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1.
PLoS Pathog ; 16(8): e1008753, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32866207

RESUMO

The induction of broad and potent immunity by vaccines is the key focus of research efforts aimed at protecting against HIV-1 infection. Soluble native-like HIV-1 envelope glycoproteins have shown promise as vaccine candidates as they can induce potent autologous neutralizing responses in rabbits and non-human primates. In this study, monoclonal antibodies were isolated and characterized from rhesus macaques immunized with the BG505 SOSIP.664 trimer to better understand vaccine-induced antibody responses. Our studies reveal a diverse landscape of antibodies recognizing immunodominant strain-specific epitopes and non-neutralizing neo-epitopes. Additionally, we isolated a subset of mAbs against an epitope cluster at the gp120-gp41 interface that recognize the highly conserved fusion peptide and the glycan at position 88 and have characteristics akin to several human-derived broadly neutralizing antibodies.


Assuntos
Vacinas contra a AIDS/imunologia , Mapeamento de Epitopos , Epitopos/imunologia , Anticorpos Anti-HIV/imunologia , Proteína gp120 do Envelope de HIV/imunologia , Proteína gp41 do Envelope de HIV/imunologia , HIV-1/imunologia , Vacinas contra a AIDS/genética , Animais , Anticorpos Monoclonais Murinos/imunologia , Epitopos/genética , Anticorpos Anti-HIV/genética , Proteína gp41 do Envelope de HIV/genética , HIV-1/genética , Macaca mulatta , Multimerização Proteica/genética , Multimerização Proteica/imunologia
2.
Proc Natl Acad Sci U S A ; 117(27): 15666-15672, 2020 07 07.
Artigo em Inglês | MEDLINE | ID: mdl-32571956

RESUMO

Muscle contraction depends on the cyclical interaction of myosin and actin filaments. Therefore, it is important to understand the mechanisms of polymerization and depolymerization of muscle myosins. Muscle myosin 2 monomers exist in two states: one with a folded tail that interacts with the heads (10S) and one with an unfolded tail (6S). It has been thought that only unfolded monomers assemble into bipolar and side-polar (smooth muscle myosin) filaments. We now show by electron microscopy that, after 4 s of polymerization in vitro in both the presence (smooth muscle myosin) and absence of ATP, skeletal, cardiac, and smooth muscle myosins form tail-folded monomers without tail-head interaction, tail-folded antiparallel dimers, tail-folded antiparallel tetramers, unfolded bipolar tetramers, and small filaments. After 4 h, the myosins form thick bipolar and, for smooth muscle myosin, side-polar filaments. Nonphosphorylated smooth muscle myosin polymerizes in the presence of ATP but with a higher critical concentration than in the absence of ATP and forms only bipolar filaments with bare zones. Partial depolymerization in vitro of nonphosphorylated smooth muscle myosin filaments by the addition of MgATP is the reverse of polymerization.


Assuntos
Citoesqueleto de Actina/química , Miosina Tipo II/química , Miosinas/química , Miosinas de Músculo Liso/química , Citoesqueleto de Actina/genética , Citoesqueleto de Actina/ultraestrutura , Animais , Galinhas , Microscopia Eletrônica , Miosina Tipo II/genética , Miosina Tipo II/ultraestrutura , Miosinas/genética , Miosinas/ultraestrutura , Fosforilação/genética , Polimerização , Conformação Proteica , Dobramento de Proteína , Multimerização Proteica/genética , Desdobramento de Proteína , Miosinas de Músculo Liso/genética , Miosinas de Músculo Liso/ultraestrutura
3.
Nat Commun ; 11(1): 2841, 2020 06 05.
Artigo em Inglês | MEDLINE | ID: mdl-32503989

RESUMO

The capsid of human papillomavirus (HPV) spontaneously arranges into a T = 7 icosahedral particle with 72 L1 pentameric capsomeres associating via disulfide bonds between Cys175 and Cys428. Here, we design a capsomere-hybrid virus-like particle (chVLP) to accommodate multiple types of L1 pentamers by the reciprocal assembly of single C175A and C428A L1 mutants, either of which alone encumbers L1 pentamer particle self-assembly. We show that co-assembly between any pair of C175A and C428A mutants across at least nine HPV genotypes occurs at a preferred equal molar stoichiometry, irrespective of the type or number of L1 sequences. A nine-valent chVLP vaccine-formed through the structural clustering of HPV epitopes-confers neutralization titers that are comparable with that of Gardasil 9 and elicits minor cross-neutralizing antibodies against some heterologous HPV types. These findings may pave the way for a new vaccine design that targets multiple pathogenic variants or cancer cells bearing diverse neoantigens.


Assuntos
Proteínas do Capsídeo/imunologia , Neoplasias/terapia , Papillomaviridae/imunologia , Infecções por Papillomavirus/terapia , Vacinas contra Papillomavirus/imunologia , Animais , Anticorpos Neutralizantes/sangue , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/sangue , Anticorpos Antivirais/imunologia , Proteínas do Capsídeo/administração & dosagem , Proteínas do Capsídeo/genética , Desenho de Fármacos , Epitopos/genética , Epitopos/imunologia , Feminino , Humanos , Imunogenicidade da Vacina , Camundongos , Modelos Animais , Mutação , Neoplasias/virologia , Testes de Neutralização , Papillomaviridae/genética , Infecções por Papillomavirus/virologia , Vacinas contra Papillomavirus/administração & dosagem , Vacinas contra Papillomavirus/genética , Multimerização Proteica/genética , Multimerização Proteica/imunologia , Vacinas de Partículas Semelhantes a Vírus/administração & dosagem , Vacinas de Partículas Semelhantes a Vírus/genética , Vacinas de Partículas Semelhantes a Vírus/imunologia
4.
Nature ; 581(7809): 480-485, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32461643

RESUMO

Most proteins associate into multimeric complexes with specific architectures1,2, which often have functional properties such as cooperative ligand binding or allosteric regulation3. No detailed knowledge is available about how any multimer and its functions arose during evolution. Here we use ancestral protein reconstruction and biophysical assays to elucidate the origins of vertebrate haemoglobin, a heterotetramer of paralogous α- and ß-subunits that mediates respiratory oxygen transport and exchange by cooperatively binding oxygen with moderate affinity. We show that modern haemoglobin evolved from an ancient monomer and characterize the historical 'missing link' through which the modern tetramer evolved-a noncooperative homodimer with high oxygen affinity that existed before the gene duplication that generated distinct α- and ß-subunits. Reintroducing just two post-duplication historical substitutions into the ancestral protein is sufficient to cause strong tetramerization by creating favourable contacts with more ancient residues on the opposing subunit. These surface substitutions markedly reduce oxygen affinity and even confer cooperativity, because an ancient linkage between the oxygen binding site and the multimerization interface was already an intrinsic feature of the protein's structure. Our findings establish that evolution can produce new complex molecular structures and functions via simple genetic mechanisms that recruit existing biophysical features into higher-level architectures.


Assuntos
Evolução Molecular , Hemoglobinas/metabolismo , Regulação Alostérica , Sítios de Ligação/genética , Heme/metabolismo , Hemoglobinas/química , Humanos , Ferro/metabolismo , Modelos Moleculares , Oxigênio/metabolismo , Multimerização Proteica/genética , Estrutura Quaternária de Proteína/genética , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo
5.
Proc Natl Acad Sci U S A ; 117(14): 7814-7823, 2020 04 07.
Artigo em Inglês | MEDLINE | ID: mdl-32198203

RESUMO

Hsp70 is a conserved molecular chaperone that plays an indispensable role in regulating protein folding, translocation, and degradation. The conformational dynamics of Hsp70 and its regulation by cochaperones are vital to its function. Using bulk and single-molecule fluorescence resonance energy transfer (smFRET) techniques, we studied the interdomain conformational distribution of human stress-inducible Hsp70A1 and the kinetics of conformational changes induced by nucleotide and the Hsp40 cochaperone Hdj1. We found that the conformations between and within the nucleotide- and substrate-binding domains show heterogeneity. The conformational distribution in the ATP-bound state can be induced by Hdj1 to form an "ADP-like" undocked conformation, which is an ATPase-stimulated state. Kinetic measurements indicate that Hdj1 binds to monomeric Hsp70 as the first step, then induces undocking of the two domains and closing of the substrate-binding cleft. Dimeric Hdj1 then facilitates dimerization of Hsp70 and formation of a heterotetrameric Hsp70-Hsp40 complex. Our results provide a kinetic view of the conformational cycle of Hsp70 and reveal the importance of the dynamic nature of Hsp70 for its function.


Assuntos
Proteínas de Choque Térmico HSP40/genética , Proteínas de Choque Térmico HSP70/ultraestrutura , Chaperonas Moleculares/ultraestrutura , Conformação Proteica , Adenosina Trifosfatases/química , Adenosina Trifosfatases/genética , Trifosfato de Adenosina/química , Transferência Ressonante de Energia de Fluorescência , Heterogeneidade Genética , Proteínas de Choque Térmico HSP40/química , Proteínas de Choque Térmico HSP70/química , Humanos , Cinética , Modelos Moleculares , Chaperonas Moleculares/química , Ligação Proteica/genética , Domínios Proteicos/genética , Dobramento de Proteína , Multimerização Proteica/genética
6.
Biochemistry ; 59(12): 1252-1260, 2020 03 31.
Artigo em Inglês | MEDLINE | ID: mdl-32176479

RESUMO

Protein kinase R (PKR) is a key antiviral component of the innate immune pathway and is activated by viral double-stranded RNAs (dsRNAs). Adenovirus-associated RNA 1 (VAI) is an abundant, noncoding viral RNA that functions as a decoy by binding PKR but not inducing activation, thereby inhibiting the antiviral response. In VAI, coaxial stacking produces an extended helix that mediates high-affinity PKR binding but is too short to result in activation. Like adenovirus, Epstein-Barr virus produces high concentrations of a noncoding RNA, EBER1. Here, we compare interactions of PKR with VAI and EBER1 and present a structural model of EBER1. Both RNAs function as inhibitors of dsRNA-mediated PKR activation. However, EBER1 weakly activates PKR whereas VAI does not. PKR binds EBER1 more weakly than VAI. Assays at physiological ion concentrations indicate that both RNAs can accommodate two PKR monomers and induce PKR dimerization. A structural model of EBER1 was obtained using constraints derived from chemical structure probing and small-angle X-ray scattering experiments. The central stem of EBER1 coaxially stacks with stem loop 4 and stem loop 1 to form an extended RNA duplex of ∼32 bp that binds PKR and promotes activation. Our observations that EBER1 binds PKR much more weakly than VAI and exhibits weak PKR activation suggest that EBER1 is less well suited to function as an RNA decoy.


Assuntos
Herpesvirus Humano 4/genética , Interações entre Hospedeiro e Microrganismos/genética , RNA Viral/metabolismo , eIF-2 Quinase/genética , Infecções por Vírus Epstein-Barr/imunologia , Infecções por Vírus Epstein-Barr/virologia , Herpesvirus Humano 4/imunologia , Humanos , Imunidade Inata/genética , Modelos Moleculares , Conformação de Ácido Nucleico , Multimerização Proteica/genética , Multimerização Proteica/imunologia , Estabilidade de RNA , RNA Viral/química , Espalhamento a Baixo Ângulo , Difração de Raios X , eIF-2 Quinase/química , eIF-2 Quinase/imunologia , eIF-2 Quinase/metabolismo
7.
PLoS One ; 15(2): e0228036, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32015565

RESUMO

Atomic Force Microscopy was utilized to study the morphology of Gag, ΨRNA, and their binding complexes with lipids in a solution environment with 0.1Å vertical and 1nm lateral resolution. TARpolyA RNA was used as a RNA control. The lipid used was phospha-tidylinositol-(4,5)-bisphosphate (PI(4,5)P2). The morphology of specific complexes Gag-ΨRNA, Gag-TARpolyA RNA, Gag-PI(4,5)P2 and PI(4,5)P2-ΨRNA-Gag were studied. They were imaged on either positively or negatively charged mica substrates depending on the net charges carried. Gag and its complexes consist of monomers, dimers and tetramers, which was confirmed by gel electrophoresis. The addition of specific ΨRNA to Gag is found to increase Gag multimerization. Non-specific TARpolyA RNA was found not to lead to an increase in Gag multimerization. The addition PI(4,5)P2 to Gag increases Gag multimerization, but to a lesser extent than ΨRNA. When both ΨRNA and PI(4,5)P2 are present Gag undergoes comformational changes and an even higher degree of multimerization.


Assuntos
Infecções por HIV/genética , HIV-1/genética , RNA Viral/genética , Produtos do Gene gag do Vírus da Imunodeficiência Humana/ultraestrutura , Membrana Celular/química , Membrana Celular/genética , Infecções por HIV/virologia , Soropositividade para HIV , HIV-1/química , HIV-1/patogenicidade , Humanos , Lipídeos/química , Microscopia de Força Atômica , Complexos Multiproteicos/química , Complexos Multiproteicos/genética , Fosfatidilinositol 4,5-Difosfato/química , Ligação Proteica , Multimerização Proteica/genética , RNA Viral/química , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/genética , Produtos do Gene gag do Vírus da Imunodeficiência Humana/química , Produtos do Gene gag do Vírus da Imunodeficiência Humana/genética
8.
Biochem J ; 477(2): 407-429, 2020 01 31.
Artigo em Inglês | MEDLINE | ID: mdl-31899476

RESUMO

Human HtrA3 (high-temperature requirement protease A3) is a trimeric multitasking propapoptotic serine protease associated with critical cellular functions and pathogenicity. Implicated in diseases including cancer and pre-eclampsia, its role as a tumor suppressor and potential therapeutic target cannot be ignored. Therefore, elucidating its mode of activation and regulatory switch becomes indispensable towards modulating its functions with desired effects for disease intervention. Using computational, biochemical and biophysical tools, we delineated the role of all domains, their combinations and the critical phenylalanine residues in regulating HtrA3 activity, oligomerization and specificity. Our findings underline the crucial roles of the N-terminus as well as the PDZ domain in oligomerization and formation of a catalytically competent enzyme, thus providing new insights into its structure-function coordination. Our study also reports an intricate ligand-induced allosteric switch, which redefines the existing hypothesis of HtrA3 activation besides opening up avenues for modulating protease activity favorably through suitable effector molecules.


Assuntos
Conformação Proteica , Serina Endopeptidases/genética , Serina Proteases/genética , Relação Estrutura-Atividade , Regulação Alostérica/genética , Sequência de Aminoácidos/genética , Catálise , Regulação Enzimológica da Expressão Gênica/genética , Humanos , Domínios PDZ/genética , Multimerização Proteica/genética , Serina Endopeptidases/química , Serina Endopeptidases/ultraestrutura , Serina Proteases/química , Serina Proteases/ultraestrutura
9.
J Biol Chem ; 295(7): 2001-2017, 2020 02 14.
Artigo em Inglês | MEDLINE | ID: mdl-31919096

RESUMO

The MAX network transcriptional repressor (MNT) is an MXD family transcription factor of the basic helix-loop-helix (bHLH) family. MNT dimerizes with another transcriptional regulator, MYC-associated factor X (MAX), and down-regulates genes by binding to E-boxes. MAX also dimerizes with MYC, an oncogenic bHLH transcription factor. Upon E-box binding, the MYC-MAX dimer activates gene expression. MNT also binds to the MAX dimerization protein MLX (MLX), and MNT-MLX and MNT-MAX dimers co-exist. However, all MNT functions have been attributed to MNT-MAX dimers, and no functions of the MNT-MLX dimer have been described. MNT's biological role has been linked to its function as a MYC oncogene modulator, but little is known about its regulation. We show here that MNT localizes to the nucleus of MAX-expressing cells and that MNT-MAX dimers bind and repress the MNT promoter, an effect that depends on one of the two E-boxes on this promoter. In MAX-deficient cells, MNT was overexpressed and redistributed to the cytoplasm. Interestingly, MNT was required for cell proliferation even in the absence of MAX. We show that in MAX-deficient cells, MNT binds to MLX, but also forms homodimers. RNA-sequencing experiments revealed that MNT regulates the expression of several genes even in the absence of MAX, with many of these genes being involved in cell cycle regulation and DNA repair. Of note, MNT-MNT homodimers regulated the transcription of some genes involved in cell proliferation. The tight regulation of MNT and its functionality even without MAX suggest a major role for MNT in cell proliferation.


Assuntos
Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/genética , Proteínas Repressoras/genética , Transcrição Genética , Sequência de Aminoácidos/genética , Animais , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/química , Proliferação de Células/genética , Regulação da Expressão Gênica/genética , Redes Reguladoras de Genes/genética , Sequências Hélice-Alça-Hélice/genética , Humanos , Regiões Promotoras Genéticas , Multimerização Proteica/genética , Proteínas Proto-Oncogênicas c-myc/química , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Repressoras/química
10.
Nucleic Acids Res ; 48(2): 847-861, 2020 01 24.
Artigo em Inglês | MEDLINE | ID: mdl-31802130

RESUMO

RNase E is a 472-kDa homo-tetrameric essential endoribonuclease involved in RNA processing and turnover in Escherichia coli. In its N-terminal half (NTH) is the catalytic active site, as also a substrate 5'-sensor pocket that renders enzyme activity maximal on 5'-monophosphorylated RNAs. The protein's non-catalytic C-terminal half (CTH) harbours RNA-binding motifs and serves as scaffold for a multiprotein degradosome complex, but is dispensable for viability. Here, we provide evidence that a full-length hetero-tetramer, composed of a mixture of wild-type and (recessive lethal) active-site mutant subunits, exhibits identical activity in vivo as the wild-type homo-tetramer itself ('recessive resurrection'). When all of the cognate polypeptides lacked the CTH, the active-site mutant subunits were dominant negative. A pair of C-terminally truncated polypeptides, which were individually inactive because of additional mutations in their active site and 5'-sensor pocket respectively, exhibited catalytic function in combination, both in vivo and in vitro (i.e. intragenic or allelic complementation). Our results indicate that adjacent subunits within an oligomer are separately responsible for 5'-sensing and cleavage, and that RNA binding facilitates oligomerization. We propose also that the CTH mediates a rate-determining initial step for enzyme function, which is likely the binding and channelling of substrate for NTH's endonucleolytic action.


Assuntos
Domínio Catalítico/genética , Endorribonucleases/genética , RNA/genética , Sítios de Ligação/genética , Catálise , Endorribonucleases/química , Escherichia coli/química , Escherichia coli/genética , Complexos Multienzimáticos/química , Complexos Multienzimáticos/genética , Mutação/genética , Peptídeos/genética , Polirribonucleotídeo Nucleotidiltransferase/química , Polirribonucleotídeo Nucleotidiltransferase/genética , Conformação Proteica , Multimerização Proteica/genética , RNA/química , RNA Helicases/química , RNA Helicases/genética , Motivos de Ligação ao RNA/genética
11.
Nucleic Acids Res ; 48(2): 788-801, 2020 01 24.
Artigo em Inglês | MEDLINE | ID: mdl-31799608

RESUMO

In all organisms, a selected type of proteins accomplishes critical roles in cellular processes that govern gene expression. The multifunctional protein Gemin5 cooperates in translation control and ribosome binding, besides acting as the RNA-binding protein of the survival of motor neuron (SMN) complex. While these functions reside on distinct domains located at each end of the protein, the structure and function of the middle region remained unknown. Here, we solved the crystal structure of an extended tetratricopeptide (TPR)-like domain in human Gemin5 that self-assembles into a previously unknown canoe-shaped dimer. We further show that the dimerization module is functional in living cells driving the interaction between the viral-induced cleavage fragment p85 and the full-length Gemin5, which anchors splicing and translation members. Disruption of the dimerization surface by a point mutation in the TPR-like domain prevents this interaction and also abrogates translation enhancement induced by p85. The characterization of this unanticipated dimerization domain provides the structural basis for a role of the middle region of Gemin5 as a central hub for protein-protein interactions.


Assuntos
Biossíntese de Proteínas , Proteínas de Ligação a RNA/genética , Ribonucleoproteínas Nucleares Pequenas/genética , Proteínas do Complexo SMN/genética , Humanos , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas/genética , Multimerização Proteica/genética , Ribonucleoproteínas Nucleares Pequenas/química , Proteínas do Complexo SMN/química
12.
Biochim Biophys Acta Mol Cell Res ; 1867(1): 118573, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31678591

RESUMO

Cytochrome c (Cyt c) released from mitochondria interacts with Apaf-1 to form the heptameric apoptosome, which initiates the caspase cascade to execute apoptosis. Although lysine residue at 72 (K72) of Cyt c plays an important role in the Cyt c-Apaf-1 interaction, the underlying mechanism of interaction between Cyt c and Apaf-1 is still not clearly defined. Here we identified multiple lysine residues including K72, which are also known to interact with ATP, to play a key role in Cyt c-Apaf-1 interaction. Mutation of these lysine residues abrogates the apoptosome formation causing inhibition of caspase activation. Using in-silico molecular docking, we have identified Cyt c-binding interface on Apaf-1. Although mutant Cyt c shows higher affinity for Apaf-1, the presence of Cyt c-WT restores the apoptosome activity. ATP addition modulates only mutant Cyt c binding to Apaf-1 but not WT Cyt c binding to Apaf-1. Using TCGA and cBioPortal, we identified multiple mutations in both Apaf-1 and Cyt c that are predicted to interfere with apoptosome assembly. We also demonstrate that transcript levels of various enzymes involved with dATP or ATP synthesis are increased in various cancers. Silencing of nucleotide metabolizing enzymes such as ribonucleotide reductase subunit M1 (RRM1) and ATP-producing glycolytic enzymes PKM2 attenuated ATP production and enhanced caspase activation. These findings suggest important role for lysine residues of Cyt c and nucleotides in the regulation of apoptosome-dependent apoptotic cell death as well as demonstrate how these mutations and nucleotides may have a pivotal role in human diseases such as cancer.


Assuntos
Apoptossomas/fisiologia , Citocromos c/química , Simulação de Acoplamento Molecular , Neoplasias/patologia , Nucleotídeos/química , Alanina/química , Alanina/genética , Substituição de Aminoácidos , Apoptossomas/química , Fator Apoptótico 1 Ativador de Proteases/química , Fator Apoptótico 1 Ativador de Proteases/metabolismo , Estudos de Casos e Controles , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/metabolismo , Células Cultivadas , Citocromos c/genética , Citocromos c/metabolismo , Feminino , Humanos , Lisina/química , Lisina/genética , Masculino , Modelos Moleculares , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Proteínas Mutantes/fisiologia , Neoplasias/genética , Neoplasias/metabolismo , Nucleotídeos/metabolismo , Células PC-3 , Ligação Proteica/genética , Mapeamento de Interação de Proteínas , Multimerização Proteica/genética , Transdução de Sinais/genética
13.
Proc Natl Acad Sci U S A ; 117(1): 733-740, 2020 01 07.
Artigo em Inglês | MEDLINE | ID: mdl-31874927

RESUMO

Vascular plants provide most of the biomass, food, and feed on earth, yet the molecular innovations that led to the evolution of their conductive tissues are unknown. Here, we reveal the evolutionary trajectory for the heterodimeric TMO5/LHW transcription factor complex, which is rate-limiting for vascular cell proliferation in Arabidopsis thaliana Both regulators have origins predating vascular tissue emergence, and even terrestrialization. We further show that TMO5 evolved its modern function, including dimerization with LHW, at the origin of land plants. A second innovation in LHW, coinciding with vascular plant emergence, conditioned obligate heterodimerization and generated the critical function in vascular development in Arabidopsis In summary, our results suggest that the division potential of vascular cells may have been an important factor contributing to the evolution of vascular plants.


Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Evolução Molecular , Regulação da Expressão Gênica no Desenvolvimento , Regulação da Expressão Gênica de Plantas , Transativadores/genética , Proteínas de Arabidopsis/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Proliferação de Células/genética , Floema/citologia , Floema/crescimento & desenvolvimento , Floema/metabolismo , Filogenia , Plantas Geneticamente Modificadas , Multimerização Proteica/genética , Transativadores/metabolismo , Xilema/citologia , Xilema/crescimento & desenvolvimento , Xilema/metabolismo
14.
Nat Chem Biol ; 16(2): 179-187, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31844302

RESUMO

Augmenting live cells with new signal transduction capabilities is a key objective in genetic engineering and synthetic biology. We showed earlier that two-component signaling pathways could function in mammalian cells, albeit while losing their ligand sensitivity. Here, we show how to transduce small-molecule ligands in a dose-dependent fashion into gene expression in mammalian cells using two-component signaling machinery. First, we engineer mutually complementing truncated mutants of a histidine kinase unable to dimerize and phosphorylate the response regulator. Next, we fuse these mutants to protein domains capable of ligand-induced dimerization, which restores the phosphoryl transfer in a ligand-dependent manner. Cytoplasmic ligands are transduced by facilitating mutant dimerization in the cytoplasm, while extracellular ligands trigger dimerization at the inner side of a plasma membrane. These findings point to the potential of two-component regulatory systems as enabling tools for orthogonal signaling pathways in mammalian cells.


Assuntos
Histidina Quinase/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Transdução de Sinais/fisiologia , Biologia Sintética/métodos , Proteínas da Membrana Bacteriana Externa/genética , Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Regulação da Expressão Gênica , Células HEK293 , Histidina Quinase/genética , Humanos , Complexos Multienzimáticos/genética , Complexos Multienzimáticos/metabolismo , Mutação , Fosforilação/genética , Domínios Proteicos , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Multimerização Proteica/genética , Receptores Acoplados a Proteínas-G/genética , Receptores Acoplados a Proteínas-G/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteína 1A de Ligação a Tacrolimo/genética , Proteína 1A de Ligação a Tacrolimo/metabolismo , beta-Arrestinas/genética , beta-Arrestinas/metabolismo
15.
J Virol ; 94(6)2020 02 28.
Artigo em Inglês | MEDLINE | ID: mdl-31852780

RESUMO

The phosphoprotein (P) of the nonsegmented negative-sense RNA viruses is a multimeric modular protein that is essential for RNA transcription and replication. Despite great variability in length and sequence, the architecture of this protein is conserved among the different viral families, with a long N-terminal intrinsically disordered region comprising a nucleoprotein chaperone module, a central multimerization domain (PMD), connected by a disordered linker to a C-terminal nucleocapsid-binding domain. The P protein of vesicular stomatitis virus (VSV) forms dimers, and here we investigate the importance of its dimerization domain, PMD, for viral gene expression and virus growth. A truncated P protein lacking the central dimerization domain (PΔMD) loses its ability to form dimers both in vitro and in a yeast two-hybrid system but conserves its ability to bind N. In a minireplicon system, the truncated monomeric protein performs almost as well as the full-length dimeric protein, while a recombinant virus harboring the same truncation in the P protein has been rescued and follows replication kinetics similar to those seen with the wild-type virus, showing that the dimerization domain of P is dispensable for viral gene expression and virus replication in cell culture. Because RNA viruses have high mutation rates, it is unlikely that a structured domain such as a VSV dimerization domain would persist in the absence of a function(s), but our work indicates that it is not required for the functioning of the RNA polymerase machinery or for the assembly of new viruses.IMPORTANCE The phosphoprotein (P) is an essential and conserved component of all nonsegmented negative-sense RNA viruses, including some major human pathogens (e.g., rabies virus, measles virus, respiratory syncytial virus [RSV], Ebola virus, and Nipah virus). P is a modular protein with intrinsically disordered regions and folded domains that plays specific and similar roles in the replication of the different viruses and, in some cases, hijacks cell components to the advantage of the virus and is involved in immune evasion. All P proteins are multimeric, but the role of this multimerization is still unclear. Here, we demonstrate that the dimerization domain of VSV P is dispensable for the expression of virally encoded proteins and for virus growth in cell culture. This provides new insights into and raises questions about the functioning of the RNA-synthesizing machinery of the nonsegmented negative-sense RNA viruses.


Assuntos
Fosfoproteínas/química , Domínios Proteicos , Multimerização Proteica , Vírus da Estomatite Vesicular Indiana/metabolismo , RNA Polimerases Dirigidas por DNA/metabolismo , Dimerização , Modelos Moleculares , Nucleocapsídeo/metabolismo , Nucleoproteínas/metabolismo , Fosfoproteínas/genética , Ligação Proteica , Conformação Proteica , Multimerização Proteica/genética , RNA Viral/genética , Alinhamento de Sequência , Estomatite Vesicular/virologia , Vírus da Estomatite Vesicular Indiana/genética , Vírus da Estomatite Vesicular Indiana/crescimento & desenvolvimento , Replicação Viral
16.
J Mol Model ; 26(1): 1, 2019 Dec 13.
Artigo em Inglês | MEDLINE | ID: mdl-31834477

RESUMO

The aggregation of proteins in the brain is one of the main features of neurodegenerative diseases. In Alzheimer's disease, the abnormal aggregation of Aß-42 is due to intrinsic and extrinsic factors. The latter is due to variations in the environment, such as temperature, salt concentration, and pH. We evaluated the effect of protonation/deprotonation of residues that are part of trimeric and pentameric oligomers at pH 5, pH 6, and pH 7. Molecular dynamics simulation at 200 ns in the canonical ensemble was implemented. The results have revealed that histidine, glutamic acid, and aspartic acid residues showed a protonation/deprotonation effect in oligomers. The root mean square deviation analysis was used to analyze the structural stability at different pHs. We found an increase in hydrophobicity in the side chains of the trimer, while in the pentamer, the structural instability of a compact structure at pH 5 caused the hydrophobic core to open, revealing the hydrophobic region to the environment. At this point, we believe that conformational changes mediated by pH are essential in the aggregation of Aß-42 oligomers.


Assuntos
Doença de Alzheimer/genética , Peptídeos beta-Amiloides/genética , Fragmentos de Peptídeos/genética , Agregação Patológica de Proteínas/genética , Doença de Alzheimer/patologia , Sequência de Aminoácidos/genética , Peptídeos beta-Amiloides/química , Ácido Aspártico/química , Ácido Aspártico/genética , Histidina/química , Histidina/genética , Humanos , Concentração de Íons de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Modelos Moleculares , Simulação de Dinâmica Molecular , Fragmentos de Peptídeos/química , Agregação Patológica de Proteínas/patologia , Conformação Proteica , Multimerização Proteica/genética
17.
Nat Commun ; 10(1): 5703, 2019 12 13.
Artigo em Inglês | MEDLINE | ID: mdl-31836707

RESUMO

The macromolecular machines of life use allosteric control to self-assemble, dissociate and change shape in response to signals. Despite enormous interest, the design of nanoscale allosteric assemblies has proven tremendously challenging. Here we present a proof of concept of allosteric assembly in which an engineered fold switch on the protein monomer triggers or blocks assembly. Our design is based on the hyper-stable, naturally monomeric protein CI2, a paradigm of simple two-state folding, and the toroidal arrangement with 6-fold symmetry that it only adopts in crystalline form. We engineer CI2 to enable a switch between the native and an alternate, latent fold that self-assembles onto hexagonal toroidal particles by exposing a favorable inter-monomer interface. The assembly is controlled on demand via the competing effects of temperature and a designed short peptide. These findings unveil a remarkable potential for structural metamorphosis in proteins and demonstrate key principles for engineering protein-based nanomachinery.


Assuntos
Engenharia de Proteínas/métodos , Dobramento de Proteína , Multimerização Proteica/genética , Proteínas/metabolismo , Inibidores de Serino Proteinase/metabolismo , Regulação Alostérica , Clonagem Molecular , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Mutação , Estrutura Secundária de Proteína/genética , Proteínas/genética , Proteínas/isolamento & purificação , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Serina Proteases/metabolismo , Inibidores de Serino Proteinase/genética , Inibidores de Serino Proteinase/isolamento & purificação
18.
Oxid Med Cell Longev ; 2019: 9186469, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31885824

RESUMO

Cardiolipin (CL) is a multifunctional dimeric phospholipid that physically interacts with electron transport chain complexes I, III, and IV, and ATP synthase (complex V). The enzyme ALCAT1 catalyzes the conversion of cardiolipin by incorporating polyunsaturated fatty acids into cardiolipin. The resulting CL species are said to be more susceptible to oxidative damage. This is thought to negatively affect the interaction of cardiolipin and electron transport chain complexes, leading to increased ROS production and mitochondrial dysfunction. Furthermore, it is discussed that ALCAT1 itself is upregulated due to oxidative stress. Here, we investigated the effects of overexpression of ALCAT1 under different metabolic conditions. ALCAT1 is located at the ER and mitochondria, probably at contact sites. We found that respiration stimulated by galactose supply promoted supercomplex assembly but also led to increased mitochondrial ROS levels. Endogeneous ALCAT1 protein expression levels showed a fairly high variability. Artificially induced ALCAT1 overexpression reduced supercomplex formation, further promoted ROS production, and prevented upregulation of coupled respiration. Taken together, our data suggest that the amount of the CL conversion enzyme ALCAT1 is critical for coupling mitochondrial respiration and metabolic plasticity.


Assuntos
1-Acilglicerol-3-Fosfato O-Aciltransferase/metabolismo , Cardiolipinas/metabolismo , Mitocôndrias/metabolismo , Complexos Multiproteicos/metabolismo , 1-Acilglicerol-3-Fosfato O-Aciltransferase/genética , Respiração Celular , Galactose/metabolismo , Células HeLa , Humanos , Potencial da Membrana Mitocondrial , Estresse Oxidativo , Multimerização Proteica/genética , Espécies Reativas de Oxigênio/metabolismo
19.
Elife ; 82019 12 24.
Artigo em Inglês | MEDLINE | ID: mdl-31873072

RESUMO

Coupling of endoplasmic reticulum (ER) stress to dimerisation-dependent activation of the UPR transducer IRE1 is incompletely understood. Whilst the luminal co-chaperone ERdj4 promotes a complex between the Hsp70 BiP and IRE1's stress-sensing luminal domain (IRE1LD) that favours the latter's monomeric inactive state and loss of ERdj4 de-represses IRE1, evidence linking these cellular and in vitro observations is presently lacking. We report that enforced loading of endogenous BiP onto endogenous IRE1α repressed UPR signalling in CHO cells and deletions in the IRE1α locus that de-repressed the UPR in cells, encode flexible regions of IRE1LD that mediated BiP-induced monomerisation in vitro. Changes in the hydrogen exchange mass spectrometry profile of IRE1LD induced by ERdj4 and BiP confirmed monomerisation and were consistent with active destabilisation of the IRE1LD dimer. Together, these observations support a competition model whereby waning ER stress passively partitions ERdj4 and BiP to IRE1LD to initiate active repression of UPR signalling.


Assuntos
Estresse do Retículo Endoplasmático/genética , Endorribonucleases/química , Proteínas de Choque Térmico HSP40/química , Proteínas de Membrana/química , Chaperonas Moleculares/química , Proteínas Serina-Treonina Quinases/química , Resposta a Proteínas não Dobradas/genética , Animais , Células CHO , Cricetinae , Cricetulus , Retículo Endoplasmático/genética , Endorribonucleases/genética , Escherichia coli/genética , Proteínas de Choque Térmico HSP40/genética , Proteínas de Choque Térmico HSP70/química , Proteínas de Choque Térmico HSP70/genética , Humanos , Proteínas de Membrana/genética , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Ligação Proteica/genética , Conformação Proteica , Multimerização Proteica/genética , Proteínas Serina-Treonina Quinases/genética
20.
Elife ; 82019 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-31573509

RESUMO

Small heat shock proteins (sHSPs) are nature's 'first responders' to cellular stress, interacting with affected proteins to prevent their aggregation. Little is known about sHSP structure beyond its structured α-crystallin domain (ACD), which is flanked by disordered regions. In the human sHSP HSPB1, the disordered N-terminal region (NTR) represents nearly 50% of the sequence. Here, we present a hybrid approach involving NMR, hydrogen-deuterium exchange mass spectrometry, and modeling to provide the first residue-level characterization of the NTR. The results support a model in which multiple grooves on the ACD interact with specific NTR regions, creating an ensemble of 'quasi-ordered' NTR states that can give rise to the known heterogeneity and plasticity of HSPB1. Phosphorylation-dependent interactions inform a mechanism by which HSPB1 is activated under stress conditions. Additionally, we examine the effects of disease-associated NTR mutations on HSPB1 structure and dynamics, leveraging our emerging structural insights.


Assuntos
Proteínas de Choque Térmico Pequenas/genética , Proteínas de Choque Térmico/genética , Chaperonas Moleculares/genética , Agregados Proteicos/genética , Domínios e Motivos de Interação entre Proteínas/genética , Sequência de Aminoácidos/genética , Humanos , Espectrometria de Massas , Modelos Moleculares , Conformação Proteica , Multimerização Proteica/genética , Espalhamento a Baixo Ângulo
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