically impinges on cargo fate, as retrograde (microtubule minus-end directed) traffic delivers vesicle contents to the lysosome for proteolysis, while the opposing anterograde (plus-end directed) movement promotes recycling and secretion. Intriguingly, the endoplasmic reticulum (ER) is emerging as a key player in spatiotemporal control of late endosome and lysosome transport, through the establishment of physical contacts with these organelles. Earlier studies have described how minus-end-directed motor proteins become discharged from vesicles engaged at such contact sites. Now, Raiborg et al. implicate ER-mediated interactions, induced by protrudin, in loading plus-end-directed motor kinesin-1 onto endosomes, thereby stimulating their transport toward the cell's periphery. In this review, we recast the prevailing concepts on bidirectional late endosome transport and discuss the emerging paradigm of inter-compartmental regulation from the ER-endosome interface viewpoint.
Assuntos
Transporte Biológico/fisiologia , Retículo Endoplasmático/fisiologia , Endossomos/fisiologia , Retículo Endoplasmático/metabolismo , Endossomos/metabolismo , Cinesinas/metabolismo , Lisossomos/metabolismo , Lisossomos/fisiologiaRESUMO
Efficacy of chemotherapy in the treatment of distinct malignancies is often hampered by drug resistance arising in the tumor. Understanding the molecular basis of drug resistance and translating this knowledge into personalized treatment decisions can enhance therapeutic efficacy and even curative outcome. Over the years, multiple drug resistance mechanisms have been identified that enable tumors to cope with the damage instigated by a specific drug or group of anti-tumor agents. Here we provide an overview of the molecular pathways leading to resistance against conventional anti-cancer drugs, with emphasis on the utility of these pathways for rational selection of treatments for individual cancer patients. We further complement the review by discussing the pitfalls and difficulties in translating these findings into novel treatment strategies for cancer patients.
Assuntos
Antineoplásicos/uso terapêutico , Biomarcadores Tumorais/genética , Resistencia a Medicamentos Antineoplásicos/genética , Regulação Neoplásica da Expressão Gênica , Proteínas de Neoplasias/antagonistas & inibidores , Neoplasias/tratamento farmacológico , Apoptose/efeitos dos fármacos , Biomarcadores Tumorais/metabolismo , Cisplatino/uso terapêutico , Doxorrubicina/uso terapêutico , Resistência a Múltiplos Medicamentos/efeitos dos fármacos , Resistência a Múltiplos Medicamentos/genética , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Humanos , Terapia de Alvo Molecular , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/patologia , Medicina de Precisão , Transdução de SinaisRESUMO
Trafficking of cargo through the endosomal system depends on endosomal fusion events mediated by SNARE proteins, Rab-GTPases, and multisubunit tethering complexes. The CORVET and HOPS tethering complexes, respectively, regulate early and late endosomal tethering and have been characterized in detail in yeast where their sequential membrane targeting and assembly is well understood. Mammalian CORVET and HOPS subunits significantly differ from their yeast homologues, and novel proteins with high homology to CORVET/HOPS subunits have evolved. However, an analysis of the molecular interactions between these subunits in mammals is lacking. Here, we provide a detailed analysis of interactions within the mammalian CORVET and HOPS as well as an additional endosomal-targeting complex (VIPAS39-VPS33B) that does not exist in yeast. We show that core interactions within CORVET and HOPS are largely conserved but that the membrane-targeting module in HOPS has significantly changed to accommodate binding to mammalian-specific RAB7 interacting lysosomal protein (RILP). Arthrogryposis-renal dysfunction-cholestasis (ARC) syndrome-associated mutations in VPS33B selectively disrupt recruitment to late endosomes by RILP or binding to its partner VIPAS39. Within the shared core of CORVET/HOPS, we find that VPS11 acts as a molecular switch that binds either CORVET-specific TGFBRAP1 or HOPS-specific VPS39/RILP thereby allowing selective targeting of these tethering complexes to early or late endosomes to time fusion events in the endo/lysosomal pathway.
Assuntos
Endossomos/metabolismo , Complexos Multiproteicos/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Substituição de Aminoácidos , Artrogripose/genética , Artrogripose/metabolismo , Artrogripose/patologia , Proteínas Relacionadas à Autofagia , Linhagem Celular , Colestase/genética , Colestase/metabolismo , Colestase/patologia , Endossomos/genética , Endossomos/patologia , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Complexos Multiproteicos/genética , Mutação de Sentido Incorreto , Insuficiência Renal/genética , Insuficiência Renal/metabolismo , Insuficiência Renal/patologia , Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/metabolismoRESUMO
Human leukocyte antigen (HLA) class-I molecules present fragments of the cellular proteome to the T cell receptor (TCR) of cytotoxic T cells to control infectious diseases and cancer. The large number of combinations of HLA class-I allotypes and peptides allows for highly specific and dedicated low-affinity interactions to a diverse array of TCRs and natural killer (NK) cell receptors. Whether the divergent HLA class-I peptide complex is exclusive for interactions with these proteins is unknown. Using genome-wide CRISPR-Cas9 activation and knockout screens, we identified peptide-specific HLA-C∗07 combinations that can interact with the surface molecules CD55 and heparan sulfate. These interactions closely resemble the HLA class-I interaction with the TCR regarding both the affinity range and the specificity of the peptide and HLA allele. These findings indicate that various proteins can specifically bind HLA class-I peptide complexes due to their polymorphic nature, which suggests there are more interactions like the ones we describe here.
RESUMO
The genetic circuits that allow cancer cells to evade immune killing via epithelial mesenchymal plasticity remain poorly understood. Here, we showed that mesenchymal-like (Mes) KPC3 pancreatic cancer cells were more resistant to cytotoxic T lymphocyte (CTL)-mediated killing than the parental epithelial-like (Epi) cells and used parallel genome-wide CRISPR screens to assess the molecular underpinnings of this difference. Core CTL-evasion genes (such as IFN-γ pathway components) were clearly evident in both types. Moreover, we identified and validated multiple Mes-specific regulators of cytotoxicity, such as Egfr and Mfge8. Both genes were significantly higher expressed in Mes cancer cells, and their depletion sensitized Mes cancer cells to CTL-mediated killing. Notably, Mes cancer cells secreted more Mfge8 to inhibit proliferation of CD8+ T cells and production of IFN-γ and TNFα. Clinically, increased Egfr and Mfge8 expression was correlated with a worse prognosis. Thus, Mes cancer cells use Egfr-mediated intrinsic and Mfge8-mediated extrinsic mechanisms to facilitate immune escape from CD8+ T cells.
Assuntos
Linfócitos T CD8-Positivos , Neoplasias Pancreáticas , Humanos , Transição Epitelial-Mesenquimal/genética , Evasão da Resposta Imune/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Receptores ErbB/genética , Linhagem Celular Tumoral , Neoplasias Pancreáticas/genéticaRESUMO
The immune checkpoint NKG2A/CD94 is a promising target for cancer immunotherapy, and its ligand major histocompatibility complex E (MHC-E) is frequently upregulated in cancer. NKG2A/CD94-mediated inhibition of lymphocytes depends on the presence of specific leader peptides in MHC-E, but when and where they are presented in situ is unknown. We apply a nanobody specific for the Qdm/Qa-1b complex, the NKG2A/CD94 ligand in mouse, and find that presentation of Qdm peptide depends on every member of the endoplasmic reticulum-resident peptide loading complex. With a turnover rate of 30 min, the Qdm peptide reflects antigen processing capacity in real time. Remarkably, Qdm/Qa-1b complexes require inflammatory signals for surface expression in situ, despite the broad presence of Qa-1b molecules in homeostasis. Furthermore, we identify LILRB1 as a functional inhibition receptor for MHC-E in steady state. These data provide a molecular understanding of NKG2A blockade in immunotherapy and assign MHC-E as a convergent ligand for multiple immune checkpoints.
Assuntos
Antígenos de Histocompatibilidade Classe I , Neoplasias , Camundongos , Animais , Antígenos de Histocompatibilidade Classe I/metabolismo , Receptor B1 de Leucócitos Semelhante a Imunoglobulina/metabolismo , Células Matadoras Naturais , Ligantes , Peptídeos/metabolismo , Neoplasias/metabolismo , Subfamília C de Receptores Semelhantes a Lectina de Células NK/metabolismoRESUMO
Epitope-tagged active-site-directed probes are widely used to visualize the activity of deubiquitinases (DUBs) in cell extracts, to investigate the specificity and potency of small-molecule DUB inhibitors, and to isolate and identify DUBs by mass spectrometry. With DUBs arising as novel potential drug targets, probes are required that can be produced in sufficient amounts and to meet the specific needs of a given experiment. The established method for the generation of DUB probes makes use of labor-intensive intein-based methods that have inherent limitations concerning the incorporation of unnatural amino acids and the amount of material that can be obtained. Here, we describe the total chemical synthesis of active-site-directed probes and their application to activity-based profiling and identification of functional DUBs. This synthetic methodology allowed the easy incorporation of desired tags for specific applications, for example, fluorescent reporters, handles for immunoprecipitation or affinity pull-down, and cleavable linkers. Additionally, the synthetic method can be scaled up to provide significant amounts of probe. Fluorescent ubiquitin probes allowed faster, in-gel detection of active DUBs, as compared to (immuno)blotting procedures. A biotinylated probe holding a photocleavable linker enabled the affinity pull-down and subsequent mild, photorelease of DUBs. Also, DUB activity levels were monitored in response to overexpression or knockdown, and to inhibition by small molecules. Furthermore, fluorescent probes revealed differential DUB activity profiles in a panel of lung and prostate cancer cells.
Assuntos
Endopeptidases/metabolismo , Corantes Fluorescentes/química , Ubiquitina/química , Ubiquitina/metabolismo , Ubiquitinação , Biotina/química , Biotinilação , Domínio Catalítico , Linhagem Celular Tumoral , Corantes Fluorescentes/síntese química , Corantes Fluorescentes/metabolismo , Humanos , Técnicas de Síntese em Fase SólidaRESUMO
Membrane contact sites (MCS) are intracellular regions where two organelles come closer to exchange information and material. The majority of the endoplasmic reticulum (ER) MCS are attributed to the ER-localized tether proteins VAPA, VAPB, and MOSPD2. These recruit other proteins to the ER by interacting with their FFAT motifs. Here, we describe MOSPD1 and MOSPD3 as ER-localized tethers interacting with FFAT motif-containing proteins. Using BioID, we identify proteins interacting with VAP and MOSPD proteins and find that MOSPD1 and MOSPD3 prefer unconventional FFAT-related FFNT (two phenylalanines [FF] in a neutral tract) motifs. Moreover, VAPA/VAPB/MOSPD2 and MOSPD1/MOSPD3 assemble into two separate ER-resident complexes to interact with FFAT and FFNT motifs, respectively. Because of their ability to interact with FFNT motifs, MOSPD1 and MOSPD3 could form MCS between the ER and other organelles. Collectively, these findings expand the VAP family of proteins and highlight two separate complexes in control of interactions between intracellular compartments.
Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Membrana/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Motivos de Aminoácidos/genética , Linhagem Celular , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Membrana Celular/fisiologia , Retículo Endoplasmático/genética , Retículo Endoplasmático/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/fisiologia , Proteínas de Membrana/fisiologia , Membranas Mitocondriais/metabolismo , Ligação Proteica/genética , Domínios e Motivos de Interação entre Proteínas/genética , Domínios e Motivos de Interação entre Proteínas/fisiologia , Mapeamento de Interação de Proteínas/métodos , Proteínas de Transporte Vesicular/fisiologiaRESUMO
C9orf82 protein, or conserved anti-apoptotic protein 1 or caspase activity and apoptosis inhibitor 1 (CAAP1) has been implicated as a negative regulator of the intrinsic apoptosis pathway by modulating caspase expression and activity. In contrast, an independent genome wide screen for factors capable of driving drug resistance to the topoisomerase II (Topo II) poisons doxorubicin and etoposide, implicated a role for the nuclear protein C9orf82 in delaying DSBs repair downstream of Topo II, hereby sensitizing cells to DSB induced apoptosis. To determine its function in a genetically defined setting in vivo and ex vivo, we here employed CRISPR/Cas9 technology in zygotes to generate a C9orf82 knockout mouse model. C9orf82ko/ko mice were born at a Mendelian ratio and did not display any overt macroscopic or histological abnormalities. DSBs repair dependent processes like lymphocyte development and class switch recombination (CSR) appeared normal, arguing against a link between the C9orf82 encoded protein and V(D)J recombination or CSR. Most relevant, primary pre-B cell cultures and Tp53 transformed mouse embryo fibroblasts (MEFs) derived from C9orf82ko/ko E14.5 and wild type embryos displayed comparable sensitivity to a number of DNA lesions, including DSBs breaks induced by the topoisomerase II inhibitors, etoposide and doxorubicin. Likewise, the kinetics of γH2AX formation and resolution in response to etoposide of C9orf82 protein proficient, deficient and overexpressing MEFs were indistinguishable. These data argue against a direct role of C9orf82 protein in delaying repair of Topo II generated DSBs and regulating apoptosis. The genetically defined systems generated in this study will be of value to determine the actual function of C9orf82 protein.
Assuntos
Proteínas Reguladoras de Apoptose/fisiologia , Quebras de DNA de Cadeia Dupla , DNA Topoisomerases Tipo II/metabolismo , Animais , Apoptose , Proteínas Reguladoras de Apoptose/genética , Linfócitos B/citologia , Linfócitos B/metabolismo , Sistemas CRISPR-Cas , Caspase 3/metabolismo , Proliferação de Células , Células Cultivadas , Dano ao DNA , Reparo do DNA , Switching de Imunoglobulina , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Linfócitos T/citologia , Linfócitos T/metabolismoRESUMO
The endosomal system is a highly dynamic multifunctional organelle, whose complexity is regulated in part by reversible ubiquitylation. Despite the wide-ranging influence of ubiquitin in endosomal processes, relatively few enzymes utilizing ubiquitin have been described to control endosome integrity and function. Here we reveal the deubiquitylating enzyme (DUB) ubiquitin-specific protease 32 (USP32) as a powerful player in this context. Loss of USP32 inhibits late endosome (LE) transport and recycling of LE cargos, resulting in dispersion and swelling of the late compartment. Using SILAC-based ubiquitome profiling we identify the small GTPase Rab7-the logistical centerpiece of LE biology-as a substrate of USP32. Mechanistic studies reveal that LE transport effector RILP prefers ubiquitylation-deficient Rab7, while retromer-mediated LE recycling benefits from an intact cycle of Rab7 ubiquitylation. Collectively, our observations suggest that reversible ubiquitylation helps switch Rab7 between its various functions, thereby maintaining global spatiotemporal order in the endosomal system.