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1.
Cell ; 154(1): 169-84, 2013 Jul 03.
Artigo em Inglês | MEDLINE | ID: mdl-23827681

RESUMO

Sixteen ovarian tumor (OTU) family deubiquitinases (DUBs) exist in humans, and most members regulate cell-signaling cascades. Several OTU DUBs were reported to be ubiquitin (Ub) chain linkage specific, but comprehensive analyses are missing, and the underlying mechanisms of linkage specificity are unclear. Using Ub chains of all eight linkage types, we reveal that most human OTU enzymes are linkage specific, preferring one, two, or a defined subset of linkage types, including unstudied atypical Ub chains. Biochemical analysis and five crystal structures of OTU DUBs with or without Ub substrates reveal four mechanisms of linkage specificity. Additional Ub-binding domains, the ubiquitinated sequence in the substrate, and defined S1' and S2 Ub-binding sites on the OTU domain enable OTU DUBs to distinguish linkage types. We introduce Ub chain restriction analysis, in which OTU DUBs are used as restriction enzymes to reveal linkage type and the relative abundance of Ub chains on substrates.


Assuntos
Endopeptidases/química , Endopeptidases/metabolismo , Neoplasias Ovarianas/enzimologia , Ubiquitinação , Catálise , Domínio Catalítico , Cristalografia por Raios X , Endopeptidases/genética , Feminino , Humanos , Modelos Moleculares , Neoplasias Ovarianas/metabolismo , Estrutura Terciária de Proteína , Tioléster Hidrolases/química , Tioléster Hidrolases/metabolismo , Ubiquitinas/metabolismo
2.
J Biol Chem ; 299(8): 105040, 2023 08.
Artigo em Inglês | MEDLINE | ID: mdl-37442237

RESUMO

Cu/Zn-superoxide dismutase (CuZnSOD) is an enzyme that binds a copper and zinc ion and also forms an intramolecular disulfide bond. Together with the copper ion as the active site, the disulfide bond is completely conserved among these proteins; indeed, the disulfide bond plays critical roles in maintaining the catalytically competent conformation of CuZnSOD. Here, we found that a CuZnSOD protein in Paenibacillus lautus (PaSOD) has no Cys residue but exhibits a significant level of enzyme activity. The crystal structure of PaSOD revealed hydrophobic and hydrogen-bonding interactions in substitution for the disulfide bond of the other CuZnSOD proteins. Also notably, we determined that PaSOD forms a homodimer through an additional domain with a novel fold at the N terminus. While the advantages of lacking Cys residues and adopting a novel dimer configuration remain obscure, PaSOD does not require a disulfide-introducing/correcting system for maturation and could also avoid misfolding caused by aberrant thiol oxidations under an oxidative environment.


Assuntos
Proteínas de Bactérias , Dissulfetos , Superóxido Dismutase-1 , Cobre , Cisteína , Dissulfetos/química , Superóxido Dismutase-1/química , Zinco , Proteínas de Bactérias/química , Paenibacillus , Dobramento de Proteína
3.
J Biol Chem ; 299(6): 104798, 2023 06.
Artigo em Inglês | MEDLINE | ID: mdl-37156398

RESUMO

Canine degenerative myelopathy (DM), a fatal neurodegenerative disease in dogs, shares clinical and genetic features with amyotrophic lateral sclerosis, a human motor neuron disease. Mutations in the SOD1 gene encoding Cu/Zn superoxide dismutase (SOD1) cause canine DM and a subset of inherited human amyotrophic lateral sclerosis. The most frequent DM causative mutation is homozygous E40K mutation, which induces the aggregation of canine SOD1 but not of human SOD1. However, the mechanism through which canine E40K mutation induces species-specific aggregation of SOD1 remains unknown. By screening human/canine chimeric SOD1s, we identified that the humanized mutation of the 117th residue (M117L), encoded by exon 4, significantly reduced aggregation propensity of canine SOD1E40K. Conversely, introducing a mutation of leucine 117 to methionine, a residue homologous to canine, promoted E40K-dependent aggregation in human SOD1. M117L mutation improved protein stability and reduced cytotoxicity of canine SOD1E40K. Furthermore, crystal structural analysis of canine SOD1 proteins revealed that M117L increased the packing within the hydrophobic core of the ß-barrel structure, contributing to the increased protein stability. Our findings indicate that the structural vulnerability derived intrinsically from Met 117 in the hydrophobic core of the ß-barrel structure induces E40K-dependent species-specific aggregation in canine SOD1.


Assuntos
Doenças do Cão , Mutação , Doenças Neurodegenerativas , Superóxido Dismutase-1 , Animais , Cães , Humanos , Esclerose Lateral Amiotrófica/genética , Esclerose Lateral Amiotrófica/metabolismo , Doenças Neurodegenerativas/genética , Doenças Neurodegenerativas/metabolismo , Doenças Neurodegenerativas/veterinária , Superóxido Dismutase-1/genética , Superóxido Dismutase-1/metabolismo , Doenças do Cão/genética , Doenças do Cão/metabolismo , Especificidade da Espécie
4.
Cell ; 136(6): 1098-109, 2009 Mar 20.
Artigo em Inglês | MEDLINE | ID: mdl-19303852

RESUMO

Activation of nuclear factor-kappaB (NF-kappaB), a key mediator of inducible transcription in immunity, requires binding of NF-kappaB essential modulator (NEMO) to ubiquitinated substrates. Here, we report that the UBAN (ubiquitin binding in ABIN and NEMO) motif of NEMO selectively binds linear (head-to-tail) ubiquitin chains. Crystal structures of the UBAN motif revealed a parallel coiled-coil dimer that formed a heterotetrameric complex with two linear diubiquitin molecules. The UBAN dimer contacted all four ubiquitin moieties, and the integrity of each binding site was required for efficient NF-kappaB activation. Binding occurred via a surface on the proximal ubiquitin moiety and the canonical Ile44 surface on the distal one, thereby providing specificity for linear chain recognition. Residues of NEMO involved in binding linear ubiquitin chains are required for NF-kappaB activation by TNF-alpha and other agonists, providing an explanation for the detrimental effect of NEMO mutations in patients suffering from X-linked ectodermal dysplasia and immunodeficiency.


Assuntos
Quinase I-kappa B/metabolismo , Subunidade p50 de NF-kappa B/metabolismo , Ubiquitina/metabolismo , Motivos de Aminoácidos , Displasia Ectodérmica/metabolismo , Humanos , Quinase I-kappa B/química , Modelos Moleculares , Ligação Proteica , Ubiquitina/química , Ubiquitinas/química , Ubiquitinas/metabolismo , Doenças por Imunodeficiência Combinada Ligada ao Cromossomo X/metabolismo
5.
Mol Cell ; 57(6): 995-1010, 2015 Mar 19.
Artigo em Inglês | MEDLINE | ID: mdl-25684205

RESUMO

The small Rho GTPase RAC1 is an essential regulator of cellular signaling that controls actin rearrangements and cell motility. Here, we identify a novel CUL3 RING ubiquitin ligase complex, containing the substrate adaptors KBTBD6 and KBTBD7, that mediates ubiquitylation and proteasomal degradation of TIAM1, a RAC1-specific GEF. Increasing the abundance of TIAM1 by depletion of KBTBD6 and/or KBTBD7 leads to elevated RAC1 activity, changes in actin morphology, loss of focal adhesions, reduced proliferation, and enhanced invasion. KBTBD6 and KBTBD7 employ ATG8 family-interacting motifs to bind preferentially to GABARAP proteins. Surprisingly, ubiquitylation and degradation of TIAM1 by CUL3(KBTBD6/KBTBD7) depends on its binding to GABARAP proteins. Our study reveals that recruitment of CUL3(KBTBD6/KBTBD7) to GABARAP-containing vesicles regulates the abundance of membrane-associated TIAM1 and subsequently spatially restricted RAC1 signaling. Besides their role in autophagy and trafficking, we uncovered a previously unknown function of GABARAP proteins as membrane-localized signaling scaffolds.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Culina/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Transativadores/metabolismo , Proteínas rac1 de Ligação ao GTP/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/química , Proteínas Adaptadoras de Transdução de Sinal/genética , Sequência de Aminoácidos , Proteínas Reguladoras de Apoptose , Família da Proteína 8 Relacionada à Autofagia , Proteínas Culina/genética , Fatores de Troca do Nucleotídeo Guanina/genética , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Proteínas dos Microfilamentos/metabolismo , Proteínas Associadas aos Microtúbulos/química , Proteínas Associadas aos Microtúbulos/genética , Modelos Moleculares , Dados de Sequência Molecular , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , Ressonância Magnética Nuclear Biomolecular , Conformação Proteica , Multimerização Proteica , Transdução de Sinais , Proteína 1 Indutora de Invasão e Metástase de Linfoma de Células T , Transativadores/genética , Ubiquitinação , Proteínas rac1 de Ligação ao GTP/genética
6.
Mol Cell ; 60(1): 89-104, 2015 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-26431026

RESUMO

Hereditary spastic paraplegias (HSPs) are a diverse group of neurodegenerative diseases that are characterized by axonopathy of the corticospinal motor neurons. A mutation in the gene encoding for Tectonin ß-propeller containing protein 2 (TECPR2) causes HSP that is complicated by neurological symptoms. While TECPR2 is a human ATG8 binding protein and positive regulator of autophagy, the exact function of TECPR2 is unknown. Here, we show that TECPR2 associates with several trafficking components, among them the COPII coat protein SEC24D. TECPR2 is required for stabilization of SEC24D protein levels, maintenance of functional ER exit sites (ERES), and efficient ER export in a manner dependent on binding to lipidated LC3C. TECPR2-deficient HSP patient cells display alterations in SEC24D abundance and ER export efficiency. Additionally, TECPR2 and LC3C are required for autophagosome formation, possibly through maintaining functional ERES. Collectively, these results reveal that TECPR2 functions as molecular scaffold linking early secretion pathway and autophagy.


Assuntos
Autofagia , Proteínas de Transporte/metabolismo , Retículo Endoplasmático/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Transporte Proteico , Paraplegia Espástica Hereditária/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Reguladoras de Apoptose , Proteínas de Transporte/genética , Células HeLa , Humanos , Mutação , Proteínas do Tecido Nervoso/genética , Paraplegia Espástica Hereditária/metabolismo , Proteínas de Transporte Vesicular/metabolismo
7.
Nature ; 538(7625): 402-405, 2016 Oct 20.
Artigo em Inglês | MEDLINE | ID: mdl-27732584

RESUMO

The post-translational modification of proteins with polyubiquitin regulates virtually all aspects of cell biology. Eight distinct chain linkage types co-exist in polyubiquitin and are independently regulated in cells. This 'ubiquitin code' determines the fate of the modified protein. Deubiquitinating enzymes of the ovarian tumour (OTU) family regulate cellular signalling by targeting distinct linkage types within polyubiquitin, and understanding their mechanisms of linkage specificity gives fundamental insights into the ubiquitin system. Here we reveal how the deubiquitinase Cezanne (also known as OTUD7B) specifically targets Lys11-linked polyubiquitin. Crystal structures of Cezanne alone and in complex with monoubiquitin and Lys11-linked diubiquitin, in combination with hydrogen-deuterium exchange mass spectrometry, enable us to reconstruct the enzymatic cycle in great detail. An intricate mechanism of ubiquitin-assisted conformational changes activates the enzyme, and while all chain types interact with the enzymatic S1 site, only Lys11-linked chains can bind productively across the active site and stimulate catalytic turnover. Our work highlights the plasticity of deubiquitinases and indicates that new conformational states can occur when a true substrate, such as diubiquitin, is bound at the active site.


Assuntos
Enzimas Desubiquitinantes/metabolismo , Endopeptidases/metabolismo , Lisina/metabolismo , Poliubiquitina/metabolismo , Biocatálise , Domínio Catalítico , Cristalografia por Raios X , Enzimas Desubiquitinantes/química , Enzimas Desubiquitinantes/genética , Medição da Troca de Deutério , Endopeptidases/química , Endopeptidases/genética , Ativação Enzimática , Humanos , Espectrometria de Massas , Modelos Moleculares , Ligação Proteica , Conformação Proteica , Especificidade por Substrato , Ubiquitinação , Ubiquitinas/metabolismo
8.
Mol Cell ; 54(3): 349-61, 2014 May 08.
Artigo em Inglês | MEDLINE | ID: mdl-24726327

RESUMO

Linear ubiquitin chains are implicated in the regulation of the NF-κB pathway, immunity, and inflammation. They are synthesized by the LUBAC complex containing the catalytic subunit HOIL-1-interacting protein (HOIP) and are disassembled by the linear ubiquitin-specific deubiquitinase OTULIN. Little is known about the regulation of these opposing activities. Here we demonstrate that HOIP and OTULIN interact and act as a bimolecular editing pair for linear ubiquitin signals in vivo. The HOIP PUB domain binds to the PUB interacting motif (PIM) of OTULIN and the chaperone VCP/p97. Structural studies revealed the basis of high-affinity interaction with the OTULIN PIM. The conserved Tyr56 of OTULIN makes critical contacts with the HOIP PUB domain, and its phosphorylation negatively regulates this interaction. Functionally, HOIP binding to OTULIN is required for the recruitment of OTULIN to the TNF receptor complex and to counteract HOIP-dependent activation of the NF-κB pathway.


Assuntos
Endopeptidases/química , NF-kappa B/metabolismo , Ubiquitina-Proteína Ligases/química , Adenosina Trifosfatases/química , Adenosina Trifosfatases/metabolismo , Sítios de Ligação , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/metabolismo , Cristalografia por Raios X , Endopeptidases/metabolismo , Células HeLa , Humanos , Ligação de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Modelos Moleculares , Fosforilação , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Processamento de Proteína Pós-Traducional , Estrutura Secundária de Proteína , Receptores do Fator de Necrose Tumoral/metabolismo , Transdução de Sinais , Termodinâmica , Ubiquitina-Proteína Ligases/metabolismo , Proteína com Valosina
9.
Nature ; 522(7556): 354-8, 2015 Jun 18.
Artigo em Inglês | MEDLINE | ID: mdl-26040720

RESUMO

The endoplasmic reticulum (ER) is the largest intracellular endomembrane system, enabling protein and lipid synthesis, ion homeostasis, quality control of newly synthesized proteins and organelle communication. Constant ER turnover and modulation is needed to meet different cellular requirements and autophagy has an important role in this process. However, its underlying regulatory mechanisms remain unexplained. Here we show that members of the FAM134 reticulon protein family are ER-resident receptors that bind to autophagy modifiers LC3 and GABARAP, and facilitate ER degradation by autophagy ('ER-phagy'). Downregulation of FAM134B protein in human cells causes an expansion of the ER, while FAM134B overexpression results in ER fragmentation and lysosomal degradation. Mutant FAM134B proteins that cause sensory neuropathy in humans are unable to act as ER-phagy receptors. Consistently, disruption of Fam134b in mice causes expansion of the ER, inhibits ER turnover, sensitizes cells to stress-induced apoptotic cell death and leads to degeneration of sensory neurons. Therefore, selective ER-phagy via FAM134 proteins is indispensable for mammalian cell homeostasis and controls ER morphology and turnover in mice and humans.


Assuntos
Autofagia/fisiologia , Retículo Endoplasmático/metabolismo , Proteínas de Membrana/metabolismo , Proteínas de Neoplasias/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Apoptose , Proteínas Reguladoras de Apoptose , Biomarcadores/metabolismo , Linhagem Celular , Retículo Endoplasmático/química , Feminino , Deleção de Genes , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Lisossomos/metabolismo , Masculino , Proteínas de Membrana/deficiência , Proteínas de Membrana/genética , Camundongos , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas de Neoplasias/deficiência , Proteínas de Neoplasias/genética , Fagossomos/metabolismo , Ligação Proteica , Células Receptoras Sensoriais/metabolismo , Células Receptoras Sensoriais/patologia
10.
Mol Cell ; 48(3): 329-42, 2012 Nov 09.
Artigo em Inglês | MEDLINE | ID: mdl-23022382

RESUMO

Autophagy protects cellular homeostasis by capturing cytosolic components and invading pathogens for lysosomal degradation. Autophagy receptors target cargo to autophagy by binding ATG8 on autophagosomal membranes. The expansion of the ATG8 family in higher eukaryotes suggests that specific interactions with autophagy receptors facilitate differential cargo handling. However, selective interactors of ATG8 orthologs are unknown. Here we show that the selectivity of the autophagy receptor NDP52 for LC3C is crucial for innate immunity since cells lacking either protein cannot protect their cytoplasm against Salmonella. LC3C is required for antibacterial autophagy because in its absence the remaining ATG8 orthologs do not support efficient antibacterial autophagy. Structural analysis revealed that the selectivity of NDP52 for LC3C is conferred by a noncanonical LIR, in which lack of an aromatic residue is balanced by LC3C-specific interactions. Our report illustrates that specificity in the interaction between autophagy receptors and autophagy machinery is of functional importance to execute selective autophagy.


Assuntos
Autofagia , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas Nucleares/metabolismo , Salmonella/metabolismo , Motivos de Aminoácidos/genética , Sequência de Aminoácidos , Western Blotting , Cristalografia por Raios X , Citoplasma/metabolismo , Citoplasma/microbiologia , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , Células HeLa , Humanos , Microscopia de Fluorescência , Proteínas Associadas aos Microtúbulos/química , Proteínas Associadas aos Microtúbulos/genética , Modelos Moleculares , Dados de Sequência Molecular , Proteínas Nucleares/química , Proteínas Nucleares/genética , Ligação Proteica , Estrutura Terciária de Proteína , Interferência de RNA , Salmonella/classificação , Salmonella typhimurium/metabolismo , Homologia de Sequência de Aminoácidos , Especificidade da Espécie
11.
J Cell Sci ; 129(5): 875-80, 2016 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-26906419

RESUMO

Ubiquitin plays an essential role in modulating protein functions, and deregulation of the ubiquitin system leads to the development of multiple human diseases. Owing to its molecular features, ubiquitin can form various homo- and heterotypic polymers on substrate proteins, thereby provoking distinct cellular responses. The concept of multifaceted ubiquitin chains encoding different functions has been substantiated in recent years. It has been established that all possible ubiquitin linkage types are utilized for chain assembly and propagation of specific signals in vivo. In addition, branched ubiquitin chains and phosphorylated ubiquitin molecules have been put under the spotlight recently. The development of novel technologies has provided detailed insights into the structure and function of previously poorly understood ubiquitin signals. In this Cell Science at a Glance article and accompanying poster, we provide an update on the complexity of ubiquitin chains and their physiological relevance.


Assuntos
Ubiquitina/química , Ubiquitinação , Sequência de Aminoácidos , Animais , Humanos , Imunidade Inata , Mitofagia , Proteólise , Transdução de Sinais , Ubiquitina/fisiologia
12.
Biochemistry ; 56(47): 6281-6291, 2017 11 28.
Artigo em Inglês | MEDLINE | ID: mdl-29094929

RESUMO

Serratia marcescens secretes a lipase, LipA, through a type I secretion system (T1SS). The T1SS for LipA, the Lip system, is composed of an inner membrane ABC transporter with its nucleotide-binding domains (NBD), LipB, a membrane fusion protein, LipC, and an outer membrane channel protein, LipD. Passenger protein secreted by this system has been functionally and structurally characterized well, but relatively little information about the transporter complex is available. Here, we report the crystallographic studies of LipC without the membrane anchor region, LipC-, and the NBD of LipB (LipB-NBD). LipC- crystallographic analysis has led to the determination of the structure of the long α-helical and lipoyl domains, but not the area where it interacts with LipB, suggesting that the region is flexible without LipB. The long α-helical domain has three α-helices, which interacts with LipD in the periplasm. LipB-NBD has the common overall architecture and ATP hydrolysis activity of ABC transporter NBDs. Using the predicted models of full-length LipB and LipD, the overall structural insight into the Lip system is discussed.


Assuntos
Proteínas de Bactérias/química , Lipase/química , Lipase/metabolismo , Proteínas de Fusão de Membrana/química , Fusão de Membrana/fisiologia , Nucleotídeos/metabolismo , Serratia marcescens/enzimologia , Proteínas de Bactérias/metabolismo , Cristalografia por Raios X , Proteínas de Fusão de Membrana/metabolismo , Nucleotídeos/química , Conformação Proteica
13.
J Biol Chem ; 291(17): 9025-41, 2016 Apr 22.
Artigo em Inglês | MEDLINE | ID: mdl-26929408

RESUMO

The covalent conjugation of ubiquitin-fold modifier 1 (UFM1) to proteins generates a signal that regulates transcription, response to cell stress, and differentiation. Ufmylation is initiated by ubiquitin-like modifier activating enzyme 5 (UBA5), which activates and transfers UFM1 to ubiquitin-fold modifier-conjugating enzyme 1 (UFC1). The details of the interaction between UFM1 and UBA5 required for UFM1 activation and its downstream transfer are however unclear. In this study, we described and characterized a combined linear LC3-interacting region/UFM1-interacting motif (LIR/UFIM) within the C terminus of UBA5. This single motif ensures that UBA5 binds both UFM1 and light chain 3/γ-aminobutyric acid receptor-associated proteins (LC3/GABARAP), two ubiquitin (Ub)-like proteins. We demonstrated that LIR/UFIM is required for the full biological activity of UBA5 and for the effective transfer of UFM1 onto UFC1 and a downstream protein substrate both in vitro and in cells. Taken together, our study provides important structural and functional insights into the interaction between UBA5 and Ub-like modifiers, improving the understanding of the biology of the ufmylation pathway.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Processamento de Proteína Pós-Traducional/fisiologia , Proteínas/metabolismo , Enzimas Ativadoras de Ubiquitina/metabolismo , Enzimas de Conjugação de Ubiquitina/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/química , Proteínas Adaptadoras de Transdução de Sinal/genética , Motivos de Aminoácidos , Proteínas Reguladoras de Apoptose , Células HEK293 , Humanos , Proteínas Associadas aos Microtúbulos/química , Proteínas Associadas aos Microtúbulos/genética , Estrutura Secundária de Proteína , Proteínas/química , Proteínas/genética , Relação Estrutura-Atividade , Enzimas Ativadoras de Ubiquitina/química , Enzimas Ativadoras de Ubiquitina/genética , Enzimas de Conjugação de Ubiquitina/química , Enzimas de Conjugação de Ubiquitina/genética
14.
Proc Natl Acad Sci U S A ; 108(6): 2228-33, 2011 Feb 08.
Artigo em Inglês | MEDLINE | ID: mdl-21266548

RESUMO

Crimean Congo hemorrhagic fever virus (CCHFV) is a deadly human pathogen that evades innate immune responses by efficiently interfering with antiviral signaling pathways mediated by NF-κB, IRF3, and IFNα/ß. These pathways rely on protein ubiquitination for their activation, and one outcome is the modification of proteins with the ubiquitin (Ub)-like modifier interferon-stimulated gene (ISG)15. CCHFV and related viruses encode a deubiquitinase (DUB) of the ovarian tumor (OTU) family, which unlike eukaryotic OTU DUBs also targets ISG15 modifications. Here we characterized the viral OTU domain of CCHFV (vOTU) biochemically and structurally, revealing that it hydrolyzes four out of six tested Ub linkages, but lacks activity against linear and K29-linked Ub chains. vOTU cleaved Ub and ISG15 with similar kinetics, and we were able to understand vOTU cross-reactivity at the molecular level from crystal structures of vOTU in complex with Ub and ISG15. An N-terminal extension in vOTU not present in eukaryotic OTU binds to the hydrophobic Ile44 patch of Ub, which results in a dramatically different Ub orientation compared to a eukaryotic OTU-Ub complex. The C-terminal Ub-like fold of ISG15 (ISG15-C) adopts an equivalent binding orientation. Interestingly, ISG15-C contains an additional second hydrophobic surface that is specifically contacted by vOTU. These subtle differences in Ub/ISG15 binding allowed the design of vOTU variants specific for either Ub or ISG15, which will be useful tools to understand the relative contribution of ubiquitination vs. ISGylation in viral infection. Furthermore, the crystal structures will allow structure-based design of antiviral agents targeting this enzyme.


Assuntos
Citocinas/imunologia , Vírus da Febre Hemorrágica da Crimeia-Congo/imunologia , Neoplasias Ovarianas , Peptídeo Hidrolases/imunologia , Ubiquitina/imunologia , Ubiquitinação/imunologia , Ubiquitinas/imunologia , Proteínas Virais/imunologia , Citocinas/genética , Citocinas/metabolismo , Feminino , Vírus da Febre Hemorrágica da Crimeia-Congo/enzimologia , Vírus da Febre Hemorrágica da Crimeia-Congo/genética , Humanos , Peptídeo Hidrolases/genética , Peptídeo Hidrolases/metabolismo , Estrutura Quaternária de Proteína , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Relação Estrutura-Atividade , Ubiquitina/genética , Ubiquitina/metabolismo , Ubiquitinas/genética , Ubiquitinas/metabolismo , Proteínas Virais/genética , Proteínas Virais/metabolismo
15.
EMBO Rep ; 12(4): 350-7, 2011 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-21399617

RESUMO

Modification of proteins by ubiquitin (Ub) and Ub-like (Ubl) modifiers regulates a variety of cellular functions. The ability of Ub to form chains of eight structurally and functionally distinct types adds further complexity to the system. Ub-specific proteases (USPs) hydrolyse polyUb chains, and some have been suggested to be cross-reactive with Ubl modifiers, such as neural precursor cell expressed, developmentally downregulated 8 (NEDD8) and interferon-stimulated gene 15 (ISG15). Here, we report that USP21 cleaves Ub polymers, and with reduced activity also targets ISG15, but is inactive against NEDD8. A crystal structure of USP21 in complex with linear diUb aldehyde shows how USP21 interacts with polyUb through a previously unidentified second Ub- and ISG15-binding surface on the USP domain core. We also rationalize the inability of USP21 to target NEDD8 and identify differences that allow USPs to distinguish between structurally related modifications.


Assuntos
Poliubiquitina/metabolismo , Ubiquitina Tiolesterase/química , Ubiquitina Tiolesterase/metabolismo , Sítios de Ligação/fisiologia , Cristalografia por Raios X , Citocinas/química , Citocinas/metabolismo , Humanos , Proteína NEDD8 , Ligação Proteica , Ubiquitinas/química , Ubiquitinas/metabolismo
16.
Nat Commun ; 13(1): 1790, 2022 04 04.
Artigo em Inglês | MEDLINE | ID: mdl-35379805

RESUMO

Despite the recent clinical success of T cell checkpoint inhibition targeting the CTLA-4 and PD-1 pathways, many patients either fail to achieve objective responses or they develop resistance to therapy. In some cases, poor responses to checkpoint blockade have been linked to suboptimal CD28 costimulation and the inability to generate and maintain a productive adaptive anti-tumor immune response. To address this, here we utilize directed evolution to engineer a CD80 IgV domain with increased PD-L1 affinity and fuse this to an immunoglobulin Fc domain, creating a therapeutic (ALPN-202, davoceticept) capable of providing CD28 costimulation in a PD-L1-dependent fashion while also antagonizing PD-1 - PD-L1 and CTLA-4-CD80/CD86 interactions. We demonstrate that by combining CD28 costimulation and dual checkpoint inhibition, ALPN-202 enhances T cell activation and anti-tumor efficacy in cell-based assays and mouse tumor models more potently than checkpoint blockade alone and thus has the potential to generate potent, clinically meaningful anti-tumor immunity in humans.


Assuntos
Antígenos CD28 , Neoplasias , Animais , Antígeno B7-1/metabolismo , Antígenos CD28/metabolismo , Humanos , Ativação Linfocitária , Camundongos , Neoplasias/tratamento farmacológico , Neoplasias/genética , Linfócitos T
17.
J Biol Chem ; 285(1): 365-72, 2010 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-19887378

RESUMO

PLAA (ortholog of yeast Doa1/Ufd3, also know as human PLAP or phospholipase A2-activating protein) has been implicated in a variety of disparate biological processes that involve the ubiquitin system. It is linked to the maintenance of ubiquitin levels, but the mechanism by which it accomplishes this is unclear. The C-terminal PUL (PLAP, Ufd3p, and Lub1p) domain of PLAA binds p97, an AAA ATPase, which among other functions helps transfer ubiquitinated proteins to the proteasome for degradation. In yeast, loss of Doa1 is suppressed by altering p97/Cdc48 function indicating that physical interaction between PLAA and p97 is functionally important. Although the overall regions of interaction between these proteins are known, the structural basis has been unavailable. We solved the high resolution crystal structure of the p97-PLAA complex showing that the PUL domain forms a 6-mer Armadillo-containing domain. Its N-terminal extension folds back onto the inner curvature forming a deep ridge that is positively charged with residues that are phylogenetically conserved. The C terminus of p97 binds in this ridge, where the side chain of p97-Tyr(805), implicated in phosphorylation-dependent regulation, is buried. Expressed in doa1Delta null cells, point mutants of the yeast ortholog Doa1 that disrupt this interaction display slightly reduced ubiquitin levels, but unlike doa1Delta null cells, showed only some of the growth phenotypes. These data suggest that the p97-PLAA interaction is important for a subset of PLAA-dependent biological processes and provides a framework to better understand the role of these complex molecules in the ubiquitin system.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/química , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Adenosina Trifosfatases/química , Adenosina Trifosfatases/metabolismo , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/metabolismo , Proteínas Nucleares/química , Proteínas/química , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Cristalografia por Raios X , Polarização de Fluorescência , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Mutação/genética , Proteínas Nucleares/metabolismo , Ligação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Proteínas/metabolismo , Saccharomyces cerevisiae/citologia , Alinhamento de Sequência , Proteína com Valosina
18.
Commun Biol ; 4(1): 1238, 2021 10 29.
Artigo em Inglês | MEDLINE | ID: mdl-34716405

RESUMO

C-phycocyanin (CPC), a blue pigment protein, is an indispensable component of giant phycobilisomes, which are light-harvesting antenna complexes in cyanobacteria that transfer energy efficiently to photosystems I and II. X-ray crystallographic and electron microscopy (EM) analyses have revealed the structure of CPC to be a closed toroidal hexamer by assembling two trimers. In this study, the structural characterization of non-conventional octameric CPC is reported for the first time. Analyses of the crystal and cryogenic EM structures of the native CPC from filamentous thermophilic cyanobacterium Thermoleptolyngbya sp. O-77 unexpectedly illustrated the coexistence of conventional hexamer and novel octamer. In addition, an unusual dimeric state, observed via analytical ultracentrifugation, was postulated to be a key intermediate structure in the assemble of the previously unobserved octamer. These observations provide new insights into the assembly processes of CPCs and the mechanism of energy transfer in the light-harvesting complexes.


Assuntos
Proteínas de Bactérias/química , Cianobactérias/química , Ficocianina/química
19.
J Med Chem ; 64(7): 3720-3746, 2021 04 08.
Artigo em Inglês | MEDLINE | ID: mdl-33769048

RESUMO

Autophagy is the common name for a number of lysosome-based degradation pathways of cytosolic cargos. The key components of autophagy are members of Atg8 family proteins involved in almost all steps of the process, from autophagosome formation to their selective fusion with lysosomes. In this study, we show that the homologous members of the human Atg8 family proteins, LC3A and LC3B, are druggable by a small molecule inhibitor novobiocin. Structure-activity relationship (SAR) studies of the 4-hydroxy coumarin core scaffold were performed, supported by a crystal structure of the LC3A dihydronovobiocin complex. The study reports the first nonpeptide inhibitors for these protein interaction targets and will lay the foundation for the development of more potent chemical probes for the Atg8 protein family which may also find applications for the development of autophagy-mediated degraders (AUTACs).


Assuntos
4-Hidroxicumarinas/farmacologia , Autofagia/efeitos dos fármacos , Proteínas Associadas aos Microtúbulos/metabolismo , Ligação Proteica/efeitos dos fármacos , Proteína Sequestossoma-1/metabolismo , 4-Hidroxicumarinas/síntese química , 4-Hidroxicumarinas/metabolismo , Células HEK293 , Humanos , Ligantes , Estrutura Molecular , Novobiocina/química , Relação Estrutura-Atividade
20.
Nat Methods ; 4(12): 1019-21, 2007 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-17982461

RESUMO

We tested the general applicability of in situ proteolysis to form protein crystals suitable for structure determination by adding a protease (chymotrypsin or trypsin) digestion step to crystallization trials of 55 bacterial and 14 human proteins that had proven recalcitrant to our best efforts at crystallization or structure determination. This is a work in progress; so far we determined structures of 9 bacterial proteins and the human aminoimidazole ribonucleotide synthetase (AIRS) domain.


Assuntos
Cristalização/métodos , Cristalografia/métodos , Peptídeo Hidrolases/química , Proteínas/química , Proteínas/ultraestrutura , Conformação Proteica
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