RESUMO
In diderm bacteria, the Lol pathway canonically mediates the periplasmic transport of lipoproteins from the inner membrane (IM) to the outer membrane (OM) and therefore plays an essential role in bacterial envelope homeostasis. After extrusion of modified lipoproteins from the IM via the LolCDE complex, the periplasmic chaperone LolA carries lipoproteins through the periplasm and transfers them to the OM lipoprotein insertase LolB, itself a lipoprotein with a LolA-like fold. Yet, LolB homologs appear restricted to γ-proteobacteria and are missing from spirochetes like the tick-borne Lyme disease pathogen Borrelia burgdorferi, suggesting a different hand-off mechanism at the OM. Here, we solved the crystal structure of the B. burgdorferi LolA homolog BB0346 (LolABb) at 1.9 Å resolution. We identified multiple structural deviations in comparative analyses to other solved LolA structures, particularly a unique LolB-like protruding loop domain. LolABb failed to complement an Escherichia coli lolA knockout, even after codon optimization, signal I peptide adaptation, and a C-terminal chimerization which had allowed for complementation with an α-proteobacterial LolA. Analysis of a conditional B. burgdorferi lolA knockout strain indicated that LolABb was essential for growth. Intriguingly, protein localization assays indicated that initial depletion of LolABb led to an emerging mislocalization of both IM and periplasmic OM lipoproteins, but not surface lipoproteins. Together, these findings further support the presence of two separate primary secretion pathways for periplasmic and surface OM lipoproteins in B. burgdorferi and suggest that the distinct structural features of LolABb allow it to function in a unique LolB-deficient lipoprotein sorting system.
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Borrelia spirochetes are unique among diderm bacteria in their lack of lipopolysaccharide (LPS) in the outer membrane (OM) and their abundance of surface-exposed lipoproteins with major roles in transmission, virulence, and pathogenesis. Despite their importance, little is known about how surface lipoproteins are translocated through the periplasm and the OM. Here, we characterized Borrelia burgdorferi BB0838, a distant homolog of the OM LPS assembly protein LptD. Using a CRISPR interference approach, we showed that BB0838 is required for cell growth and envelope stability. Upon BB0838 knockdown, surface lipoprotein OspA was retained in the inner leaflet of the OM, as determined by its inaccessibility to in situ proteolysis but its presence in OM vesicles. The topology of the OM porin/adhesin P66 remained unaffected. Quantitative mass spectrometry of the B. burgdorferi membrane-associated proteome confirmed the selective periplasmic retention of surface lipoproteins under BB0838 knockdown conditions. Additional analysis identified a single in situ protease-accessible BB0838 peptide that mapped to a predicted ß-barrel surface loop. Alphafold Multimer modeled a B. burgdorferi LptB2 FGCAD complex spanning the periplasm. Together, this suggests that BB0838/LptDBb facilitates the essential terminal step in spirochetal surface lipoprotein secretion, using an orthologous OM component of a pathway that secretes LPS in proteobacteria.
Assuntos
Borrelia burgdorferi , Borrelia burgdorferi/metabolismo , Proteínas da Membrana Bacteriana Externa/metabolismo , Lipopolissacarídeos/metabolismo , Bactérias/metabolismo , Lipoproteínas/metabolismoRESUMO
Ribosomal RNA (rRNA) transcription by RNA polymerase I (Pol I) is a critical rate-limiting step in ribosome biogenesis, which is essential for cell survival. Despite its global function, disruptions in ribosome biogenesis cause tissue-specific birth defects called ribosomopathies, which frequently affect craniofacial development. Here, we describe a cellular and molecular mechanism underlying the susceptibility of craniofacial development to disruptions in Pol I transcription. We show that Pol I subunits are highly expressed in the neuroepithelium and neural crest cells (NCCs), which generate most of the craniofacial skeleton. High expression of Pol I subunits sustains elevated rRNA transcription in NCC progenitors, which supports their high tissue-specific levels of protein translation, but also makes NCCs particularly sensitive to rRNA synthesis defects. Consistent with this model, NCC-specific deletion of Pol I subunits Polr1a, Polr1c, and associated factor Tcof1 in mice cell-autonomously diminishes rRNA synthesis, which leads to p53 protein accumulation, resulting in NCC apoptosis and craniofacial anomalies. Furthermore, compound mutations in Pol I subunits and associated factors specifically exacerbate the craniofacial anomalies characteristic of the ribosomopathies Treacher Collins syndrome and Acrofacial Dysostosis-Cincinnati type. Mechanistically, we demonstrate that diminished rRNA synthesis causes an imbalance between rRNA and ribosomal proteins. This leads to increased binding of ribosomal proteins Rpl5 and Rpl11 to Mdm2 and concomitantly diminished binding between Mdm2 and p53. Altogether, our results demonstrate a dynamic spatiotemporal requirement for rRNA transcription during mammalian cranial NCC development and corresponding tissue-specific threshold sensitivities to disruptions in rRNA transcription in the pathogenesis of congenital craniofacial disorders.
Assuntos
Anormalidades Craniofaciais , RNA Polimerase I , RNA Ribossômico , Proteínas Ribossômicas , Crânio , Transcrição Gênica , Animais , Anormalidades Craniofaciais/genética , Disostose Mandibulofacial/genética , Camundongos , Crista Neural/embriologia , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , RNA Polimerase I/metabolismo , RNA Ribossômico/genética , Proteínas Ribossômicas/metabolismo , Crânio/embriologia , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismoRESUMO
Changes in available nutrients are inevitable events for most living organisms. Upon nutritional stress, several signaling pathways cooperate to change the transcription program through chromatin regulation to rewire cellular metabolism. In budding yeast, histone H3 threonine 11 phosphorylation (H3pT11) acts as a marker of low glucose stress and regulates the transcription of nutritional stress-responsive genes. Understanding how this histone modification 'senses' external glucose changes remains elusive. Here, we show that Tda1, the yeast ortholog of human Nuak1, is a direct kinase for H3pT11 upon low glucose stress. Yeast AMP-activated protein kinase (AMPK) directly phosphorylates Tda1 to govern Tda1 activity, while CK2 regulates Tda1 nuclear localization. Collectively, AMPK and CK2 signaling converge on histone kinase Tda1 to link external low glucose stress to chromatin regulation.
Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Caseína Quinase II/metabolismo , Histonas/metabolismo , Proteínas Quinases/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Glucose/metabolismo , Fosforilação , Proteínas de Transporte VesicularRESUMO
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
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Merkel cell carcinoma (MCC)-a neuroendocrine cancer of the skin-is caused by the integration of Merkel cell polyomavirus and persistent expression of large T antigen and small T antigen. We report that small T antigen in complex with MYCL and the EP400 complex activates the expression of LSD1 (KDM1A), RCOR2 and INSM1 to repress gene expression by the lineage transcription factor ATOH1. LSD1 inhibition reduces the growth of MCC in vitro and in vivo. Through a forward-genetics CRISPR-Cas9 screen, we identified an antagonistic relationship between LSD1 and the non-canonical BAF (ncBAF) chromatin remodelling complex. Changes in gene expression and chromatin accessibility caused by LSD1 inhibition were partially rescued by BRD9 inhibition, revealing that LSD1 and ncBAF antagonistically regulate an overlapping set of genes. Our work provides mechanistic insight into the dependence of MCC on LSD1 and a tumour suppressor role for ncBAF in cancer.
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Proper orientation of the mitotic spindle is essential for cell fate determination, tissue morphogenesis, and homeostasis. During epithelial proliferation, planar spindle alignment ensures the maintenance of polarized tissue architecture, and aberrant spindle orientation can disrupt epithelial integrity. Nevertheless, in vivo mechanisms that restrict the mitotic spindle to the plane of the epithelium remain poorly understood. Here we show that the junction-localized tumor suppressors Scribbled (Scrib) and Discs large (Dlg) control planar spindle orientation via Mud and 14-3-3 proteins in the Drosophila wing disc epithelium. During mitosis, Scrib is required for the junctional localization of Dlg, and both affect mitotic spindle movements. Using coimmunoprecipitation and mass spectrometry, we identify 14-3-3 proteins as Dlg-interacting partners and further report that loss of 14-3-3s causes both abnormal spindle orientation and disruption of epithelial architecture as a consequence of basal cell delamination and apoptosis. Combined, these biochemical and genetic analyses indicate that 14-3-3s function together with Scrib, Dlg, and Mud during planar cell division.
Assuntos
Proteínas 14-3-3/metabolismo , Polaridade Celular , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/citologia , Fuso Acromático/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Asas de Animais/citologia , Proteínas 14-3-3/genética , Animais , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Morfogênese , Fuso Acromático/genética , Proteínas Supressoras de Tumor/genética , Asas de Animais/metabolismoRESUMO
Human Dual-specificity tyrosine (Y) Regulated Kinase 1A (DYRK1A) is encoded by a dosage dependent gene whereby either trisomy or haploinsufficiency result in developmental abnormalities. However, the function and regulation of this important protein kinase are not fully understood. Here, we report proteomic analysis of DYRK1A in human cells that revealed a novel role of DYRK1A in DNA double-strand breaks (DSBs) repair, mediated in part by its interaction with the ubiquitin-binding protein RNF169 that accumulates at the DSB sites and promotes homologous recombination repair (HRR) by displacing 53BP1, a key mediator of non-homologous end joining (NHEJ). We found that overexpression of active, but not the kinase inactive DYRK1A in U-2 OS cells inhibits accumulation of 53BP1 at the DSB sites in the RNF169-dependent manner. DYRK1A phosphorylates RNF169 at two sites that influence its ability to displace 53BP1 from the DSBs. Although DYRK1A is not required for the recruitment of RNF169 to the DSB sites and 53BP1 displacement, inhibition of DYRK1A or mutation of the DYRK1A phosphorylation sites in RNF169 decreases its ability to block accumulation of 53BP1 at the DSB sites. Interestingly, CRISPR-Cas9 knockout of DYRK1A in human and mouse cells also diminished the 53BP1 DSB recruitment in a manner that did not require RNF169, suggesting that dosage of DYRK1A can influence the DNA repair processes through both RNF169-dependent and independent mechanisms. Human U-2 OS cells devoid of DYRK1A display an increased HRR efficiency and resistance to DNA damage, therefore our findings implicate DYRK1A in the DNA repair processes.
Assuntos
Dano ao DNA , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Tirosina Quinases/metabolismo , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Animais , Sistemas CRISPR-Cas/genética , Pontos de Checagem do Ciclo Celular/efeitos da radiação , Linhagem Celular Tumoral , Dano ao DNA/efeitos da radiação , Reparo do DNA , Raios gama , Edição de Genes , Humanos , Redes e Vias Metabólicas , Camundongos , Fosforilação , Ligação Proteica , Proteínas Serina-Treonina Quinases/deficiência , Proteínas Serina-Treonina Quinases/genética , Proteínas Tirosina Quinases/deficiência , Proteínas Tirosina Quinases/genética , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Ubiquitina-Proteína Ligases/antagonistas & inibidores , Ubiquitina-Proteína Ligases/genética , Quinases DyrkRESUMO
The primary envelopment/de-envelopment of Herpes viruses during nuclear exit is poorly understood. In Herpes simplex virus type-1 (HSV-1), proteins pUL31 and pUL34 are critical, while pUS3 and some others contribute; however, efficient membrane fusion may require additional host proteins. We postulated that vesicle fusion proteins present in the nuclear envelope might facilitate primary envelopment and/or de-envelopment fusion with the outer nuclear membrane. Indeed, a subpopulation of vesicle-associated membrane protein-associated protein B (VAPB), a known vesicle trafficking protein, was present in the nuclear membrane co-locating with pUL34. VAPB knockdown significantly reduced both cell-associated and supernatant virus titers. Moreover, VAPB depletion reduced cytoplasmic accumulation of virus particles and increased levels of nuclear encapsidated viral DNA. These results suggest that VAPB is an important player in the exit of primary enveloped HSV-1 virions from the nucleus. Importantly, VAPB knockdown did not alter pUL34, calnexin or GM-130 localization during infection, arguing against an indirect effect of VAPB on cellular vesicles and trafficking. Immunogold-labelling electron microscopy confirmed VAPB presence in nuclear membranes and moreover associated with primary enveloped HSV-1 particles. These data suggest that VAPB could be a cellular component of a complex that facilitates UL31/UL34/US3-mediated HSV-1 nuclear egress.
Assuntos
Núcleo Celular/metabolismo , Herpesvirus Humano 1/fisiologia , Fusão de Membrana , Proteínas de Transporte Vesicular/metabolismo , Liberação de Vírus/fisiologia , Replicação Viral/fisiologia , Animais , Núcleo Celular/ultraestrutura , Chlorocebus aethiops , Células HeLa , Herpes Simples/metabolismo , Herpes Simples/virologia , Herpesvirus Humano 1/ultraestrutura , Humanos , Membranas Intracelulares/metabolismo , Membranas Intracelulares/ultraestrutura , Microssomos/metabolismo , Microssomos/ultraestrutura , Membrana Nuclear/metabolismo , Células Vero , Proteínas Virais/metabolismo , Vírion/metabolismo , Vírion/ultraestruturaRESUMO
Metazoans contain two homologs of the Gcn5-binding protein Ada2, Ada2a and Ada2b, which nucleate formation of the ATAC and SAGA complexes, respectively. In Drosophila melanogaster, there are two splice isoforms of Ada2b: Ada2b-PA and Ada2b-PB. Here, we show that only the Ada2b-PB isoform is in SAGA; in contrast, Ada2b-PA associates with Gcn5, Ada3, Sgf29 and Chiffon, forming the Chiffon histone acetyltransferase (CHAT) complex. Chiffon is the Drosophila ortholog of Dbf4, which binds and activates the cell cycle kinase Cdc7 to initiate DNA replication. In flies, Chiffon and Cdc7 are required in ovary follicle cells for gene amplification, a specialized form of DNA re-replication. Although chiffon was previously reported to be dispensable for viability, here, we find that Chiffon is required for both histone acetylation and viability in flies. Surprisingly, we show that chiffon is a dicistronic gene that encodes distinct Cdc7- and CHAT-binding polypeptides. Although the Cdc7-binding domain of Chiffon is not required for viability in flies, the CHAT-binding domain is essential for viability, but is not required for gene amplification, arguing against a role in DNA replication.
Assuntos
Proteínas de Drosophila/metabolismo , Proteínas do Ovo/metabolismo , Histona Acetiltransferases/metabolismo , Histonas/metabolismo , Acetilação , Animais , Linhagem Celular , Proteínas de Drosophila/genética , Drosophila melanogaster , Proteínas do Ovo/genética , Histona Acetiltransferases/genética , Histonas/genética , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismoRESUMO
Metabolic disorder has been suggested to underlie Alzheimer's disease (AD). However, the decisive molecular linkages remain unclear. We discovered that human Molybdopterin Synthase Associating Complex, MPTAC, promotes sulfur amino acid catabolism to prevent oxidative damage from excess sulfur amino acids, which, in turn, advances fatty acid oxidation and acetyl coenzyme A (acetyl-CoA) synthesis. The association of MPTAC with Protein arginine (R) Methyltransferase 5 (PRMT5) complex and small nuclear ribonucleoprotein (SNRP) splicing factors enables SNRPs to sense metabolic states through their methylation. This promotes the splicing fidelity of amyloid precursor protein (APP) pre-mRNA and proper APP fragmentation, abnormalities of which have been observed in the platelets of AD patients. The functions of MPTAC are crucial to maintain expression of drebrin 1, which is required for synaptic plasticity, through prevention from oxidative damage. Thus, adjustment of sulfur amino acid catabolism by MPTAC prevents events that occur early in the onset of AD.
Assuntos
Doença de Alzheimer/metabolismo , Aminoácidos Sulfúricos/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Sulfurtransferases/metabolismo , Células HEK293 , Humanos , Plasticidade NeuronalRESUMO
Laminopathies yield tissue-specific pathologies, yet arise from mutation of ubiquitously-expressed genes. A little investigated hypothesis to explain this is that the mutated proteins or their partners have tissue-specific splice variants. To test this, we analyzed RNA-Seq datasets, finding novel isoforms or isoform tissue-specificity for: Lap2, linked to cardiomyopathy; Nesprin 2, linked to Emery-Dreifuss muscular dystrophy and Lmo7, that regulates the Emery-Dreifuss muscular dystrophy linked emerin gene. Interestingly, the muscle-specific Lmo7 exon is rich in serine phosphorylation motifs, suggesting regulatory function. Muscle-specific splice variants in non-nuclear envelope proteins linked to other muscular dystrophies were also found. Nucleoporins tissue-specific variants were found for Nup54, Nup133, Nup153 and Nup358/RanBP2. RT-PCR confirmed novel Lmo7 and RanBP2 variants and specific knockdown of the Lmo7 variantreduced myogenic index. Nuclear envelope proteins were enriched for tissue-specific splice variants compared to the rest of the genome, suggesting that splice variants contribute to its tissue-specific functions.
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Processamento Alternativo/genética , Bases de Dados de Ácidos Nucleicos , Conjuntos de Dados como Assunto , Músculo Esquelético/metabolismo , Membrana Nuclear/genética , Proteínas Nucleares/genética , RNA Mensageiro/análise , RNA Mensageiro/genética , Animais , Células Cultivadas , Camundongos , Membrana Nuclear/metabolismo , Proteínas Nucleares/metabolismo , Especificidade de Órgãos/genéticaRESUMO
Merkel cell carcinoma (MCC) frequently contains integrated copies of Merkel cell polyomavirus DNA that express a truncated form of Large T antigen (LT) and an intact Small T antigen (ST). While LT binds RB and inactivates its tumor suppressor function, it is less clear how ST contributes to MCC tumorigenesis. Here we show that ST binds specifically to the MYC homolog MYCL (L-MYC) and recruits it to the 15-component EP400 histone acetyltransferase and chromatin remodeling complex. We performed a large-scale immunoprecipitation for ST and identified co-precipitating proteins by mass spectrometry. In addition to protein phosphatase 2A (PP2A) subunits, we identified MYCL and its heterodimeric partner MAX plus the EP400 complex. Immunoprecipitation for MAX and EP400 complex components confirmed their association with ST. We determined that the ST-MYCL-EP400 complex binds together to specific gene promoters and activates their expression by integrating chromatin immunoprecipitation with sequencing (ChIP-seq) and RNA-seq. MYCL and EP400 were required for maintenance of cell viability and cooperated with ST to promote gene expression in MCC cell lines. A genome-wide CRISPR-Cas9 screen confirmed the requirement for MYCL and EP400 in MCPyV-positive MCC cell lines. We demonstrate that ST can activate gene expression in a EP400 and MYCL dependent manner and this activity contributes to cellular transformation and generation of induced pluripotent stem cells.
Assuntos
Antígenos Virais de Tumores/metabolismo , Carcinoma de Célula de Merkel/virologia , Transformação Celular Viral/fisiologia , DNA Helicases/metabolismo , Proteínas de Ligação a DNA/metabolismo , Regulação Neoplásica da Expressão Gênica/fisiologia , Proteínas Proto-Oncogênicas c-myc/metabolismo , Antígenos Transformantes de Poliomavirus/metabolismo , Carcinoma de Célula de Merkel/genética , Carcinoma de Célula de Merkel/metabolismo , Linhagem Celular Tumoral , Humanos , Immunoblotting , Imunoprecipitação , Poliomavírus das Células de Merkel , Infecções por Polyomavirus/complicações , Infecções por Polyomavirus/genética , Infecções por Polyomavirus/metabolismo , Infecções Tumorais por Vírus/complicações , Infecções Tumorais por Vírus/genética , Infecções Tumorais por Vírus/metabolismoRESUMO
In human cells, cytoplasmic dynein-1 is essential for long-distance transport of many cargos, including organelles, RNAs, proteins, and viruses, towards microtubule minus ends. To understand how a single motor achieves cargo specificity, we identified the human dynein interactome by attaching a promiscuous biotin ligase ('BioID') to seven components of the dynein machinery, including a subunit of the essential cofactor dynactin. This method reported spatial information about the large cytosolic dynein/dynactin complex in living cells. To achieve maximal motile activity and to bind its cargos, human dynein/dynactin requires 'activators', of which only five have been described. We developed methods to identify new activators in our BioID data, and discovered that ninein and ninein-like are a new family of dynein activators. Analysis of the protein interactomes for six activators, including ninein and ninein-like, suggests that each dynein activator has multiple cargos.
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Proteínas de Transporte/metabolismo , Movimento Celular , Dineínas do Citoplasma/metabolismo , Complexo Dinactina/metabolismo , Linhagem Celular , Técnicas Citológicas/métodos , Humanos , Microtúbulos/metabolismo , Coloração e Rotulagem/métodosRESUMO
The Lyme disease spirochete Borrelia burgdorferi is unique among bacteria in its large number of lipoproteins that are encoded by a small, exceptionally fragmented, and predominantly linear genome. Peripherally anchored in either the inner or outer membrane and facing either the periplasm or the external environment, these lipoproteins assume varied roles. A prominent subset of lipoproteins functioning as the apparent linchpins of the enzootic tick-vertebrate infection cycle have been explored as vaccine targets. Yet, most of the B. burgdorferi lipoproteome has remained uncharacterized. Here, we comprehensively and conclusively localize the B. burgdorferi lipoproteome by applying established protein localization assays to a newly generated epitope-tagged lipoprotein expression library and by validating the obtained individual protein localization results using a sensitive global mass spectrometry approach. The derived consensus localization data indicate that 86 of the 125 analyzed lipoproteins encoded by B. burgdorferi are secreted to the bacterial surface. Thirty-one of the remaining 39 periplasmic lipoproteins are retained in the inner membrane, with only 8 lipoproteins being anchored in the periplasmic leaflet of the outer membrane. The localization of 10 lipoproteins was further defined or revised, and 52 surface and 23 periplasmic lipoproteins were newly localized. Cross-referencing prior studies revealed that the borrelial surface lipoproteome contributing to the host-pathogen interface is encoded predominantly by plasmids. Conversely, periplasmic lipoproteins are encoded mainly by chromosomal loci. These studies close a gap in our understanding of the functional lipoproteome of an important human pathogen and set the stage for more in-depth studies of thus-far-neglected spirochetal lipoproteins.IMPORTANCE The small and exceptionally fragmented genome of the Lyme disease spirochete Borrelia burgdorferi encodes over 120 lipoproteins. Studies in the field have predominantly focused on a relatively small number of surface lipoproteins that play important roles in the transmission and pathogenesis of this global human pathogen. Yet, a comprehensive spatial assessment of the entire borrelial lipoproteome has been missing. The current study newly identifies 52 surface and 23 periplasmic lipoproteins. Overall, two-thirds of the B. burgdorferi lipoproteins localize to the surface, while outer membrane lipoproteins facing the periplasm are rare. This analysis underscores the dominant contribution of lipoproteins to the spirochete's rather complex and adaptable host-pathogen interface, and it encourages further functional exploration of its lipoproteome.
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Proteínas de Bactérias/metabolismo , Borrelia burgdorferi/metabolismo , Regulação Bacteriana da Expressão Gênica/fisiologia , Lipoproteínas/metabolismo , Proteínas de Membrana/metabolismo , Proteínas de Bactérias/genética , Borrelia burgdorferi/genética , Epitopos , Escherichia coli/metabolismo , Biblioteca Gênica , Lipoproteínas/genética , Espectrometria de Massas , Proteínas de Membrana/genética , Transporte ProteicoRESUMO
Synaptic vesicles (SVs) fuse with the plasma membrane at a precise location called the presynaptic active zone (AZ). This fusion is coordinated by proteins embedded within a cytoskeletal matrix assembled at the AZ (CAZ). In the present study, we have identified a novel binding partner for the CAZ proteins Piccolo and Bassoon. This interacting protein, Trio, is a member of the Dbl family of guanine nucleotide exchange factors (GEFs) known to regulate the dynamic assembly of actin and growth factor dependent axon guidance and synaptic growth. Trio was found to interact with the C-terminal PBH 9/10 domains of Piccolo and Bassoon via its own N-terminal Spectrin repeats, a domain that is also critical for its localization to the CAZ. Moreover, our data suggest that regions within the C-terminus of Trio negatively regulate its interactions with Piccolo/Bassoon. These findings provide a mechanism for the presynaptic targeting of Trio and support a model in which Piccolo and Bassoon play a role in regulating neurotransmission through interactions with proteins, including Trio, that modulate the dynamic assembly of F-actin during cycles of synaptic vesicle exo- and endocytosis.
Assuntos
Proteínas do Citoesqueleto/genética , Fatores de Troca do Nucleotídeo Guanina/genética , Proteínas do Tecido Nervoso/genética , Neurônios/metabolismo , Neuropeptídeos/genética , Terminações Pré-Sinápticas/metabolismo , Proteínas Serina-Treonina Quinases/genética , Transmissão Sináptica/genética , Actinas/genética , Actinas/metabolismo , Animais , Sítios de Ligação , Células COS , Chlorocebus aethiops , Proteínas do Citoesqueleto/metabolismo , Embrião de Mamíferos , Endocitose , Regulação da Expressão Gênica , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Hipocampo/citologia , Hipocampo/metabolismo , Humanos , Proteínas do Tecido Nervoso/metabolismo , Neurônios/ultraestrutura , Neuropeptídeos/metabolismo , Terminações Pré-Sinápticas/ultraestrutura , Cultura Primária de Células , Proteínas Serina-Treonina Quinases/metabolismo , Ratos , Ratos Sprague-Dawley , Vesículas Sinápticas/metabolismo , Vesículas Sinápticas/ultraestruturaRESUMO
The SAGA complex contains two enzymatic modules, which house histone acetyltransferase (HAT) and deubiquitinase (DUB) activities. USP22 is the catalytic subunit of the DUB module, but two adaptor proteins, ATXN7L3 and ENY2, are necessary for DUB activity toward histone H2Bub1 and other substrates. ATXN7L3B shares 74% identity with the N-terminal region of ATXN7L3, but the functions of ATXN7L3B are not known. Here we report that ATXN7L3B interacts with ENY2 but not other SAGA components. Even though ATXN7L3B localizes in the cytoplasm, ATXN7L3B overexpression increases H2Bub1 levels, while overexpression of ATXN7L3 decreases H2Bub1 levels. In vitro, ATXN7L3B competes with ATXN7L3 to bind ENY2, and in vivo, knockdown of ATXN7L3B leads to concomitant loss of ENY2. Unlike the ATXN7L3 DUB complex, a USP22-ATXN7L3B-ENY2 complex cannot deubiquitinate H2Bub1 efficiently in vitro Moreover, ATXN7L3B knockdown inhibits migration of breast cancer cells in vitro and limits expression of ER target genes. Collectively, our studies suggest that ATXN7L3B regulates H2Bub1 levels and SAGA DUB activity through competition for ENY2 binding.
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Neoplasias da Mama/metabolismo , Citoplasma/metabolismo , Histonas/metabolismo , Tioléster Hidrolases/metabolismo , Fatores de Transcrição/metabolismo , Movimento Celular , Núcleo Celular/enzimologia , Feminino , Técnicas de Silenciamento de Genes , Células HEK293 , Humanos , Células MCF-7 , Fatores de Transcrição/genética , Ubiquitina Tiolesterase , Regulação para CimaRESUMO
HIV replication in nondividing host cells occurs in the presence of high concentrations of noncanonical dUTP, apolipoprotein B mRNA-editing, enzyme-catalytic, polypeptide-like 3 (APOBEC3) cytidine deaminases, and SAMHD1 (a cell cycle-regulated dNTP triphosphohydrolase) dNTPase, which maintains low concentrations of canonical dNTPs in these cells. These conditions favor the introduction of marks of DNA damage into viral cDNA, and thereby prime it for processing by DNA repair enzymes. Accessory protein Vpr, found in all primate lentiviruses, and its HIV-2/simian immunodeficiency virus (SIV) SIVsm paralogue Vpx, hijack the CRL4(DCAF1) E3 ubiquitin ligase to alleviate some of these conditions, but the extent of their interactions with DNA repair proteins has not been thoroughly characterized. Here, we identify HLTF, a postreplication DNA repair helicase, as a common target of HIV-1/SIVcpz Vpr proteins. We show that HIV-1 Vpr reprograms CRL4(DCAF1) E3 to direct HLTF for proteasome-dependent degradation independent from previously reported Vpr interactions with base excision repair enzyme uracil DNA glycosylase (UNG2) and crossover junction endonuclease MUS81, which Vpr also directs for degradation via CRL4(DCAF1) E3. Thus, separate functions of HIV-1 Vpr usurp CRL4(DCAF1) E3 to remove key enzymes in three DNA repair pathways. In contrast, we find that HIV-2 Vpr is unable to efficiently program HLTF or UNG2 for degradation. Our findings reveal complex interactions between HIV-1 and the DNA repair machinery, suggesting that DNA repair plays important roles in the HIV-1 life cycle. The divergent interactions of HIV-1 and HIV-2 with DNA repair enzymes and SAMHD1 imply that these viruses use different strategies to guard their genomes and facilitate their replication in the host.
Assuntos
DNA Glicosilases/metabolismo , Proteínas de Ligação a DNA/metabolismo , HIV-1/fisiologia , HIV-2/fisiologia , Fatores de Transcrição/metabolismo , Produtos do Gene vpr do Vírus da Imunodeficiência Humana/metabolismo , Sequência de Aminoácidos , Ciclo Celular , DNA Helicases/metabolismo , Endonucleases/metabolismo , Células HEK293 , Humanos , Dados de Sequência Molecular , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Proteômica , Proteína 1 com Domínio SAM e Domínio HD , Proteínas de Saccharomyces cerevisiae/metabolismo , Ubiquitina-Proteína Ligases/metabolismoRESUMO
KAT6 histone acetyltransferases (HATs) are highly conserved in eukaryotes and are involved in cell cycle regulation. However, information regarding their roles in regulating cell cycle progression is limited. Here, we report the identification of subunits of the Drosophila Enok complex and demonstrate that all subunits are important for its HAT activity. We further report a novel interaction between the Enok complex and the Elg1 proliferating cell nuclear antigen (PCNA)-unloader complex. Depletion of Enok in S2 cells resulted in a G1/S cell cycle block, and this block can be partially relieved by depleting Elg1. Furthermore, depletion of Enok reduced the chromatin-bound levels of PCNA in both S2 cells and early embryos, suggesting that the Enok complex may interact with the Elg1 complex and down-regulate its PCNA-unloading function to promote the G1/S transition. Supporting this hypothesis, depletion of Enok also partially rescued the endoreplication defects in Elg1-depleted nurse cells. Taken together, our study provides novel insights into the roles of KAT6 HATs in cell cycle regulation through modulating PCNA levels on chromatin.