Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 26
Filtrar
1.
Cell ; 171(4): 918-933.e20, 2017 Nov 02.
Artigo em Inglês | MEDLINE | ID: mdl-29033132

RESUMO

Posttranslational modification with ubiquitin chains controls cell fate in all eukaryotes. Depending on the connectivity between subunits, different ubiquitin chain types trigger distinct outputs, as seen with K48- and K63-linked conjugates that drive protein degradation or complex assembly, respectively. Recent biochemical analyses also suggested roles for mixed or branched ubiquitin chains, yet without a method to monitor endogenous conjugates, the physiological significance of heterotypic polymers remained poorly understood. Here, we engineered a bispecific antibody to detect K11/K48-linked chains and identified mitotic regulators, misfolded nascent polypeptides, and pathological Huntingtin variants as their endogenous substrates. We show that K11/K48-linked chains are synthesized and processed by essential ubiquitin ligases and effectors that are mutated across neurodegenerative diseases; accordingly, these conjugates promote rapid proteasomal clearance of aggregation-prone proteins. By revealing key roles of K11/K48-linked chains in cell-cycle and quality control, we establish heterotypic ubiquitin conjugates as important carriers of biological information.


Assuntos
Anticorpos Biespecíficos/análise , Transdução de Sinais , Ubiquitina/metabolismo , Ciclossomo-Complexo Promotor de Anáfase/metabolismo , Ciclo Celular , Humanos , Mitose , Biossíntese de Proteínas , Ubiquitinação
2.
Cell ; 167(2): 525-538.e14, 2016 Oct 06.
Artigo em Inglês | MEDLINE | ID: mdl-27716508

RESUMO

The ubiquitin ligase CUL3 is an essential regulator of neural crest specification whose aberrant activation has been linked to autism, schizophrenia, and hypertension. CUL3 exerts its roles by pairing with ∼90 distinct substrate adaptors, yet how the different CUL3-complexes are activated is poorly understood. Here, we show that CUL3 and its adaptor KLHL12 require two calcium-binding proteins, PEF1 and ALG2, for recognition of their substrate SEC31. PEF1 and ALG2 form a target-specific co-adaptor that translates a transient rise in cytosolic calcium levels into more persistent SEC31 ubiquitylation, which in turn triggers formation of large COPII coats and promotes collagen secretion. As calcium also instructs chondrocyte differentiation and collagen synthesis, calcium-dependent control of CUL3KLHL12 integrates collagen secretion into broader programs of craniofacial bone formation. Our work, therefore, identifies both calcium and CUL3 co-adaptors as important regulators of ubiquitylation events that control human development.


Assuntos
Proteínas de Ligação ao Cálcio/metabolismo , Cálcio/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas Culina/metabolismo , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Proteínas dos Microfilamentos/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Vesículas Revestidas pelo Complexo de Proteína do Envoltório/metabolismo , Colágeno/metabolismo , Células HEK293 , Humanos , Especificidade por Substrato , Ubiquitinação , Proteínas de Transporte Vesicular/metabolismo
3.
Mol Cell ; 83(6): 927-941.e8, 2023 03 16.
Artigo em Inglês | MEDLINE | ID: mdl-36898370

RESUMO

Mitophagy is a form of selective autophagy that disposes of superfluous and potentially damage-inducing organelles in a tightly controlled manner. While the machinery involved in mitophagy induction is well known, the regulation of the components is less clear. Here, we demonstrate that TNIP1 knockout in HeLa cells accelerates mitophagy rates and that ectopic TNIP1 negatively regulates the rate of mitophagy. These functions of TNIP1 depend on an evolutionarily conserved LIR motif as well as an AHD3 domain, which are required for binding to the LC3/GABARAP family of proteins and the autophagy receptor TAX1BP1, respectively. We further show that phosphorylation appears to regulate its association with the ULK1 complex member FIP200, allowing TNIP1 to compete with autophagy receptors, which provides a molecular rationale for its inhibitory function during mitophagy. Taken together, our findings describe TNIP1 as a negative regulator of mitophagy that acts at the early steps of autophagosome biogenesis.


Assuntos
Proteínas Relacionadas à Autofagia , Autofagia , Mitofagia , Humanos , Proteínas Reguladoras de Apoptose/genética , Proteínas Reguladoras de Apoptose/metabolismo , Autofagia/genética , Família da Proteína 8 Relacionada à Autofagia/metabolismo , Proteínas de Ligação a DNA/metabolismo , Células HeLa , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Mitofagia/genética , Proteínas de Neoplasias/metabolismo
4.
EMBO J ; 43(10): 1919-1946, 2024 May.
Artigo em Inglês | MEDLINE | ID: mdl-38360993

RESUMO

Most cellular ubiquitin signaling is initiated by UBA1, which activates and transfers ubiquitin to tens of E2 enzymes. Clonally acquired UBA1 missense mutations cause an inflammatory-hematologic overlap disease called VEXAS (vacuoles, E1, X-linked, autoinflammatory, somatic) syndrome. Despite extensive clinical investigation into this lethal disease, little is known about the underlying molecular mechanisms. Here, by dissecting VEXAS-causing UBA1 mutations, we discovered that p.Met41 mutations alter cytoplasmic isoform expression, whereas other mutations reduce catalytic activity of nuclear and cytoplasmic isoforms by diverse mechanisms, including aberrant oxyester formation. Strikingly, non-p.Met41 mutations most prominently affect transthioesterification, revealing ubiquitin transfer to cytoplasmic E2 enzymes as a shared property of pathogenesis amongst different VEXAS syndrome genotypes. A similar E2 charging bottleneck exists in some lung cancer-associated UBA1 mutations, but not in spinal muscular atrophy-causing UBA1 mutations, which instead, render UBA1 thermolabile. Collectively, our results highlight the precision of conformational changes required for faithful ubiquitin transfer, define distinct and shared mechanisms of UBA1 inactivation in diverse diseases, and suggest that specific E1-E2 modules control different aspects of tissue differentiation and maintenance.


Assuntos
Enzimas Ativadoras de Ubiquitina , Enzimas Ativadoras de Ubiquitina/metabolismo , Enzimas Ativadoras de Ubiquitina/genética , Humanos , Mutação de Sentido Incorreto , Ubiquitina/metabolismo , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/metabolismo
5.
EMBO J ; 42(17): e114931, 2023 09 04.
Artigo em Inglês | MEDLINE | ID: mdl-37528760

RESUMO

The CUL4 paralogs CUL4A and CUL4B assemble into structurally similar multisubunit ubiquitin E3 ligases (CRL4A/B) that regulate diverse aspects of cell biology. New work in this issue of The EMBO Journal shows that the longer N-terminal tail of CUL4B tells these molecular twins apart, by promoting the formation of paralog-specific CRL4B complexes that control cytoskeletal processes during mitosis and brain development.


Assuntos
Ubiquitina-Proteína Ligases , Ubiquitina , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , Mitose , Encéfalo/metabolismo , Proteínas Culina/genética , Proteínas Culina/metabolismo , Ubiquitinação
6.
EMBO J ; 42(22): e113491, 2023 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-37621214

RESUMO

Nix is a membrane-anchored outer mitochondrial protein that induces mitophagy. While Nix has an LC3-interacting (LIR) motif that binds to ATG8 proteins, it also contains a minimal essential region (MER) that induces mitophagy through an unknown mechanism. We used chemically induced dimerization (CID) to probe the mechanism of Nix-mediated mitophagy and found that both the LIR and MER are required for robust mitophagy. We find that the Nix MER interacts with the autophagy effector WIPI2 and recruits WIPI2 to mitochondria. The Nix LIR motif is also required for robust mitophagy and converts a homogeneous WIPI2 distribution on the surface of the mitochondria into puncta, even in the absence of ATG8s. Together, this work reveals unanticipated mechanisms in Nix-induced mitophagy and the elusive role of the MER, while also describing an interesting example of autophagy induction that acts downstream of the canonical initiation complexes.


Assuntos
Autofagia , Mitofagia , Mitocôndrias/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Família da Proteína 8 Relacionada à Autofagia/metabolismo , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo
7.
Blood ; 140(13): 1496-1506, 2022 09 29.
Artigo em Inglês | MEDLINE | ID: mdl-35793467

RESUMO

Somatic mutations in UBA1 cause vacuoles, E1 ubiquitin-activating enzyme, X-linked, autoinflammatory somatic (VEXAS) syndrome, an adult-onset inflammatory disease with an overlap of hematologic manifestations. VEXAS syndrome is characterized by a high mortality rate and significant clinical heterogeneity. We sought to determine independent predictors of survival in VEXAS and to understand the mechanistic basis for these factors. We analyzed 83 patients with somatic pathogenic variants in UBA1 at p.Met41 (p.Met41Leu/Thr/Val), the start codon for translation of the cytoplasmic isoform of UBA1 (UBA1b). Patients with the p.Met41Val genotype were most likely to have an undifferentiated inflammatory syndrome. Multivariate analysis showed ear chondritis was associated with increased survival, whereas transfusion dependence and the p.Met41Val variant were independently associated with decreased survival. Using in vitro models and patient-derived cells, we demonstrate that p.Met41Val variant supports less UBA1b translation than either p.Met41Leu or p.Met41Thr, providing a molecular rationale for decreased survival. In addition, we show that these 3 canonical VEXAS variants produce more UBA1b than any of the 6 other possible single-nucleotide variants within this codon. Finally, we report a patient, clinically diagnosed with VEXAS syndrome, with 2 novel mutations in UBA1 occurring in cis on the same allele. One mutation (c.121 A>T; p.Met41Leu) caused severely reduced translation of UBA1b in a reporter assay, but coexpression with the second mutation (c.119 G>C; p.Gly40Ala) rescued UBA1b levels to those of canonical mutations. We conclude that regulation of residual UBA1b translation is fundamental to the pathogenesis of VEXAS syndrome and contributes to disease prognosis.


Assuntos
Nucleotídeos , Enzimas Ativadoras de Ubiquitina , Códon de Iniciação , Humanos , Mutação , Enzimas Ativadoras de Ubiquitina/genética , Ubiquitinação
8.
Exp Cell Res ; 396(2): 112300, 2020 11 15.
Artigo em Inglês | MEDLINE | ID: mdl-32986984

RESUMO

Metazoan development relies on intricate cell differentiation, communication, and migration pathways, which ensure proper formation of specialized cell types, tissues, and organs. These pathways are crucially controlled by ubiquitylation, a reversible post-translational modification that regulates the stability, activity, localization, or interaction landscape of substrate proteins. Specificity of ubiquitylation is ensured by E3 ligases, which bind substrates and co-operate with E1 and E2 enzymes to mediate ubiquitin transfer. Cullin3-RING ligases (CRL3s) are a large class of multi-subunit E3s that have emerged as important regulators of cell differentiation and development. In particular, recent evidence from human disease genetics, animal models, and mechanistic studies have established their involvement in the control of craniofacial and brain development. Here, we summarize regulatory principles of CRL3 assembly, substrate recruitment, and ubiquitylation that allow this class of E3s to fulfill their manifold functions in development. We further review our current mechanistic understanding of how specific CRL3 complexes orchestrate neuroectodermal differentiation and highlight diseases associated with their dysregulation. Based on evidence from human disease genetics, we propose that other unknown CRL3 complexes must help coordinate craniofacial and brain development and discuss how combining emerging strategies from the field of disease gene discovery with biochemical and human pluripotent stem cell approaches will likely facilitate their identification.


Assuntos
Encéfalo/embriologia , Proteínas Culina/metabolismo , Doença/genética , Face/embriologia , Crânio/embriologia , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitina/metabolismo , Animais , Humanos , Ubiquitina-Proteína Ligases/química
9.
Nature ; 525(7570): 523-7, 2015 Sep 24.
Artigo em Inglês | MEDLINE | ID: mdl-26399832

RESUMO

Metazoan development depends on the accurate execution of differentiation programs that allow pluripotent stem cells to adopt specific fates. Differentiation requires changes to chromatin architecture and transcriptional networks, yet whether other regulatory events support cell-fate determination is less well understood. Here we identify the ubiquitin ligase CUL3 in complex with its vertebrate-specific substrate adaptor KBTBD8 (CUL3(KBTBD8)) as an essential regulator of human and Xenopus tropicalis neural crest specification. CUL3(KBTBD8) monoubiquitylates NOLC1 and its paralogue TCOF1, the mutation of which underlies the neurocristopathy Treacher Collins syndrome. Ubiquitylation drives formation of a TCOF1-NOLC1 platform that connects RNA polymerase I with ribosome modification enzymes and remodels the translational program of differentiating cells in favour of neural crest specification. We conclude that ubiquitin-dependent regulation of translation is an important feature of cell-fate determination.


Assuntos
Diferenciação Celular , Crista Neural/citologia , Crista Neural/metabolismo , Biossíntese de Proteínas , Ubiquitina/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Diferenciação Celular/genética , Proteínas Culina/metabolismo , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Humanos , Disostose Mandibulofacial/genética , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Proteômica , RNA Polimerase I/metabolismo , Ribossomos/química , Ribossomos/metabolismo , Ubiquitinação , Xenopus
10.
Mol Cell ; 49(4): 591-600, 2013 Feb 21.
Artigo em Inglês | MEDLINE | ID: mdl-23438855

RESUMO

Ubiquitylation is an essential posttranslational modification that can regulate the stability, activity, and localization of thousands of proteins. The reversible attachment of ubiquitin as well as interpretation of the ubiquitin signal depends on dynamic protein networks that are challenging to analyze. In this perspective, we discuss tools of the trade that have recently been developed to dissect mechanisms of ubiquitin-dependent signaling, thereby revealing the critical features of an important cellular code.


Assuntos
Proteínas Ubiquitinadas/metabolismo , Ubiquitinação , Animais , Humanos , Processamento de Proteína Pós-Traducional , Estabilidade Proteica , Proteólise , Transdução de Sinais , Ubiquitina-Proteína Ligases/fisiologia
12.
Biotechnol Bioeng ; 110(11): 2882-93, 2013 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-23616347

RESUMO

Microalgae are discussed as a potential renewable feedstock for biofuel production. The production of highly concentrated algae biomass with a high fatty acid content, accompanied by high productivity with the use of natural sunlight is therefore of great interest. In the current study an outdoor pilot plant with five 30 L Flat Panel Airlift reactors (FPA) installed southwards were operated in 2011 in Stuttgart, Germany. The patented FPA reactor works on the basis of an airlift loop reactor and offers efficient intermixing for homogeneous light distribution. A lipid production process with the microalgae Chlorella vulgaris (SAG 211-12), under nitrogen and phosphorous deprivation, was established and evaluated in regard to the fatty acid content, fatty acid productivity and light yield. In the first set of experiments limitations caused by restricted CO2 availability were excluded by enriching the media with NaOH. The higher alkalinity allows a higher CO2 content of supplied air and leads to doubling of fatty acid productivity. The second set of experiments focused on how the ratio of light intensity to biomass concentration in the reactor impacts fatty acid content, productivity and light yield. The specific light availability was specified as mol photons on the reactor surface per gram biomass in the reactor. This is the first publication based on experimental data showing the quantitative correlation between specific light availability, fatty acid content and biomass light yield for a lipid production process under nutrient deprivation and outdoor conditions. High specific light availability leads to high fatty acid contents. Lower specific light availability increases fatty acid productivity and biomass light yield. An average fatty acid productivity of 0.39 g L⁻¹ day⁻¹ for a 12 days batch process with a final fatty acid content of 44.6% [w/w] was achieved. Light yield of 0.4 g mol photons⁻¹ was obtained for the first 6 days of cultivation.


Assuntos
Reatores Biológicos/microbiologia , Chlorella vulgaris/crescimento & desenvolvimento , Chlorella vulgaris/metabolismo , Lipídeos/biossíntese , Biomassa , Dióxido de Carbono/metabolismo , Meios de Cultura/química , Fermentação , Alemanha , Luz
13.
bioRxiv ; 2023 Oct 16.
Artigo em Inglês | MEDLINE | ID: mdl-37873486

RESUMO

STING activation by cyclic dinucleotides in mammals induces interferon- and NFκB -related gene expression, and the lipidation of LC3B at Golgi membranes. While mechanisms of the interferon response are well understood, the mechanisms of NFκB activation mediated by STING remain unclear. We report that STING activation induces K63- and M1-linked/linear ubiquitin chain formation at LC3B-associated Golgi membranes. Loss of the LUBAC E3 ubiquitin ligase prevents formation of linear, but not K63-linked ubiquitin chains or STING activation and inhibits STING-induced NFκB and IRF3-mediated signaling in monocytic THP1 cells. The proton channel activity of STING is also important for both K63 and linear ubiquitin chain formation, and NFκB- and interferon-related gene expression. Thus, LUBAC synthesis of linear ubiquitin chains regulates STING-mediated innate immune signaling.

14.
Nat Commun ; 14(1): 4499, 2023 07 26.
Artigo em Inglês | MEDLINE | ID: mdl-37495603

RESUMO

The molecular mechanisms that coordinate patterning of the embryonic ectoderm into spatially distinct lineages to form the nervous system, epidermis, and neural crest-derived craniofacial structures are unclear. Here, biochemical disease-variant profiling reveals a posttranslational pathway that drives early ectodermal differentiation in the vertebrate head. The anteriorly expressed ubiquitin ligase CRL3-KLHL4 restricts signaling of the ubiquitous cytoskeletal regulator CDC42. This regulation relies on the CDC42-activating complex GIT1-ßPIX, which CRL3-KLHL4 exploits as a substrate-specific co-adaptor to recognize and monoubiquitylate PAK1. Surprisingly, we find that ubiquitylation converts the canonical CDC42 effector PAK1 into a CDC42 inhibitor. Loss of CRL3-KLHL4 or a disease-associated KLHL4 variant reduce PAK1 ubiquitylation causing overactivation of CDC42 signaling and defective ectodermal patterning and neurulation. Thus, tissue-specific restriction of CDC42 signaling by a ubiquitin-based effector-to-inhibitor is essential for early face, brain, and skin formation, revealing how cell-fate and morphometric changes are coordinated to ensure faithful organ development.


Assuntos
Crista Neural , Ubiquitina , Encéfalo , Ectoderma , Transdução de Sinais
15.
Arthritis Rheumatol ; 75(7): 1285-1290, 2023 07.
Artigo em Inglês | MEDLINE | ID: mdl-36762418

RESUMO

OBJECTIVE: Somatic mutations in UBA1 have recently been causally linked to a severe adult-onset inflammatory condition referred to as VEXAS (vacuoles, E1 enzyme, X-linked, autoinflammatory, somatic) syndrome. Ubiquitin-activating enzyme E1 (UBA-1) is of fundamental importance to the modulation of ubiquitin homeostasis and to the majority of downstream ubiquitylation-dependent cellular processes. Direct sequencing analysis of exon 3 containing the prevalent variants p.Met41Leu, p.Met41Val, and/or p.Met41Thr is usually used to confirm the disease-associated mutations. METHODS: We studied the clinical, biochemical, and molecular genetic characteristics of a 59-year-old man with a 2-year history of arthritis, fever, night sweats, nonspecific skin rash, lymphadenopathy, and myelodysplastic syndrome with multilineage dysplasia. RESULTS: The mutational analysis revealed a previously undescribed sequence variant c.1430G>C in exon 14 (p.Gly477Ala) in the gene UBA1. In vitro enzymatic analyses showed that p.Gly477Ala led to both decreased E1 ubiquitin thioester formation and E2 enzyme charging. CONCLUSION: We report a case of a patient of European ancestry with clinical manifestations of VEXAS syndrome associated with a newly identified dysfunctional UBA-1 enzyme variant. Due to the patient's insufficient response to various immunosuppressive treatments, allogeneic hematopoietic stem cell transplantation was performed, which resulted in significant improvement of clinical and laboratory manifestations of the disease.


Assuntos
Síndromes Mielodisplásicas , Enzimas Ativadoras de Ubiquitina , Adulto , Masculino , Humanos , Pessoa de Meia-Idade , Enzimas Ativadoras de Ubiquitina/genética , Pacientes , Ubiquitinas , Mutação
16.
bioRxiv ; 2023 Oct 10.
Artigo em Inglês | MEDLINE | ID: mdl-37873213

RESUMO

Most cellular ubiquitin signaling is initiated by UBA1, which activates and transfers ubiquitin to tens of E2 enzymes. Clonally acquired UBA1 missense mutations cause an inflammatory-hematologic overlap disease called VEXAS (vacuoles, E1, X-linked, autoinflammatory, somatic) syndrome. Despite extensive clinical investigation into this lethal disease, little is known about the underlying molecular mechanisms. Here, by dissecting VEXAS-causing UBA1 mutations, we discovered that p.Met41 mutations alter cytoplasmic isoform expression, whereas other mutations reduce catalytic activity of nuclear and cytoplasmic isoforms by diverse mechanisms, including aberrant oxyester formation. Strikingly, non-p.Met41 mutations most prominently affect transthioesterification, revealing ubiquitin transfer to cytoplasmic E2 enzymes as a shared property of pathogenesis amongst different VEXAS syndrome genotypes. A similar E2 charging bottleneck exists in some lung cancer-associated UBA1 mutations, but not in spinal muscular atrophy-causing UBA1 mutations, which instead, render UBA1 thermolabile. Collectively, our results highlight the precision of conformational changes required for faithful ubiquitin transfer, define distinct and shared mechanisms of UBA1 inactivation in diverse diseases, and suggest that specific E1-E2 modules control different aspects of tissue differentiation and maintenance.

17.
Nat Rev Rheumatol ; 18(8): 435-447, 2022 08.
Artigo em Inglês | MEDLINE | ID: mdl-35523963

RESUMO

Ubiquitylation is an essential post-translational modification that regulates intracellular signalling networks by triggering proteasomal substrate degradation, changing the activity of substrates or mediating changes in proteins that interact with substrates. Hundreds of enzymes participate in reversible ubiquitylation of proteins, some acting globally and others targeting specific proteins. Ubiquitylation is essential for innate immune responses, as it facilitates rapid regulation of inflammatory pathways, thereby ensuring sufficient but not excessive responses. A growing number of inborn errors of immunity are attributed to dysregulated ubiquitylation. These genetic disorders exhibit broad clinical manifestations, ranging from susceptibility to infection to autoinflammatory and/or autoimmune features, lymphoproliferation and propensity to malignancy. Many autoinflammatory disorders result from disruption of components of the ubiquitylation machinery and lead to overactivation of innate immune cells. An understanding of the disorders of ubiquitylation in autoinflammatory diseases could enable the development of novel management strategies.


Assuntos
Inflamação , Ubiquitina , Humanos , Imunidade Inata , Processamento de Proteína Pós-Traducional , Ubiquitina/genética , Ubiquitina/metabolismo , Ubiquitinação
18.
Hypertension ; 79(1): 60-75, 2022 01.
Artigo em Inglês | MEDLINE | ID: mdl-34878901

RESUMO

Familial hyperkalemic hypertension is caused by pathogenic variants in genes of the CUL3 (cullin-3)-KLHL3 (kelch-like-family-member-3)-WNK (with no-lysine [K] kinase) pathway, manifesting clinically as hyperkalemia, metabolic acidosis, and high systolic blood pressure. The ubiquitin E3 ligase CUL3-KLHL3 targets WNK kinases for degradation to limit activation of the thiazide-sensitive NCC (Na-Cl cotransporter). All known variants in CUL3 lead to exon 9 skipping (CUL3Δ9) and typically result in severe familial hyperkalemic hypertension and growth disturbances in patients. Whether other variants in CUL3 cause familial hyperkalemic hypertension is unknown. Here, we identify a novel de novo heterozygous CUL3 variant (CUL3Δ474-477) in a pediatric familial hyperkalemic hypertension patient with multiple congenital anomalies and reveal molecular mechanisms by which CUL3Δ474-477 leads to dysregulation of the CUL3-KLHL3-WNK signaling axis. Using patient-derived urinary extracellular vesicles and dermal fibroblasts, in vitro assays, and cultured kidney cells, we demonstrate that CUL3Δ474-477 causes reduced total CUL3 levels due to increased autoubiquitination. The CUL3Δ474-477 that escapes autodegradation shows enhanced modification with NEDD8 (neural precursor cell expressed developmentally down-regulated protein 8) and increased formation of CUL3-KLHL3 complexes that are impaired in ubiquitinating WNK4. Proteomic analysis of CUL3 complexes revealed that, in addition to increased KLHL3 binding, the CUL3Δ474-477 variant also exhibits increased interactions with other BTB (Bric-a-brac, Tramtrack, and Broad complex) substrate adaptors, providing a rationale for the patient's diverse phenotypes. We conclude that the pathophysiological effects of CUL3Δ474-477 are caused by reduced CUL3 levels and formation of catalytically impaired CUL3 ligase complexes.


Assuntos
Proteínas Culina/genética , Pseudo-Hipoaldosteronismo/genética , Proteínas Ligases SKP Culina F-Box/metabolismo , Pré-Escolar , Proteínas Culina/metabolismo , Humanos , Masculino , Proteômica , Pseudo-Hipoaldosteronismo/metabolismo , Transdução de Sinais/genética
19.
Traffic ; 10(10): 1543-59, 2009 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-19624487

RESUMO

SNARE proteins mediate membrane fusion in the secretory pathway of eukaryotic cells. Genetic deletion and siRNA-based knockdown have been instrumental in assigning given SNAREs to defined intracellular transport steps. However, SNARE depletion occasionally results in barely detectable phenotypes. To understand how cells cope with SNARE loss, we have knocked down several SNAREs functioning in early endosome fusion. Surprisingly, knockdown of syntaxin 13, syntaxin 6 and vti1a, alone or in combinations, did not result in measurable changes of endosomal trafficking or fusion. We found that the residual SNARE levels (typically approximately 10%) were sufficient for a substantial amount of SNARE-SNARE interactions. Conversely, in wild-type cells, most SNARE molecules were concentrated in clusters, constituting a spare pool not readily available for interactions. Additionally, the knockdown organelles exhibited enhanced docking. We conclude that SNAREs are expressed at much higher levels than needed for maintenance of organelle fusion, and that loss of SNAREs is compensated for by the co-regulation of the docking machinery.


Assuntos
Endossomos/metabolismo , Membranas Intracelulares/metabolismo , Fusão de Membrana , Proteínas SNARE/genética , Animais , Western Blotting , Toxina da Cólera/metabolismo , Regulação para Baixo , Inativação Gênica , Imunoprecipitação , Fusão de Membrana/genética , Microscopia de Fluorescência , Células PC12 , Transporte Proteico , RNA Interferente Pequeno/genética , Ratos , Transfecção
20.
Cell Death Differ ; 28(2): 538-556, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33335288

RESUMO

Metazoan development from a one-cell zygote to a fully formed organism requires complex cellular differentiation and communication pathways. To coordinate these processes, embryos frequently encode signaling information with the small protein modifier ubiquitin, which is typically attached to lysine residues within substrates. During ubiquitin signaling, a three-step enzymatic cascade modifies specific substrates with topologically unique ubiquitin modifications, which mediate changes in the substrate's stability, activity, localization, or interacting proteins. Ubiquitin signaling is critically regulated by deubiquitylases (DUBs), a class of ~100 human enzymes that oppose the conjugation of ubiquitin. DUBs control many essential cellular functions and various aspects of human physiology and development. Recent genetic studies have identified mutations in several DUBs that cause developmental disorders. Here we review principles controlling DUB activity and substrate recruitment that allow these enzymes to regulate ubiquitin signaling during development. We summarize key mechanisms of how DUBs control embryonic and postnatal differentiation processes, highlight developmental disorders that are caused by mutations in particular DUB members, and describe our current understanding of how these mutations disrupt development. Finally, we discuss how emerging tools from human disease genetics will enable the identification and study of novel congenital disease-causing DUBs.


Assuntos
Anormalidades Congênitas/enzimologia , Enzimas Desubiquitinantes/metabolismo , Enzimas Desubiquitinantes/fisiologia , Animais , Humanos , Processamento de Proteína Pós-Traducional , Transdução de Sinais , Ubiquitina/metabolismo , Ubiquitinação
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA