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1.
Front Immunol ; 12: 626308, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33854501

RESUMO

We have previously shown that conformational change in the ß2-integrin is a very early activation marker that can be detected with fluorescent multimers of its ligand intercellular adhesion molecule (ICAM)-1 for rapid assessment of antigen-specific CD8+ T cells. In this study, we describe a modified protocol of this assay for sensitive detection of functional antigen-specific CD4+ T cells using a monoclonal antibody (clone m24 Ab) specific for the open, high-affinity conformation of the ß2-integrin. The kinetics of ß2-integrin activation was different on CD4+ and CD8+ T cells (several hours vs. few minutes, respectively); however, m24 Ab readily stained both cell types 4-6 h after antigen stimulation. With this protocol, we were able to monitor ex vivo effector and memory CD4+ and CD8+ T cells specific for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), cytomegalovirus (CMV), Epstein-Barr virus (EBV), and hepatitis B virus (HBV) in whole blood or cryopreserved peripheral blood mononuclear cells (PBMCs) of infected or vaccinated individuals. By costaining ß2-integrin with m24 and CD154 Abs, we assessed extremely low frequencies of polyfunctional CD4+ T cell responses. The novel assay used in this study allows very sensitive and simultaneous screening of both CD4+ and CD8+ T cell reactivities, with versatile applicability in clinical and vaccination studies.


Assuntos
Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Interações Hospedeiro-Patógeno/imunologia , Integrinas/metabolismo , Adulto , Idoso , Sequência de Aminoácidos , Sítios de Ligação , /imunologia , /virologia , Proteínas de Transporte/química , Citocinas/metabolismo , Citomegalovirus/imunologia , Epitopos de Linfócito T/química , Epitopos de Linfócito T/imunologia , Feminino , Antígenos HLA/química , Antígenos HLA/imunologia , Interações Hospedeiro-Patógeno/genética , Humanos , Imuno-Histoquímica , Imunofenotipagem , Integrinas/genética , Molécula 1 de Adesão Intercelular/química , Molécula 1 de Adesão Intercelular/metabolismo , Ativação Linfocitária/imunologia , Masculino , Pessoa de Meia-Idade , Ligação Proteica , Multimerização Proteica , Especificidade do Receptor de Antígeno de Linfócitos T , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo
2.
Int J Mol Sci ; 22(6)2021 Mar 14.
Artigo em Inglês | MEDLINE | ID: mdl-33799326

RESUMO

The human zinc transporter ZnT8 provides the granules of pancreatic ß-cells with zinc (II) ions for assembly of insulin hexamers for storage. Until recently, the structure and function of human ZnTs have been modelled on the basis of the 3D structures of bacterial zinc exporters, which form homodimers with each monomer having six transmembrane α-helices harbouring the zinc transport site and a cytosolic domain with an α,ß structure and additional zinc-binding sites. However, there are important differences in function as the bacterial proteins export an excess of zinc ions from the bacterial cytoplasm, whereas ZnT8 exports zinc ions into subcellular vesicles when there is no apparent excess of cytosolic zinc ions. Indeed, recent structural investigations of human ZnT8 show differences in metal binding in the cytosolic domain when compared to the bacterial proteins. Two common variants, one with tryptophan (W) and the other with arginine (R) at position 325, have generated considerable interest as the R-variant is associated with a higher risk of developing type 2 diabetes. Since the mutation is at the apex of the cytosolic domain facing towards the cytosol, it is not clear how it can affect zinc transport through the transmembrane domain. We expressed the cytosolic domain of both variants of human ZnT8 and have begun structural and functional studies. We found that (i) the metal binding of the human protein is different from that of the bacterial proteins, (ii) the human protein has a C-terminal extension with three cysteine residues that bind a zinc(II) ion, and (iii) there are small differences in stability between the two variants. In this investigation, we employed nickel(II) ions as a probe for the spectroscopically silent Zn(II) ions and utilised colorimetric and fluorimetric indicators for Ni(II) ions to investigate metal binding. We established Ni(II) coordination to the C-terminal cysteines and found differences in metal affinity and coordination in the two ZnT8 variants. These structural differences are thought to be critical for the functional differences regarding the diabetes risk. Further insight into the assembly of the metal centres in the cytosolic domain was gained from potentiometric investigations of zinc binding to synthetic peptides corresponding to N-terminal and C-terminal sequences of ZnT8 bearing the metal-coordinating ligands. Our work suggests the involvement of the C-terminal cysteines, which are part of the cytosolic domain, in a metal chelation and/or acquisition mechanism and, as now supported by the high-resolution structural work, provides the first example of metal-thiolate coordination chemistry in zinc transporters.


Assuntos
Proteínas de Transporte/ultraestrutura , Insulina/genética , Relação Estrutura-Atividade , Transportador 8 de Zinco/ultraestrutura , Proteínas de Transporte/química , Proteínas de Transporte/genética , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/patologia , Humanos , Insulina/metabolismo , Células Secretoras de Insulina/metabolismo , Conformação Molecular , Níquel/química , Conformação Proteica em alfa-Hélice/genética , Domínios Proteicos/genética , Zinco/química , Transportador 8 de Zinco/química , Transportador 8 de Zinco/genética
3.
Nat Commun ; 12(1): 1543, 2021 03 09.
Artigo em Inglês | MEDLINE | ID: mdl-33750839

RESUMO

Protein engineering has great potential for devising multifunctional recombinant proteins to serve as next-generation protein therapeutics, but it often requires drastic modifications of the parental protein scaffolds e.g., additional domains at the N/C-terminus or replacement of a domain by another. A discovery platform system, called RaPID (Random non-standard Peptides Integrated Discovery) system, has enabled rapid discovery of small de novo macrocyclic peptides that bind a target protein with high binding specificity and affinity. Capitalizing on the optimized binding properties of the RaPID-derived peptides, here we show that RaPID-derived pharmacophore sequences can be readily implanted into surface-exposed loops on recombinant proteins and maintain both the parental peptide binding function(s) and the host protein function. We refer to this protein engineering method as lasso-grafting and demonstrate that it can endow specific binding capacity toward various receptors into a diverse set of scaffolds that includes IgG, serum albumin, and even capsid proteins of adeno-associated virus, enabling us to rapidly formulate and produce bi-, tri-, and even tetra-specific binder molecules.


Assuntos
Peptídeos/química , Peptídeos/farmacologia , Engenharia de Proteínas/métodos , Proteínas do Capsídeo/química , Proteínas de Transporte/química , Linhagem Celular , Dependovirus , Humanos , Imunoglobulina G/química , Modelos Moleculares , Albumina Sérica/química , Bibliotecas de Moléculas Pequenas
4.
Nat Commun ; 12(1): 1379, 2021 03 02.
Artigo em Inglês | MEDLINE | ID: mdl-33654074

RESUMO

Many immune responses depend upon activation of NF-κB, an important transcription factor in the elicitation of a cytokine response. Here we show that N4BP1 inhibits TLR-dependent activation of NF-κB by interacting with the NF-κB signaling essential modulator (NEMO, also known as IκB kinase γ) to attenuate NEMO-NEMO dimerization or oligomerization. The UBA-like (ubiquitin associated-like) and CUE-like (ubiquitin conjugation to ER degradation-like) domains in N4BP1 mediate interaction with the NEMO COZI domain. Both in vitro and in mice, N4bp1 deficiency specifically enhances TRIF-independent (TLR2, TLR7, or TLR9-mediated) but not TRIF-dependent (TLR3 or TLR4-mediated) NF-κB activation, leading to increased production of proinflammatory cytokines. In response to TLR4 or TLR3 activation, TRIF causes activation of caspase-8, which cleaves N4BP1 distal to residues D424 and D490 and abolishes its inhibitory effect. N4bp1-/- mice also have diminished numbers of T cells in the peripheral blood. Our work identifies N4BP1 as an inhibitory checkpoint protein that must be overcome to activate NF-κB, and a TRIF-initiated caspase-8-dependent mechanism by which this is accomplished.


Assuntos
Proteínas de Transporte/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , NF-kappa B/metabolismo , Multimerização Proteica , Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Animais , Proteínas de Transporte/química , Proteínas de Transporte/genética , Caspase 8/metabolismo , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Células HEK293 , Herpesvirus Humano 1/fisiologia , Humanos , Interleucina-6/sangue , Macrófagos Peritoneais/efeitos dos fármacos , Macrófagos Peritoneais/metabolismo , Camundongos Endogâmicos C57BL , Mutação/genética , Inibidor de NF-kappaB alfa/metabolismo , Oligodesoxirribonucleotídeos/farmacologia , Fosforilação/efeitos dos fármacos , Ligação Proteica/efeitos dos fármacos , Domínios Proteicos , Multimerização Proteica/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Ubiquitina/metabolismo
5.
Mol Cell ; 81(6): 1246-1259.e8, 2021 03 18.
Artigo em Inglês | MEDLINE | ID: mdl-33548203

RESUMO

The Integrator is a specialized 3' end-processing complex involved in cleavage and transcription termination of a subset of nascent RNA polymerase II transcripts, including small nuclear RNAs (snRNAs). We provide evidence of the modular nature of the Integrator complex by biochemically characterizing its two subcomplexes, INTS5/8 and INTS10/13/14. Using cryoelectron microscopy (cryo-EM), we determined a 3.5-Å-resolution structure of the INTS4/9/11 ternary complex, which constitutes Integrator's catalytic core. Our structure reveals the spatial organization of the catalytic nuclease INTS11, bound to its catalytically impaired homolog INTS9 via several interdependent interfaces. INTS4, a helical repeat protein, plays a key role in stabilizing nuclease domains and other components. In this assembly, all three proteins form a composite electropositive groove, suggesting a putative RNA binding path within the complex. Comparison with other 3' end-processing machineries points to distinct features and a unique architecture of the Integrator's catalytic module.


Assuntos
Complexos Multiproteicos , Terminação da Transcrição Genética , Proteínas de Transporte/química , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Microscopia Crioeletrônica , Células HEK293 , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/química , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Complexos Multiproteicos/química , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , Complexos Multiproteicos/ultraestrutura , Subunidades Proteicas/química , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo
6.
Int J Mol Sci ; 22(4)2021 Feb 04.
Artigo em Inglês | MEDLINE | ID: mdl-33557270

RESUMO

ß(1,3)-glucans are a component of fungal and plant cell walls. The ß-glucan of pathogens is recognized as a non-self-component in the host defense system. Long ß-glucan chains are capable of forming a triple helix structure, and the tertiary structure may profoundly affect the interaction with ß-glucan-binding proteins. Although the atomic details of ß-glucan binding and signaling of cognate receptors remain mostly unclear, X-ray crystallography and NMR analyses have revealed some aspects of ß-glucan structure and interaction. Here, we will review three-dimensional (3D) structural characteristics of ß-glucans and the modes of interaction with ß-glucan-binding proteins.


Assuntos
Proteínas de Transporte/química , Proteínas de Transporte/metabolismo , Lectinas/química , Lectinas/metabolismo , beta-Glucanas/química , beta-Glucanas/metabolismo , Animais , Sítios de Ligação , Cristalografia por Raios X , Humanos , Modelos Moleculares , Ligação Proteica
7.
Int J Mol Sci ; 22(4)2021 Feb 07.
Artigo em Inglês | MEDLINE | ID: mdl-33562355

RESUMO

Synphilin-1 has previously been identified as an interaction partner of α-Synuclein (αSyn), a primary constituent of neurodegenerative disease-linked Lewy bodies. In this study, the repercussions of a disrupted glyoxalase system and aldose reductase function on Synphilin-1 inclusion formation characteristics and cell growth were investigated. To this end, either fluorescent dsRed-tagged or non-tagged human SNCAIP, which encodes the Synphilin-1 protein, was expressed in Saccharomyces cerevisiae and Schizosaccharomyces pombe yeast strains devoid of enzymes Glo1, Glo2, and Gre3. Presented data shows that lack of Glo2 and Gre3 activity in S. cerevisiae increases the formation of large Synphilin-1 inclusions. This correlates with enhanced oxidative stress levels and an inhibitory effect on exponential growth, which is most likely caused by deregulation of autophagic degradation capacity, due to excessive Synphilin-1 aggresome build-up. These findings illustrate the detrimental impact of increased oxidation and glycation on Synphilin-1 inclusion formation. Similarly, polar-localised inclusions were observed in wild-type S. pombe cells and strains deleted for either glo1+ or glo2+. Contrary to S. cerevisiae, however, no growth defects were observed upon expression of SNCAIP. Altogether, our findings show the relevance of yeasts, especially S. cerevisiae, as complementary models to unravel mechanisms contributing to Synphilin-1 pathology in the context of neurodegenerative diseases.


Assuntos
Aldeído Redutase/antagonistas & inibidores , Proteínas de Transporte/química , Proteínas de Transporte/metabolismo , Lactoilglutationa Liase/antagonistas & inibidores , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/metabolismo , Estresse Oxidativo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/crescimento & desenvolvimento , Oxirredutases do Álcool/genética , Oxirredutases do Álcool/metabolismo , Aldeído Redutase/genética , Aldeído Redutase/metabolismo , Glicosilação , Humanos , Corpos de Inclusão , Lactoilglutationa Liase/genética , Lactoilglutationa Liase/metabolismo , Oxirredução , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética
8.
Nature ; 591(7848): 157-161, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33597751

RESUMO

Citrate is best known as an intermediate in the tricarboxylic acid cycle of the cell. In addition to this essential role in energy metabolism, the tricarboxylate anion also acts as both a precursor and a regulator of fatty acid synthesis1-3. Thus, the rate of fatty acid synthesis correlates directly with the cytosolic concentration of citrate4,5. Liver cells import citrate through the sodium-dependent citrate transporter NaCT (encoded by SLC13A5) and, as a consequence, this protein is a potential target for anti-obesity drugs. Here, to understand the structural basis of its inhibition mechanism, we determined cryo-electron microscopy structures of human NaCT in complexes with citrate or a small-molecule inhibitor. These structures reveal how the inhibitor-which binds to the same site as citrate-arrests the transport cycle of NaCT. The NaCT-inhibitor structure also explains why the compound selectively inhibits NaCT over two homologous human dicarboxylate transporters, and suggests ways to further improve the affinity and selectivity. Finally, the NaCT structures provide a framework for understanding how various mutations abolish the transport activity of NaCT in the brain and thereby cause epilepsy associated with mutations in SLC13A5 in newborns (which is known as SLC13A5-epilepsy)6-8.


Assuntos
Proteínas de Transporte/antagonistas & inibidores , Proteínas de Transporte/química , Ácido Cítrico/metabolismo , Microscopia Crioeletrônica , Malatos/farmacologia , Fenilbutiratos/farmacologia , Simportadores/antagonistas & inibidores , Simportadores/química , Sítios de Ligação , Encéfalo/metabolismo , Proteínas de Transporte/genética , Proteínas de Transporte/ultraestrutura , Ácido Cítrico/química , Transportadores de Ácidos Dicarboxílicos/química , Transportadores de Ácidos Dicarboxílicos/metabolismo , Epilepsia/genética , Epilepsia/metabolismo , Humanos , Malatos/química , Modelos Moleculares , Mutação , Fenilbutiratos/química , Multimerização Proteica , Sódio/metabolismo , Especificidade por Substrato/efeitos dos fármacos , Especificidade por Substrato/genética , Simportadores/genética , Simportadores/ultraestrutura
9.
Nat Commun ; 12(1): 1230, 2021 02 23.
Artigo em Inglês | MEDLINE | ID: mdl-33623002

RESUMO

The recently discovered lytic polysaccharide monooxygenases (LPMOs), which cleave polysaccharides by oxidation, have been associated with bacterial virulence, but supporting functional data is scarce. Here we show that CbpD, the LPMO of Pseudomonas aeruginosa, is a chitin-oxidizing virulence factor that promotes survival of the bacterium in human blood. The catalytic activity of CbpD was promoted by azurin and pyocyanin, two redox-active virulence factors also secreted by P. aeruginosa. Homology modeling, molecular dynamics simulations, and small angle X-ray scattering indicated that CbpD is a monomeric tri-modular enzyme with flexible linkers. Deletion of cbpD rendered P. aeruginosa unable to establish a lethal systemic infection, associated with enhanced bacterial clearance in vivo. CbpD-dependent survival of the wild-type bacterium was not attributable to dampening of pro-inflammatory responses by CbpD ex vivo or in vivo. Rather, we found that CbpD attenuates the terminal complement cascade in human serum. Studies with an active site mutant of CbpD indicated that catalytic activity is crucial for virulence function. Finally, profiling of the bacterial and splenic proteomes showed that the lack of this single enzyme resulted in substantial re-organization of the bacterial and host proteomes. LPMOs similar to CbpD occur in other pathogens and may have similar immune evasive functions.


Assuntos
Proteínas de Bactérias/metabolismo , Proteínas de Transporte/metabolismo , Oxigenases de Função Mista/metabolismo , Polissacarídeos/metabolismo , Infecções por Pseudomonas/enzimologia , Infecções por Pseudomonas/microbiologia , Pseudomonas aeruginosa/enzimologia , Pseudomonas aeruginosa/patogenicidade , Animais , Proteínas de Bactérias/química , Proteínas de Transporte/química , Morte Celular , Proteínas do Sistema Complemento/metabolismo , Humanos , Camundongos , Viabilidade Microbiana , Oxirredução , Domínios Proteicos , Proteoma/metabolismo , Proteômica , Infecções por Pseudomonas/sangue , Especificidade por Substrato , Transcrição Genética , Virulência , Fatores de Virulência/metabolismo
10.
Nat Commun ; 12(1): 1028, 2021 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-33589610

RESUMO

Upon binding to DNA breaks, poly(ADP-ribose) polymerase 1 (PARP1) ADP-ribosylates itself and other factors to initiate DNA repair. Serine is the major residue for ADP-ribosylation upon DNA damage, which strictly depends on HPF1. Here, we report the crystal structures of human HPF1/PARP1-CAT ΔHD complex at 1.98 Å resolution, and mouse and human HPF1 at 1.71 Å and 1.57 Å resolution, respectively. Our structures and mutagenesis data confirm that the structural insights obtained in a recent HPF1/PARP2 study by Suskiewicz et al. apply to PARP1. Moreover, we quantitatively characterize the key residues necessary for HPF1/PARP1 binding. Our data show that through salt-bridging to Glu284/Asp286, Arg239 positions Glu284 to catalyze serine ADP-ribosylation, maintains the local conformation of HPF1 to limit PARP1 automodification, and facilitates HPF1/PARP1 binding by neutralizing the negative charge of Glu284. These findings, along with the high-resolution structural data, may facilitate drug discovery targeting PARP1.


Assuntos
Proteínas de Transporte/química , DNA/química , Histonas/química , Proteínas Nucleares/química , Poli(ADP-Ribose) Polimerase-1/química , Serina/metabolismo , ADP-Ribosilação , Sequência de Aminoácidos , Animais , Sítios de Ligação , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Clonagem Molecular , Cristalografia por Raios X , DNA/genética , DNA/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Glutamina/metabolismo , Histonas/genética , Histonas/metabolismo , Humanos , Camundongos , Modelos Moleculares , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Poli(ADP-Ribose) Polimerase-1/genética , Poli(ADP-Ribose) Polimerase-1/metabolismo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Eletricidade Estática
11.
Molecules ; 26(2)2021 Jan 06.
Artigo em Inglês | MEDLINE | ID: mdl-33419110

RESUMO

(1) Background: Non-specific lipid transfer proteins (nsLTPs), which belong to the prolamin superfamily, are potent allergens. While the biological role of LTPs is still not well understood, it is known that these proteins bind lipids. Allergen nsLTPs are characterized by significant stability and resistance to digestion. (2) Methods: nsLTPs from gold kiwifruit (Act c 10.0101) and pomegranate (Pun g 1.0101) were isolated from their natural sources and structurally characterized using X-ray crystallography (3) Results: Both proteins crystallized and their crystal structures were determined. The proteins have a very similar overall fold with characteristic compact, mainly α-helical structures. The C-terminal sequence of Act c 10.0101 was updated based on our structural and mass spectrometry analysis. Information on proteins' sequences and structures was used to estimate the risk of cross-reactive reactions between Act c 10.0101 or Pun g 1.0101 and other allergens from this family of proteins. (4) Conclusions: Structural studies indicate a conformational flexibility of allergens from the nsLTP family and suggest that immunoglobulin E binding to some surface regions of these allergens may depend on ligand binding. Both Act c 10.0101 and Pun g 1.0101 are likely to be involved in cross-reactive reactions involving other proteins from the nsLTP family.


Assuntos
Actinidia/química , Alérgenos/química , Antígenos de Plantas/química , Proteínas de Transporte/química , Proteínas de Plantas/química , Romã (Fruta)/química , Sementes/química , Alérgenos/isolamento & purificação , Antígenos de Plantas/isolamento & purificação , Proteínas de Transporte/isolamento & purificação , Cristalografia por Raios X , Proteínas de Plantas/isolamento & purificação , Conformação Proteica em alfa-Hélice
12.
Mol Cell ; 81(6): 1231-1245.e8, 2021 03 18.
Artigo em Inglês | MEDLINE | ID: mdl-33503405

RESUMO

ATR checkpoint signaling is crucial for cellular responses to DNA replication impediments. Using an optogenetic platform, we show that TopBP1, the main activator of ATR, self-assembles extensively to yield micrometer-sized condensates. These opto-TopBP1 condensates are functional entities organized in tightly packed clusters of spherical nano-particles. TopBP1 condensates are reversible, occasionally fuse, and co-localize with TopBP1 partner proteins. We provide evidence that TopBP1 condensation is a molecular switch that amplifies ATR activity to phosphorylate checkpoint kinase 1 (Chk1) and slow down replication forks. Single amino acid substitutions of key residues in the intrinsically disordered ATR activation domain disrupt TopBP1 condensation and consequently ATR/Chk1 signaling. In physiologic salt concentration and pH, purified TopBP1 undergoes liquid-liquid phase separation in vitro. We propose that the actuation mechanism of ATR signaling is the assembly of TopBP1 condensates driven by highly regulated multivalent and cooperative interactions.


Assuntos
Proteínas Mutadas de Ataxia Telangiectasia , Proteínas de Transporte , Núcleo Celular , Proteínas de Ligação a DNA , Mutação de Sentido Incorreto , Proteínas Nucleares , Transdução de Sinais , Substituição de Aminoácidos , Animais , Proteínas Mutadas de Ataxia Telangiectasia/química , Proteínas Mutadas de Ataxia Telangiectasia/genética , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Proteínas de Transporte/química , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Núcleo Celular/química , Núcleo Celular/genética , Núcleo Celular/metabolismo , Quinase 1 do Ponto de Checagem/química , Quinase 1 do Ponto de Checagem/genética , Quinase 1 do Ponto de Checagem/metabolismo , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Células HeLa , Humanos , Proteínas Nucleares/química , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Células Sf9 , Spodoptera
13.
Nat Chem Biol ; 17(3): 254-262, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33398168

RESUMO

Degrons are elements within protein substrates that mediate the interaction with specific degradation machineries to control proteolysis. Recently, a few classes of C-terminal degrons (C-degrons) that are recognized by dedicated cullin-RING ligases (CRLs) have been identified. Specifically, CRL2 using the related substrate adapters FEM1A/B/C was found to recognize C degrons ending with arginine (Arg/C-degron). Here, we uncover the molecular mechanism of Arg/C-degron recognition by solving a subset of structures of FEM1 proteins in complex with Arg/C-degron-bearing substrates. Our structural research, complemented by binding assays and global protein stability (GPS) analyses, demonstrates that FEM1A/C and FEM1B selectively target distinct classes of Arg/C-degrons. Overall, our study not only sheds light on the molecular mechanism underlying Arg/C-degron recognition for precise control of substrate turnover, but also provides valuable information for development of chemical probes for selectively regulating proteostasis.


Assuntos
Arginina/química , Proteínas de Transporte/química , Proteínas de Ciclo Celular/química , Complexo de Endopeptidases do Proteassoma/metabolismo , Complexos Ubiquitina-Proteína Ligase/química , Sequência de Aminoácidos , Arginina/metabolismo , Sítios de Ligação , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Clonagem Molecular , Cristalografia por Raios X , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Células HEK293 , Humanos , Modelos Moleculares , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Proteólise , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Especificidade por Substrato , Complexos Ubiquitina-Proteína Ligase/genética , Complexos Ubiquitina-Proteína Ligase/metabolismo
14.
Nat Chem Biol ; 17(3): 263-271, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33398170

RESUMO

Proteome integrity depends on the ubiquitin-proteasome system to degrade unwanted or abnormal proteins. In addition to the N-degrons, C-terminal residues of proteins can also serve as degradation signals (C-degrons) that are recognized by specific cullin-RING ubiquitin ligases (CRLs) for proteasomal degradation. FEM1C is a CRL2 substrate receptor that targets the C-terminal arginine degron (Arg/C-degron), but the molecular mechanism of substrate recognition remains largely elusive. Here, we present crystal structures of FEM1C in complex with Arg/C-degron and show that FEM1C utilizes a semi-open binding pocket to capture the C-terminal arginine and that the extreme C-terminal arginine is the major structural determinant in recognition by FEM1C. Together with biochemical and mutagenesis studies, we provide a framework for understanding molecular recognition of the Arg/C-degron by the FEM family of proteins.


Assuntos
Arginina/química , Proteínas de Transporte/química , Proteínas de Ciclo Celular/química , Complexo de Endopeptidases do Proteassoma/metabolismo , Complexos Ubiquitina-Proteína Ligase/química , Sequência de Aminoácidos , Arginina/metabolismo , Sítios de Ligação , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Clonagem Molecular , Cristalografia por Raios X , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Células HEK293 , Humanos , Modelos Moleculares , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Proteólise , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Especificidade por Substrato , Complexos Ubiquitina-Proteína Ligase/genética , Complexos Ubiquitina-Proteína Ligase/metabolismo
15.
Am J Hum Genet ; 108(2): 357-367, 2021 02 04.
Artigo em Inglês | MEDLINE | ID: mdl-33508234

RESUMO

Focal segmental glomerulosclerosis (FSGS) is the main pathology underlying steroid-resistant nephrotic syndrome (SRNS) and a leading cause of chronic kidney disease. Monogenic forms of pediatric SRNS are predominantly caused by recessive mutations, while the contribution of de novo variants (DNVs) to this trait is poorly understood. Using exome sequencing (ES) in a proband with FSGS/SRNS, developmental delay, and epilepsy, we discovered a nonsense DNV in TRIM8, which encodes the E3 ubiquitin ligase tripartite motif containing 8. To establish whether TRIM8 variants represent a cause of FSGS, we aggregated exome/genome-sequencing data for 2,501 pediatric FSGS/SRNS-affected individuals and 48,556 control subjects, detecting eight heterozygous TRIM8 truncating variants in affected subjects but none in control subjects (p = 3.28 × 10-11). In all six cases with available parental DNA, we demonstrated de novo inheritance (p = 2.21 × 10-15). Reverse phenotyping revealed neurodevelopmental disease in all eight families. We next analyzed ES from 9,067 individuals with epilepsy, yielding three additional families with truncating TRIM8 variants. Clinical review revealed FSGS in all. All TRIM8 variants cause protein truncation clustering within the last exon between residues 390 and 487 of the 551 amino acid protein, indicating a correlation between this syndrome and loss of the TRIM8 C-terminal region. Wild-type TRIM8 overexpressed in immortalized human podocytes and neuronal cells localized to nuclear bodies, while constructs harboring patient-specific variants mislocalized diffusely to the nucleoplasm. Co-localization studies demonstrated that Gemini and Cajal bodies frequently abut a TRIM8 nuclear body. Truncating TRIM8 DNVs cause a neuro-renal syndrome via aberrant TRIM8 localization, implicating nuclear bodies in FSGS and developmental brain disease.


Assuntos
Proteínas de Transporte/genética , Deficiências do Desenvolvimento/genética , Epilepsia/genética , Glomerulosclerose Segmentar e Focal/genética , Espaço Intranuclear/metabolismo , Síndrome Nefrótica/genética , Síndrome Nefrótica/metabolismo , Proteínas do Tecido Nervoso/genética , Adulto , Animais , Proteínas de Transporte/química , Proteínas de Transporte/metabolismo , Linhagem Celular , Criança , Pré-Escolar , Códon sem Sentido , Deficiências do Desenvolvimento/metabolismo , Epilepsia/metabolismo , Feminino , Glomerulosclerose Segmentar e Focal/metabolismo , Humanos , Rim/metabolismo , Masculino , Camundongos , Mutação , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/metabolismo , Fenótipo , Podócitos/metabolismo , Sequenciamento Completo do Exoma
16.
Int J Mol Sci ; 22(2)2021 Jan 13.
Artigo em Inglês | MEDLINE | ID: mdl-33450960

RESUMO

This work investigated in vitro aggregation and amyloid properties of skeletal myosin binding protein-C (sMyBP-C) interacting in vivo with proteins of thick and thin filaments in the sarcomeric A-disc. Dynamic light scattering (DLS) and transmission electron microscopy (TEM) found a rapid (5-10 min) formation of large (>2 µm) aggregates. sMyBP-C oligomers formed both at the initial 5-10 min and after 16 h of aggregation. Small angle X-ray scattering (SAXS) and DLS revealed sMyBP-C oligomers to consist of 7-10 monomers. TEM and atomic force microscopy (AFM) showed sMyBP-C to form amorphous aggregates (and, to a lesser degree, fibrillar structures) exhibiting no toxicity on cell culture. X-ray diffraction of sMyBP-C aggregates registered reflections attributed to a cross-ß quaternary structure. Circular dichroism (CD) showed the formation of the amyloid-like structure to occur without changes in the sMyBP-C secondary structure. The obtained results indicating a high in vitro aggregability of sMyBP-C are, apparently, a consequence of structural features of the domain organization of proteins of this family. Formation of pathological amyloid or amyloid-like sMyBP-C aggregates in vivo is little probable due to amino-acid sequence low identity (<26%), alternating ordered/disordered regions in the protein molecule, and S-S bonds providing for general stability.


Assuntos
Amiloide/química , Amiloide/metabolismo , Proteínas de Transporte/química , Proteínas de Transporte/metabolismo , Agregados Proteicos , Sequência de Aminoácidos , Amiloide/ultraestrutura , Cromatografia Líquida de Alta Pressão , Dicroísmo Circular , Difusão Dinâmica da Luz , Técnicas In Vitro , Cinética , Espectrometria de Massas , Modelos Moleculares , Agregação Patológica de Proteínas , Conformação Proteica , Relação Estrutura-Atividade , Difração de Raios X
17.
Int J Mol Sci ; 22(3)2021 Jan 25.
Artigo em Inglês | MEDLINE | ID: mdl-33503999

RESUMO

Inherited retinal degenerative diseases (IRDs), which ultimately lead to photoreceptor cell death, are characterized by high genetic heterogeneity. Many IRD-associated genetic defects affect 3',5'-cyclic guanosine monophosphate (cGMP) levels. cGMP-dependent protein kinases (PKGI and PKGII) have emerged as novel targets, and their inhibition has shown functional protection in IRDs. The development of such novel neuroprotective compounds warrants a better understanding of the pathways downstream of PKGs that lead to photoreceptor degeneration. Here, we used human recombinant PKGs in combination with PKG activity modulators (cGMP, 3',5'-cyclic adenosine monophosphate (cAMP), PKG activator, and PKG inhibitors) on a multiplex peptide microarray to identify substrates for PKGI and PKGII. In addition, we applied this technology in combination with PKG modulators to monitor kinase activity in a complex cell system, i.e. the retinal cell line 661W, which is used as a model system for IRDs. The high-throughput method allowed quick identification of bona fide substrates for PKGI and PKGII. The response to PKG modulators helped us to identify, in addition to ten known substrates, about 50 novel substrates for PKGI and/or PKGII which are either specific for one enzyme or common to both. Interestingly, both PKGs are able to phosphorylate the regulatory subunit of PKA, whereas only PKGII can phosphorylate the catalytic subunit of PKA. In 661W cells, the results suggest that PKG activators cause minor activation of PKG, but a prominent increase in the activity of cAMP-dependent protein kinase (PKA). However, the literature suggests an important role for PKG in IRDs. This conflicting information could be reconciled by cross-talk between PKG and PKA in the retinal cells. This must be explored further to elucidate the role of PKGs in IRDs.


Assuntos
Proteínas de Transporte/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Suscetibilidade a Doenças , Degeneração Retiniana/etiologia , Degeneração Retiniana/metabolismo , Sequência de Aminoácidos , Biomarcadores , Proteínas de Transporte/química , AMP Cíclico/metabolismo , GMP Cíclico/metabolismo , Ativação Enzimática , Predisposição Genética para Doença , Humanos , Cinética , Ligação Proteica , Degeneração Retiniana/patologia , Especificidade por Substrato
18.
Biochemistry ; 60(2): 135-151, 2021 01 19.
Artigo em Inglês | MEDLINE | ID: mdl-33406357

RESUMO

σB, an alternative sigma factor, is usually employed to tackle the general stress response in Staphylococcus aureus and other Gram-positive bacteria. This protein, involved in S. aureus-mediated pathogenesis, is typically blocked by RsbW, an antisigma factor having serine kinase activity. σB, a σ70-like sigma factor, harbors three conserved domains designated σB2, σB3, and σB4. To better understand the interaction between RsbW and σB or its domains, we have studied their recombinant forms, rRsbW, rσB, rσB2, rσB3, and rσB4, using different probes. The results show that none of the rσB domains, unlike rσB, showed binding to a cognate DNA in the presence of a core RNA polymerase. However, both rσB2 and rσB3, like rσB, interacted with rRsbW, and the order of their rRsbW binding affinity looks like rσB > rσB3 > rσB2. Furthermore, the reaction between rRsbW and rσB or rσB3 was exothermic and occurred spontaneously. rRsbW and rσB3 also associate with each other at a stoichiometry of 2:1, and different types of noncovalent bonds might be responsible for their interaction. A structural model of the RsbW-σB3 complex that has supported our experimental results indicated the binding of rσB3 at the putative dimeric interface of RsbW. A genetic study shows that the tentative dimer-forming region of RsbW is crucial for preserving its rσB binding ability, serine kinase activity, and dimerization ability. Additionally, a urea-induced equilibrium unfolding study indicated a notable thermodynamic stabilization of σB3 in the presence of RsbW. Possible implications of the stabilization data in drug discovery were discussed at length.


Assuntos
Proteínas de Bactérias/metabolismo , Proteínas de Transporte/metabolismo , Domínios e Motivos de Interação entre Proteínas , Fator sigma/metabolismo , Staphylococcus aureus/metabolismo , Proteínas de Bactérias/química , Proteínas de Transporte/química , RNA Polimerases Dirigidas por DNA/metabolismo , Fosforilação , Ligação Proteica , Conformação Proteica , Fator sigma/química
19.
Arterioscler Thromb Vasc Biol ; 41(3): 1092-1104, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33472402

RESUMO

OBJECTIVE: GPVI (glycoprotein VI) is a key molecular player in collagen-induced platelet signaling and aggregation. Recent evidence indicates that it also plays important role in platelet aggregation and thrombus growth through interaction with fibrin(ogen). However, there are discrepancies in the literature regarding whether the monomeric or dimeric form of GPVI binds to fibrinogen at high affinity. The mechanisms of interaction are also not clear, including which region of fibrinogen is responsible for GPVI binding. We aimed to gain further understanding of the mechanisms of interaction at molecular level and to identify the regions on fibrinogen important for GPVI binding. Approach and Results: Using multiple surface- and solution-based protein-protein interaction methods, we observe that dimeric GPVI binds to fibrinogen with much higher affinity and has a slower dissociation rate constant than the monomer due to avidity effects. Moreover, our data show that the highest affinity interaction of GPVI is with the αC-region of fibrinogen. We further show that GPVI interacts with immobilized fibrinogen and fibrin variants at a similar level, including a nonpolymerizing fibrin variant, suggesting that GPVI binding is independent of fibrin polymerization. CONCLUSIONS: Based on the above findings, we conclude that the higher affinity of dimeric GPVI over the monomer for fibrinogen interaction is achieved by avidity. The αC-region of fibrinogen appears essential for GPVI binding. We propose that fibrin polymerization into fibers during coagulation will cluster GPVI through its αC-region, leading to downstream signaling, further activation of platelets, and potentially stimulating clot growth. Graphic Abstract: A graphic abstract is available for this article.


Assuntos
Fibrinogênio/metabolismo , Fragmentos de Peptídeos/sangue , Glicoproteínas da Membrana de Plaquetas/metabolismo , Animais , Proteínas de Transporte/química , Proteínas de Transporte/metabolismo , Produtos de Degradação da Fibrina e do Fibrinogênio/química , Produtos de Degradação da Fibrina e do Fibrinogênio/metabolismo , Fibrinogênio/química , Humanos , Técnicas In Vitro , Camundongos , Microscopia de Força Atômica , Fragmentos de Peptídeos/química , Peptídeos/química , Peptídeos/metabolismo , Agregação Plaquetária/fisiologia , Glicoproteínas da Membrana de Plaquetas/química , Domínios e Motivos de Interação entre Proteínas , Estrutura Quaternária de Proteína , Transdução de Sinais , Ressonância de Plasmônio de Superfície
20.
Arch Insect Biochem Physiol ; 106(2): e21763, 2021 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-33426694

RESUMO

Peptidoglycan recognition proteins (PGRPs) are well known for their abilities to recognize or hydrolyze peptidoglycan (PGN), one of the major bacterial cell wall components. However, much less is known about their antifungal activities. PGRP-S1 was previously identified from a crop pest, Mythimna separata (Walker) (Lepidoptera: Noctuidae). PGRP-S1 showed bacteriolytic activities against Gram-positive and Gram-negative bacteria. In this study, tissue expression analysis showed that PGRP-S1 was mainly expressed in the midgut of naïve larvae. The induction analysis showed that it was significantly induced in the larval midgut 12 h post the injection of Beauveria bassiana conidia. To identify the key residues that are related to its microbicidal activities, the structure of PGPR-S1 was predicted for structural comparison and molecular docking analysis. Six residues (H61, H62, Y97, H171, T175, and C179) were mutated to Ala individually by site-directed mutagenesis. The recombinant wild-type (WT) and mutant proteins were expressed and purified. The recombinant proteins bound to different polysaccharides, PGNs, and bacteria. H61A, Y97A, H171A, and C179A lost amidase activity. Accordingly, antibacterial assay and scanning electron microscopy confirmed that only H62A and T175A retained bacteriolytic activities. The germination of B. bassiana conidia was significantly inhibited by WT, H61A, Y97A, T175A, and C179A mutants. Electron microscopy showed that some conidia became ruptured after treatment. The growth of hyphae was inhibited by the WT, H61A, H62A, and T175A. In summary, our data showed that different residues of PGRP-S1 are involved in the antibacterial and antifungal activities.


Assuntos
Beauveria/fisiologia , Proteínas de Transporte/genética , Proteínas de Insetos/genética , Mariposas/genética , Sequência de Aminoácidos , Animais , Proteínas de Transporte/química , Proteínas de Transporte/metabolismo , Imunidade Inata , Proteínas de Insetos/química , Proteínas de Insetos/metabolismo , Larva/crescimento & desenvolvimento , Simulação de Acoplamento Molecular , Mariposas/crescimento & desenvolvimento , Mariposas/metabolismo , Filogenia , Estrutura Secundária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Esporos Fúngicos/fisiologia
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