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1.
Biochim Biophys Acta Biomembr ; 1862(6): 183230, 2020 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-32126233

RESUMO

Changes in membrane curvature are required to control the function of subcellular compartments; malfunctions of such processes are associated with a wide range of human diseases. Membrane remodeling often depends upon the presence of phosphoinositides, which recruit protein effectors for a variety of cellular functions. Phafin2 is a phosphatidylinositol 3-phosphate (PtdIns3P)-binding effector involved in endosomal and lysosomal membrane-associated signaling. Both the Phafin2 PH and the FYVE domains bind PtdIns3P, although their redundant function in the protein is unclear. Through a combination of lipid-binding assays, we found that, unlike the FYVE domain, recognition of the PH domain to PtdIns3P requires a lipid bilayer. Using site-directed mutagenesis and truncation constructs, we discovered that the Phafin2 FYVE domain is constitutive for PtdIns3P binding, whereas PH domain binding to PtdIns3P is autoinhibited by a conserved C-terminal acidic motif. These findings suggest that binding of the Phafin2 PH domain to PtdIns3P in membrane compartments occurs through a highly regulated mechanism. Potential mechanisms are discussed throughout this report.


Assuntos
Motivos de Aminoácidos/fisiologia , Fosfatos de Fosfatidilinositol/metabolismo , Proteínas de Transporte Vesicular/química , Membrana Celular/ultraestrutura , Humanos , Bicamadas Lipídicas/metabolismo , Fosfatos de Fosfatidilinositol/antagonistas & inibidores , Ligação Proteica , Domínios Proteicos , Proteínas de Transporte Vesicular/metabolismo
2.
BMB Rep ; 53(3): 160-165, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-32172729

RESUMO

The root meristem of Arabidopsis thaliana is protected by the root cap, the size of which is tightly regulated by the balance between the formative cell divisions and the dispersal of the outermost cells. We isolated an enhancer-tagged dominant mutant displaying the short and twisted root by the overexpression of ZINC-FINGER OF ARABIDOPSIS THALIANA1 (ZAT1) encoding an EAR motif-containing zinc-finger protein. The growth inhibition by ZAT1 was shared by ZAT4 and ZAT9, the ZAT1 homologues. The ZAT1 promoter was specifically active in the outermost cells of the root cap, in which ZAT1-GFP was localized when expressed by the ZAT1 promoter. The outermost cell-specific expression pattern of ZAT1 was not altered in the sombrero (smb) or smb bearskin1 (brn1) brn2 accumulating additional root-cap layers. In contrast, ZAT4-GFP and ZAT9- GFP fusion proteins were distributed to the inner root-cap cells in addition to the outermost cells where ZAT4 and ZAT9 promoters were active. Overexpression of ZAT1 induced the ectopic expression of PUTATIVE ASPARTIC PROTEASE3 involved in the programmed cell death. The EAR motif was essential for the growth inhibition by ZAT1. These results suggest that the three related ZATs might regulate the maturation of the outermost cells of the root cap. [BMB Reports 2020; 53(3): 160-165].


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/metabolismo , Proteínas de Transporte de Cátions/metabolismo , Motivos de Aminoácidos/fisiologia , Proteínas de Arabidopsis/genética , Proteínas de Transporte de Cátions/genética , Divisão Celular/fisiologia , Meristema/metabolismo , Coifa/genética , Coifa/metabolismo , Raízes de Plantas/metabolismo , Fatores de Transcrição/metabolismo , Zinco/metabolismo , Dedos de Zinco/fisiologia
3.
PLoS One ; 15(2): e0228874, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32049977

RESUMO

Matriptase plays important roles in epithelial integrity and function, which depend on its sorting to the basolateral surface of cells, where matriptase zymogen is converted to an active enzyme in order to act on its substrates. After activation, matriptase undergoes HAI-1-mediated inhibition, internalization, transcytosis, and secretion from the apical surface into the lumen. Matriptase is a mosaic protein with several distinct protein domains and motifs, which are a reflection of matriptase's complex cellular itinerary, life cycle, and the tight control of its enzymatic activity. While the molecular determinants for various matriptase regulatory events have been identified, the motif(s) required for translocation of human matriptase to the basolateral plasma membrane is unknown. The motif previously identified in rat matriptase is not conserved between the rodent and the primate. We, here, revisit the question for human matriptase through the use of a fusion protein containing a green fluorescent protein linked to the matriptase N-terminal fragment ending at Gly-149. A conserved seven amino acid motif EEGEVFL, which is similar to the monoleucine C-terminal to an acidic cluster motif involved in the basolateral targeting for some growth factors, has been shown to be required for matriptase translocation to the basolateral plasma membrane of polarized MDCK cells. Furthermore, time-lapse video microscopy showed that the motif appears to be required for entry into the correct transport vesicles, by which matriptase can undergo rapid trafficking and translocate to the plasma membrane. Our study reveals that the EEGEVFL motif is necessary, but may not be sufficient, for matriptase basolateral membrane targeting and serves as the basis for further research on its pathophysiological roles.


Assuntos
Motivos de Aminoácidos/fisiologia , Membrana Celular/metabolismo , Transporte Proteico/fisiologia , Serina Endopeptidases/metabolismo , Animais , Linhagem Celular , Estruturas da Membrana Celular/metabolismo , Polaridade Celular/fisiologia , Citoplasma/metabolismo , Cães , Precursores Enzimáticos/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , Humanos , Células Madin Darby de Rim Canino , Glicoproteínas de Membrana/metabolismo , Proteínas Secretadas Inibidoras de Proteinases/metabolismo
4.
Nat Commun ; 10(1): 3863, 2019 08 27.
Artigo em Inglês | MEDLINE | ID: mdl-31455778

RESUMO

The anaphase-promoting complex/cyclosome (APC/C) orchestrates cell cycle progression by controlling the temporal degradation of specific cell cycle regulators. Although cyclin A2 and cyclin B1 are both targeted for degradation by the APC/C, during the spindle assembly checkpoint (SAC), the mitotic checkpoint complex (MCC) represses APC/C's activity towards cyclin B1, but not cyclin A2. Through structural, biochemical and in vivo analysis, we identify a non-canonical D box (D2) that is critical for cyclin A2 ubiquitination in vitro and degradation in vivo. During the SAC, cyclin A2 is ubiquitinated by the repressed APC/C-MCC, mediated by the cooperative engagement of its KEN and D2 boxes, ABBA motif, and the cofactor Cks. Once the SAC is satisfied, cyclin A2 binds APC/C-Cdc20 through two mutually exclusive binding modes, resulting in differential ubiquitination efficiency. Our findings reveal that a single substrate can engage an E3 ligase through multiple binding modes, affecting its degradation timing and efficiency.


Assuntos
Ciclossomo-Complexo Promotor de Anáfase/metabolismo , Ciclina A2/metabolismo , Pontos de Checagem da Fase M do Ciclo Celular/fisiologia , Ubiquitinação/fisiologia , Motivos de Aminoácidos/fisiologia , Ciclossomo-Complexo Promotor de Anáfase/ultraestrutura , CDC2-CDC28 Quinases/metabolismo , CDC2-CDC28 Quinases/ultraestrutura , Proteínas Cdc20/metabolismo , Proteínas Cdc20/ultraestrutura , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/ultraestrutura , Microscopia Crioeletrônica , Ciclina A2/ultraestrutura , Células HEK293 , Humanos , Microscopia Intravital , Modelos Moleculares , Ligação Proteica/fisiologia , Proteólise , Fuso Acromático/metabolismo , Especificidade por Substrato/fisiologia
5.
Nat Commun ; 10(1): 2055, 2019 05 03.
Artigo em Inglês | MEDLINE | ID: mdl-31053714

RESUMO

Autophagy is an essential recycling and quality control pathway. Mammalian ATG8 proteins drive autophagosome formation and selective removal of protein aggregates and organelles by recruiting autophagy receptors and adaptors that contain a LC3-interacting region (LIR) motif. LIR motifs can be highly selective for ATG8 subfamily proteins (LC3s/GABARAPs), however the molecular determinants regulating these selective interactions remain elusive. Here we show that residues within the core LIR motif and adjacent C-terminal region as well as ATG8 subfamily-specific residues in the LIR docking site are critical for binding of receptors and adaptors to GABARAPs. Moreover, rendering GABARAP more LC3B-like impairs autophagy receptor degradation. Modulating LIR binding specificity of the centriolar satellite protein PCM1, implicated in autophagy and centrosomal function, alters its dynamics in cells. Our data provides new mechanistic insight into how selective binding of LIR motifs to GABARAPs is achieved, and elucidate the overlapping and distinct functions of ATG8 subfamily proteins.


Assuntos
Motivos de Aminoácidos/fisiologia , Família da Proteína 8 Relacionada à Autofagia/metabolismo , Autofagia , Ligação Proteica/fisiologia , Autoantígenos/isolamento & purificação , Autoantígenos/metabolismo , Família da Proteína 8 Relacionada à Autofagia/genética , Família da Proteína 8 Relacionada à Autofagia/isolamento & purificação , Proteína Homóloga à Proteína-1 Relacionada à Autofagia/isolamento & purificação , Proteína Homóloga à Proteína-1 Relacionada à Autofagia/metabolismo , Proteínas de Ciclo Celular/isolamento & purificação , Proteínas de Ciclo Celular/metabolismo , Centríolos/metabolismo , Técnicas de Inativação de Genes , Células HEK293 , Células HeLa , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/isolamento & purificação , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Nucleares/isolamento & purificação , Proteínas Nucleares/metabolismo , Proteólise , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo
6.
Nat Commun ; 10(1): 1261, 2019 03 19.
Artigo em Inglês | MEDLINE | ID: mdl-30890705

RESUMO

Cellular functions of arrestins are determined in part by the pattern of phosphorylation on the G protein-coupled receptors (GPCRs) to which arrestins bind. Despite high-resolution structural data of arrestins bound to phosphorylated receptor C-termini, the functional role of each phosphorylation site remains obscure. Here, we employ a library of synthetic phosphopeptide analogues of the GPCR rhodopsin C-terminus and determine the ability of these peptides to bind and activate arrestins using a variety of biochemical and biophysical methods. We further characterize how these peptides modulate the conformation of arrestin-1 by nuclear magnetic resonance (NMR). Our results indicate different functional classes of phosphorylation sites: 'key sites' required for arrestin binding and activation, an 'inhibitory site' that abrogates arrestin binding, and 'modulator sites' that influence the global conformation of arrestin. These functional motifs allow a better understanding of how different GPCR phosphorylation patterns might control how arrestin functions in the cell.


Assuntos
Arrestina/metabolismo , Fosforilação/fisiologia , Rodopsina/metabolismo , beta-Arrestina 1/metabolismo , beta-Arrestina 2/metabolismo , Motivos de Aminoácidos/fisiologia , Animais , Arrestina/química , Arrestina/genética , Arrestina/isolamento & purificação , Bioensaio , Bovinos , Membrana Celular/metabolismo , Mutação , Ressonância Magnética Nuclear Biomolecular , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Rodopsina/química , Segmento Externo da Célula Bastonete/metabolismo , beta-Arrestina 1/química , beta-Arrestina 1/isolamento & purificação , beta-Arrestina 2/química , beta-Arrestina 2/isolamento & purificação
7.
J Cell Physiol ; 234(10): 17749-17756, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-30820965

RESUMO

Cardiac fibrosis is a pathophysiological process characterized by excessive deposition of extracellular matrix. We developed a cardiac hypertrophy model using transverse aortic constriction (TAC) to uncover mechanisms relevant to excessive deposition of extracellular matrix in mouse myocardial cells. TAC caused upregulation of Tripartite motif protein 72 (TRIM72), a tripartite motif-containing protein that is critical for proliferation and migration. Importantly, in vivo silencing of TRIM72 reversed TAC-induced cardiac fibrosis, as indicated by markedly increased left ventricular systolic pressure and decreased left ventricular end-diastolic pressure. TRIM72 knockdown also attenuated deposition of fibrosis marker collagen type I and α-smooth muscle actin (α-SMA). In an in vitro study, TRIM72 was similarly upregulated in cardiac fibroblasts. Knockdown of TRIM72 markedly suppressed collagen type I and α-SMA expression and significantly decreased the proliferation and migration of cardiac fibroblasts. However, TRIM72 overexpression markedly increased collagen type I and α-SMA expression and increased the proliferation and migration of cardiac fibroblasts. Further study demonstrated that TRIM72 increased phosphorylated STAT3 in cardiac fibroblasts. TRIM72 knockdown in cardiac fibroblasts resulted in increased expression of Notch ligand Jagged-1 and its downstream gene and Notch-1 intracellular domain. Inhibition of Notch-1 abrogated sh-TRIM72-induced cardiac fibrosis. Together, our results support a novel role for TRIM72 in maintaining fibroblast-to-myofibroblast transition and suppressing fibroblast growth by regulating the STAT3/Notch-1 pathway.


Assuntos
Fibrose/metabolismo , Proteínas de Membrana/metabolismo , Receptor Notch1/metabolismo , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais/fisiologia , Motivos de Aminoácidos/fisiologia , Animais , Movimento Celular/fisiologia , Proliferação de Células/fisiologia , Colágeno Tipo I/metabolismo , Fibroblastos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Miocárdio/metabolismo , Miócitos Cardíacos/metabolismo , Miofibroblastos/metabolismo , Fosforilação/fisiologia , Domínios Proteicos/fisiologia , Fator de Crescimento Transformador beta/metabolismo , Regulação para Cima/fisiologia
8.
Biochem Pharmacol ; 164: 188-204, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-30905657

RESUMO

A Disintegrin and Metalloproteinase (ADAM) is a family of proteolytic enzymes that possess sheddase function and regulate shedding of membrane-bound proteins, growth factors, cytokines, ligands and receptors. Typically, ADAMs have a pro-domain, and a metalloproteinase, disintegrin, cysteine-rich and a characteristic transmembrane domain. Most ADAMs are activated by proprotein convertases, but can also be regulated by G-protein coupled receptor agonists, Ca2+ ionophores and protein kinase C activators. A Disintegrin and Metalloproteinase with Thrombospondin Motifs (ADAMTS) is a family of secreted enzymes closely related to ADAMs. Like ADAMs, ADAMTS members have a pro-domain, and a metalloproteinase, disintegrin, and cysteine-rich domain, but they lack a transmembrane domain and instead have characteristic thrombospondin motifs. Activated ADAMs perform several functions and participate in multiple cardiovascular processes including vascular smooth muscle cell proliferation and migration, angiogenesis, vascular cell apoptosis, cell survival, tissue repair, and wound healing. ADAMs may also be involved in pathological conditions and cardiovascular diseases such as atherosclerosis, hypertension, aneurysm, coronary artery disease, myocardial infarction and heart failure. Like ADAMs, ADAMTS have a wide-spectrum role in vascular biology and cardiovascular pathophysiology. ADAMs and ADAMTS activity is naturally controlled by endogenous inhibitors such as tissue inhibitors of metalloproteinases (TIMPs), and their activity can also be suppressed by synthetic small molecule inhibitors. ADAMs and ADAMTS can serve as important diagnostic biomarkers and potential therapeutic targets for cardiovascular disorders. Natural and synthetic inhibitors of ADAMs and ADAMTS could be potential therapeutic tools for the management of cardiovascular diseases.


Assuntos
Proteínas ADAM/metabolismo , Desintegrinas/metabolismo , Endotélio Vascular/metabolismo , Trombospondinas/metabolismo , Doenças Vasculares/metabolismo , Proteínas ADAM/antagonistas & inibidores , Motivos de Aminoácidos/efeitos dos fármacos , Motivos de Aminoácidos/fisiologia , Animais , Desintegrinas/antagonistas & inibidores , Endotélio Vascular/efeitos dos fármacos , Humanos , Inibidores de Metaloproteinases de Matriz/administração & dosagem , Trombospondinas/antagonistas & inibidores , Doenças Vasculares/tratamento farmacológico
9.
Proc Natl Acad Sci U S A ; 116(16): 8054-8059, 2019 04 16.
Artigo em Inglês | MEDLINE | ID: mdl-30926664

RESUMO

Phytophthora are eukaryotic pathogens that cause enormous losses in agriculture and forestry. Each Phytophthora species encodes hundreds of effector proteins that collectively have essential roles in manipulating host cellular processes and facilitating disease development. Here we report the crystal structure of the effector Phytophthora suppressor of RNA silencing 2 (PSR2). PSR2 produced by the soybean pathogen Phytophthora sojae (PsPSR2) consists of seven tandem repeat units, including one W-Y motif and six L-W-Y motifs. Each L-W-Y motif forms a highly conserved fold consisting of five α-helices. Adjacent units are connected through stable, directional linkages between an internal loop at the C terminus of one unit and a hydrophobic pocket at the N terminus of the following unit. This unique concatenation results in an overall stick-like structure of PsPSR2. Genome-wide analyses reveal 293 effectors from five Phytophthora species that have the PsPSR2-like arrangement, that is, containing a W-Y motif as the "start" unit, various numbers of L-W-Y motifs as the "middle" units, and a degenerate L-W-Y as the "end" unit. Residues involved in the interunit interactions show significant conservation, suggesting that these effectors also use the conserved concatenation mechanism. Furthermore, functional analysis demonstrates differential contributions of individual units to the virulence activity of PsPSR2. These findings suggest that the L-W-Y fold is a basic structural and functional module that may serve as a "building block" to accelerate effector evolution in Phytophthora.


Assuntos
Proteínas de Bactérias/química , Phytophthora/patogenicidade , Motivos de Aminoácidos/genética , Motivos de Aminoácidos/fisiologia , Proteínas de Bactérias/genética , Modelos Moleculares , Phytophthora/química , Phytophthora/genética , Doenças das Plantas/microbiologia , Sequências de Repetição em Tandem/genética
10.
Methods Mol Biol ; 1877: 1-21, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30535995

RESUMO

BCL-2 family proteins interact in a network that regulates apoptosis. The BH3 amino acid sequence motif serves to bind together this conglomerate protein family, both literally and figuratively. BH3 motifs are present in antiapoptotic and proapoptotic BCL-2 homologs, and in a separate group of unrelated BH3-only proteins often appended to the BCL-2 family. BH3-containing helices mediate many of their physical interactions to determine cell death versus survival, leading to the development of BH3 mimetics as therapeutics. Here we provide an overview of BCL-2 family interactions, their relevance in health and disease, and the progress toward regulating their interactions therapeutically.


Assuntos
Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Motivos de Aminoácidos/fisiologia , Aminoácidos/metabolismo , Animais , Apoptose/fisiologia , Humanos
11.
Artigo em Inglês | MEDLINE | ID: mdl-29990265

RESUMO

The essential role of small evolutionarily conserved structural units in proteins has been extensively researched and validated. A popular example are serine proteases, where the peptide cleavage reaction is realized by a configuration of only three residues. Brought to spatial proximity during the protein folding process, such structural motifs are often long-range contacts and usually hard to detect at sequence level. Due to the constantly increasing resource of protein 3D structure data, the computational identification of structural motifs can contribute significantly to the understanding of protein fold and function. Thus, we propose a method to discover structural motifs of high geometrical similarity and desired sequence separation in protein 3D structure data. By utilizing methods originated from data mining, no a priori knowledge is required. The applicability of the method is demonstrated by the identification of the catalytic unit of serine proteases and the ion-coordination center of cupredoxins. Furthermore, large-scale analysis of the entire Protein Data Bank points towards the presence of ubiquitous structural motifs, independent of any specific fold or function. We envision that our method is suitable to uncover functional mechanisms and to derive fingerprint libraries of structural motifs, which could be used to assess protein family association.


Assuntos
Biologia Computacional/métodos , Reconhecimento Automatizado de Padrão/métodos , Proteínas , Algoritmos , Motivos de Aminoácidos/genética , Motivos de Aminoácidos/fisiologia , Bases de Dados de Proteínas , Proteínas/química , Proteínas/genética , Proteínas/fisiologia
12.
Nat Struct Mol Biol ; 25(12): 1093-1102, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30455435

RESUMO

The cell division cycle consists of a series of temporally ordered events. Cell cycle kinases and phosphatases provide key regulatory input, but how the correct substrate phosphorylation and dephosphorylation timing is achieved is incompletely understood. Here we identify a PxL substrate recognition motif that instructs dephosphorylation by the budding yeast Cdc14 phosphatase during mitotic exit. The PxL motif was prevalent in Cdc14-binding peptides enriched in a phage display screen of native disordered protein regions. PxL motif removal from the Cdc14 substrate Cbk1 delays its dephosphorylation, whereas addition of the motif advances dephosphorylation of otherwise late Cdc14 substrates. Crystal structures of Cdc14 bound to three PxL motif substrate peptides provide a molecular explanation for PxL motif recognition on the phosphatase surface. Our results illustrate the sophistication of phosphatase-substrate interactions and identify them as an important determinant of ordered cell cycle progression.


Assuntos
Motivos de Aminoácidos/fisiologia , Divisão Celular , Saccharomyces cerevisiae/citologia , Proteínas de Ciclo Celular , Mitose , Modelos Moleculares , Fosforilação , Proteínas Tirosina Fosfatases , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae , Análise de Sequência de Proteína
13.
Int J Mol Sci ; 19(7)2018 Jul 20.
Artigo em Inglês | MEDLINE | ID: mdl-30036999

RESUMO

Although the N-terminal domain of vertebrate small heat shock proteins (sHsp) is poorly conserved, it contains a core motif preserved in many members of the sHsp family. The role of this RLFDQxFG motif remains elusive. We analyzed the specific role of the first arginine residue of this conserved octet sequence in five human sHsps (HspB1, HspB4, HspB5, HspB6, and HspB8). Substitution of this arginine with an alanine induced changes in thermal stability and/or intrinsic fluorescence of the related HspB1 and HspB8, but yielded only modest changes in the same biophysical properties of HspB4, HspB5, and HspB6 which together belong to another clade of vertebrate sHsps. Removal of the positively charged Arg side chain resulted in destabilization of the large oligomers of HspB1 and formation of smaller size oligomers of HspB5. The mutation induced only minor changes in the structure of HspB4 and HspB6. In contrast, the mutation in HspB8 was accompanied by shifting the equilibrium from dimers towards the formation of larger oligomers. We conclude that the RLFDQxFG motif plays distinct roles in the structure of several sHsp orthologs. This role correlates with the evolutionary relationship of the respective sHsps, but ultimately, it reflects the sequence context of this motif.


Assuntos
Motivos de Aminoácidos/fisiologia , Arginina/química , Cristalinas/química , Proteínas de Choque Térmico HSP20/química , Proteínas de Choque Térmico HSP27/química , Proteínas de Choque Térmico Pequenas/química , Proteínas de Choque Térmico Pequenas/metabolismo , Proteínas de Choque Térmico/química , Proteínas Serina-Treonina Quinases/química , Cadeia B de alfa-Cristalina/química , Motivos de Aminoácidos/genética , Sequência de Aminoácidos , Arginina/genética , Cromatografia em Gel , Cristalinas/genética , Cristalinas/metabolismo , Proteínas de Choque Térmico HSP20/genética , Proteínas de Choque Térmico HSP20/metabolismo , Proteínas de Choque Térmico HSP27/genética , Proteínas de Choque Térmico HSP27/metabolismo , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Proteínas de Choque Térmico Pequenas/genética , Humanos , Chaperonas Moleculares , Dados de Sequência Molecular , Mutação Puntual , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Cadeia B de alfa-Cristalina/genética , Cadeia B de alfa-Cristalina/metabolismo
14.
PLoS Genet ; 14(7): e1007514, 2018 07.
Artigo em Inglês | MEDLINE | ID: mdl-29985927

RESUMO

The stringent response is characterized by (p)ppGpp synthesis resulting in repression of translation and reprogramming of the transcriptome. In Staphylococcus aureus, (p)ppGpp is synthesized by the long RSH (RelA/SpoT homolog) enzyme, RelSau or by one of the two short synthetases (RelP, RelQ). RSH enzymes are characterized by an N-terminal enzymatic domain bearing distinct motifs for (p)ppGpp synthetase or hydrolase activity and a C-terminal regulatory domain (CTD) containing conserved motifs (TGS, DC and ACT). The intramolecular switch between synthetase and hydrolase activity of RelSau is crucial for the adaption of S. aureus to stress (stringent) or non-stress (relaxed) conditions. We elucidated the role of the CTD in the enzymatic activities of RelSau. Growth pattern, transcriptional analyses and in vitro assays yielded the following results: i) in vivo, under relaxed conditions, as well as in vitro, the CTD inhibits synthetase activity but is not required for hydrolase activity; ii) under stringent conditions, the CTD is essential for (p)ppGpp synthesis; iii) RelSau lacking the CTD exhibits net hydrolase activity when expressed in S. aureus but net (p)ppGpp synthetase activity when expressed in E. coli; iv) the TGS and DC motifs within the CTD are required for correct stringent response, whereas the ACT motif is dispensable, v) Co-immunoprecipitation indicated that the CTD interacts with the ribosome, which is largely dependent on the TGS motif. In conclusion, RelSau primarily exists in a synthetase-OFF/hydrolase-ON state, the TGS motif within the CTD is required to activate (p)ppGpp synthesis under stringent conditions.


Assuntos
Proteínas de Bactérias/genética , Regulação Bacteriana da Expressão Gênica , Hidrolases/genética , Ligases/genética , Staphylococcus aureus/fisiologia , Adaptação Fisiológica/genética , Motivos de Aminoácidos/fisiologia , Proteínas de Bactérias/metabolismo , Hidrolases/metabolismo , Ligases/metabolismo , Ribossomos/metabolismo , Estresse Fisiológico/fisiologia
15.
Nat Commun ; 9(1): 2220, 2018 06 07.
Artigo em Inglês | MEDLINE | ID: mdl-29880797

RESUMO

The first RNA recognition motif of the Drosophila SNF protein is an example of an RNA binding protein with multi-specificity. It binds different RNA hairpin loops in spliceosomal U1 or U2 small nuclear RNAs, and only in the latter case requires the auxiliary U2A' protein. Here we investigate its functions by crystal structures of SNF alone and bound to U1 stem-loop II, U2A' or U2 stem-loop IV and U2A', SNF dynamics from NMR spectroscopy, and structure-guided mutagenesis in binding studies. We find that different loop-closing base pairs and a nucleotide exchange at the tips of the loops contribute to differential SNF affinity for the RNAs. U2A' immobilizes SNF and RNA residues to restore U2 stem-loop IV binding affinity, while U1 stem-loop II binding does not require such adjustments. Our findings show how U2A' can modulate RNA specificity of SNF without changing SNF conformation or relying on direct RNA contacts.


Assuntos
Proteínas de Drosophila/metabolismo , RNA Nuclear Pequeno/metabolismo , Ribonucleoproteína Nuclear Pequena U1/metabolismo , Ribonucleoproteína Nuclear Pequena U2/metabolismo , Motivos de Aminoácidos/genética , Motivos de Aminoácidos/fisiologia , Sítios de Ligação/genética , Cristalografia por Raios X , Proteínas de Drosophila/química , Proteínas de Drosophila/genética , Proteínas de Drosophila/isolamento & purificação , Modelos Moleculares , Mutagênese Sítio-Dirigida , Ressonância Magnética Nuclear Biomolecular , Ligação Proteica/fisiologia , Domínios Proteicos/fisiologia , RNA Nuclear Pequeno/química , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Ribonucleoproteína Nuclear Pequena U1/química , Ribonucleoproteína Nuclear Pequena U1/genética , Ribonucleoproteína Nuclear Pequena U1/isolamento & purificação , Ribonucleoproteína Nuclear Pequena U2/química , Especificidade por Substrato/fisiologia
16.
Plant Cell Rep ; 37(8): 1101-1112, 2018 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-29846768

RESUMO

KEY MESSAGE: Two LysM-containing proteins, namely, MmLYP1 and MmLYK2, were identified in mulberry. These proteins might be involved in chitin signaling. The LysM1 of MmLYK2 is critical for their interactions. Chitin is a major component of fungal cell walls and acts as an elicitor in plant innate immunity. Lysin motif (LysM)-containing proteins are essential for chitin recognition. However, related studies have been rarely reported in woody plants. In this study, in mulberry, the expression of a LysM-containing protein, MmLYP1, was significantly up-regulated after treatment with chitin and pathogenic fungi. In addition, MmLYP1 has an affinity for insoluble chitin polymers. Thus, MmLYP1 might function in chitin signaling. Since MmLYP1 lacks an intracellular domain, additional protein kinases are required for this signaling. An LysM-containing kinase, MmLYK2, was then identified. Expression of the MmLYK2 did not change significantly after chitin treatment, and the affinity of MmLYK2 for insoluble chitin was not high. The structure of MmLYP1 is similar to that of the chitin elicitor-binding proteins in rice and Arabidopsis. However, MmLYK2 has two LysM motifs, while the chitin elicitor receptor kinase 1 proteins in rice and Arabidopsis have one and three LysM motifs, respectively. The LysM1 of MmLYK2 interacted with all four LysM motifs in MmLYP1 and MmLYK2 in yeast. The chimera lacking the LysM1 of MmLYK2 did not interact with MmLYP1 and MmLYK2 in yeast and Nicotiana benthamiana cells. The LysM1 in MmLYK2 is the key motif in the interaction between MmLYP1 and MmLYK2, which may be involved in chitin signaling.


Assuntos
Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Quitina/metabolismo , Motivos de Aminoácidos/genética , Motivos de Aminoácidos/fisiologia , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Ligação Proteica , Transdução de Sinais/genética , Transdução de Sinais/fisiologia
17.
Microbes Infect ; 20(5): 302-307, 2018 05.
Artigo em Inglês | MEDLINE | ID: mdl-29331581

RESUMO

Coxiella burnetii is an obligate intracellular pathogen that replicates in an endolysosome-like compartment termed the Coxiella-containing vacuole (CCV). Formation of this unique replicative niche requires delivery of bacterial effector proteins into the host cytosol where they mediate crucial interactions with the host. We previously identified an essential Dot/Icm effector, CirA that is required for intracellular replication and CCV formation. Furthermore, CirA was shown to stimulate the GTPase activity of RhoA in vitro. In the current study, we used a bioinformatics-guided approach and identified three arginine finger-like motifs, often found in Rho GTPase-activating proteins (GAPs) and endosome-lysosome basolateral sorting signals associated with vesicle trafficking. When expressed in mammalian cells, mutation of either endosome-lysosome-basolateral sorting signals or the arginine finger-like motifs rescued stress phenotypes and decreased plasma membrane localization of ectopically expressed CirA. We further demonstrate that endosome-lysosome sorting signals are required for co-localization with Rab5 and Rab7. Collectively our data indicate that arginine finger-like motifs and endosome-lysosome-basolateral sorting signals within CirA are essential for interaction with the host cytoskeleton.


Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Coxiella burnetii/metabolismo , Endossomos/metabolismo , Lisossomos/metabolismo , Sistemas de Secreção Tipo IV/metabolismo , Motivos de Aminoácidos/genética , Motivos de Aminoácidos/fisiologia , Proteínas de Bactérias/genética , Membrana Celular/metabolismo , Coxiella burnetii/genética , Células HeLa , Interações Hospedeiro-Patógeno , Humanos , Transporte Proteico , Febre Q/metabolismo , Febre Q/microbiologia , Saccharomyces cerevisiae/metabolismo , Sistemas de Secreção Tipo IV/genética , Vacúolos/metabolismo , Vacúolos/microbiologia , Proteínas rab de Ligação ao GTP/metabolismo , Proteína rhoA de Ligação ao GTP/metabolismo
18.
Neuropharmacology ; 128: 132-141, 2018 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-28986281

RESUMO

Amphetamine (AMPH)-mediated norepinephrine transporter (NET) downregulation requires NET-T258/S259 trafficking motif. The present study utilizes cell permeable NET-T258/S259 motif interfering peptide, which blocks AMPH-induced NET downregulation, to explore the role of this form of NET regulation in AMPH-mediated behaviors. In rats receiving intra-accumbal microinjections of TAT-conjugated peptides encompassing NET-T258/S259 motif, acute systemic AMPH failed to inhibit NE transport in the TAT-NET-T258/S259 wild-type (WT) peptide injected hemisphere but not in the vehicle or scrambled peptide injected hemisphere. Acute AMPH-induced hyperactivity was significantly reduced in rats receiving intra-accumbal TAT-NET-T258/S259 WT peptide compared to those receiving intra-accumbal vehicle or TAT-NET-T258A/S259A mutant peptide or corresponding TAT-conjugated scrambled peptide. Basal locomotor activity was not altered by peptide infusions alone. Similarly AMPH-induced locomotor sensitization was significantly reduced in rats receiving intra-accumbal TAT-NET-T258/S259 WT peptide prior to AMPH challenge and not in rats receiving the mutant or scrambled peptide. In conditioned place preference (CPP) paradigm, a single bilateral intra-accumbal microinjection of TAT-NET-T258/S259 WT peptide prior to CPP testing significantly reduced AMPH-induced CPP expression. Likewise, a single bilateral intra-accumbal microinjection of TAT-NET-T258/S259 WT peptide prior to drug-challenge significantly attenuated AMPH-primed CPP reinstatement. On the other hand, bilateral intra-accumbal microinjection of scrambled peptide did not affect AMPH-induced CPP expression or reinstatement. These data demonstrate a role for T258/S259-dependent NET regulation in AMPH-induced hyperactivity and sensitization as well as AMPH-induced CPP expression and reinstatement.


Assuntos
Anfetamina/toxicidade , Estimulantes do Sistema Nervoso Central/toxicidade , Condicionamento Operante/efeitos dos fármacos , Hipercinese/induzido quimicamente , Proteínas da Membrana Plasmática de Transporte de Norepinefrina/metabolismo , Transporte Proteico/efeitos dos fármacos , Motivos de Aminoácidos/efeitos dos fármacos , Motivos de Aminoácidos/fisiologia , Animais , Comportamento Exploratório/efeitos dos fármacos , Locomoção/efeitos dos fármacos , Masculino , Microinjeções , Ratos , Ratos Sprague-Dawley , Reforço Psicológico , Sinaptossomos/efeitos dos fármacos , Sinaptossomos/metabolismo
19.
Blood ; 130(25): 2799-2807, 2017 12 21.
Artigo em Inglês | MEDLINE | ID: mdl-29089309

RESUMO

The first case of hereditary fibrinogen Aα-chain amyloidosis was recognized >20 years ago, but disease mechanisms still remain unknown. Here we report detailed clinical and proteomics studies of a French kindred with a novel amyloidogenic fibrinogen Aα-chain frameshift variant, Phe521Leufs, causing a severe familial form of renal amyloidosis. Next, we focused our investigations to elucidate the molecular basis that render this Aα-chain variant amyloidogenic. We show that a 49-mer peptide derived from the C-terminal part of the Phe521Leufs chain is deposited as fibrils in the patient's kidneys, establishing that only a small portion of Phe521Leufs directly contributes to amyloid formation in vivo. In silico analysis indicated that this 49-mer Aα-chain peptide contained a motif (VLITL), with a high intrinsic propensity for ß-aggregation at residues 44 to 48 of human renal fibrils. To experimentally verify the amyloid propensity of VLITL, we generated synthetic Phe521Leufs-derived peptides and compared their capacity for fibril formation in vitro with that of their VLITL-deleted counterparts. We show that VLITL forms typical amyloid fibrils in vitro and is a major signal for cross-ß-sheet self-association of the 49-mer Phe521Leufs peptide identified in vivo, whereas its absence abrogates fibril formation. This study provides compelling evidence that VLITL confers amyloidogenic properties to Aα-chain frameshift variants, yielding a previously unknown molecular basis for the pathogenesis of Aα-chain amyloidosis.


Assuntos
Motivos de Aminoácidos/fisiologia , Amiloidose Familiar/genética , Fibrinogênio/genética , Mutação da Fase de Leitura , Sequência de Aminoácidos , Amiloide/genética , Amiloidose Familiar/patologia , Humanos , Rim/patologia , Conformação Proteica em Folha beta
20.
Proc Natl Acad Sci U S A ; 114(45): 12039-12044, 2017 11 07.
Artigo em Inglês | MEDLINE | ID: mdl-29078338

RESUMO

Membrane contact sites (MCS) are zones of contact between the membranes of two organelles. At MCS, specific proteins tether the organelles in close proximity and mediate the nonvesicular trafficking of lipids and ions between the two organelles. The endoplasmic reticulum (ER) integral membrane protein VAP is a common component of MCS involved in both tethering and lipid transfer by binding directly to proteins containing a FFAT [two phenylalanines (FF) in an acidic tract (AT)] motif. In addition to maintaining cell homeostasis, MCS formation recently emerged as a mechanism by which intracellular pathogens hijack cellular resources and establish their replication niche. Here, we investigated the mechanism by which the Chlamydia-containing vacuole, termed the inclusion, establishes direct contact with the ER. We show that the Chlamydia protein IncV, which is inserted into the inclusion membrane, displays one canonical and one noncanonical FFAT motif that cooperatively mediated the interaction of IncV with VAP. IncV overexpression was sufficient to bring the ER in close proximity of IncV-containing membranes. Although IncV deletion partially decreased VAP association with the inclusion, it did not suppress the formation of ER-inclusion MCS, suggesting the existence of redundant mechanisms in MCS formation. We propose a model in which IncV acts as one of the primary tethers that contribute to the formation of ER-inclusion MCS. Our results highlight a previously unidentified mechanism of bacterial pathogenesis and support the notion that cooperation of two FFAT motifs may be a common feature of VAP-mediated MCS formation. Chlamydia-host cell interaction therefore constitutes a unique system to decipher the molecular mechanisms underlying MCS formation.


Assuntos
Motivos de Aminoácidos/fisiologia , Proteínas de Bactérias/metabolismo , Chlamydia/metabolismo , Retículo Endoplasmático/metabolismo , Vacúolos/metabolismo , Sítios de Ligação/fisiologia , Proteínas de Transporte/metabolismo , Linhagem Celular , Linhagem Celular Tumoral , Retículo Endoplasmático/microbiologia , Células HEK293 , Células HeLa , Humanos , Corpos de Inclusão/metabolismo , Corpos de Inclusão/microbiologia , Proteínas de Membrana/metabolismo , Ligação Proteica/fisiologia , Transporte Proteico/fisiologia , Vacúolos/microbiologia , Proteínas de Transporte Vesicular/metabolismo
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