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1.
Nat Commun ; 11(1): 5736, 2020 11 12.
Artigo em Inglês | MEDLINE | ID: mdl-33184256

RESUMO

Highly charged intrinsically disordered proteins can form complexes with very high affinity in which both binding partners fully retain their disorder and dynamics, exemplified by the positively charged linker histone H1.0 and its chaperone, the negatively charged prothymosin α. Their interaction exhibits another surprising feature: The association/dissociation kinetics switch from slow two-state-like exchange at low protein concentrations to fast exchange at higher, physiologically relevant concentrations. Here we show that this change in mechanism can be explained by the formation of transient ternary complexes favored at high protein concentrations that accelerate the exchange between bound and unbound populations by orders of magnitude. Molecular simulations show how the extreme disorder in such polyelectrolyte complexes facilitates (i) diffusion-limited binding, (ii) transient ternary complex formation, and (iii) fast exchange of monomers by competitive substitution, which together enable rapid kinetics. Biological polyelectrolytes thus have the potential to keep regulatory networks highly responsive even for interactions with extremely high affinities.


Assuntos
Proteínas Intrinsicamente Desordenadas/química , Polieletrólitos/química , Cinética , Espectroscopia de Ressonância Magnética , Chaperonas Moleculares/química , Simulação de Dinâmica Molecular , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Precursores de Proteínas/química , Coloração e Rotulagem , Timosina/análogos & derivados
2.
Science ; 369(6507)2020 08 28.
Artigo em Inglês | MEDLINE | ID: mdl-32855309

RESUMO

Neuronal synapses undergo structural and functional changes throughout life, which are essential for nervous system physiology. However, these changes may also perturb the excitatory-inhibitory neurotransmission balance and trigger neuropsychiatric and neurological disorders. Molecular tools to restore this balance are highly desirable. Here, we designed and characterized CPTX, a synthetic synaptic organizer combining structural elements from cerebellin-1 and neuronal pentraxin-1. CPTX can interact with presynaptic neurexins and postsynaptic AMPA-type ionotropic glutamate receptors and induced the formation of excitatory synapses both in vitro and in vivo. CPTX restored synaptic functions, motor coordination, spatial and contextual memories, and locomotion in mouse models for cerebellar ataxia, Alzheimer's disease, and spinal cord injury, respectively. Thus, CPTX represents a prototype for structure-guided biologics that can efficiently repair or remodel neuronal circuits.


Assuntos
Proteína C-Reativa/farmacologia , Proteínas do Tecido Nervoso/farmacologia , Vias Neurais/efeitos dos fármacos , Precursores de Proteínas/farmacologia , Receptores de AMPA/metabolismo , Proteínas Recombinantes/farmacologia , Sinapses/efeitos dos fármacos , Doença de Alzheimer/terapia , Animais , Proteína C-Reativa/química , Proteína C-Reativa/uso terapêutico , Ataxia Cerebelar/terapia , Modelos Animais de Doenças , Células HEK293 , Hipocampo , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/uso terapêutico , Domínios Proteicos , Precursores de Proteínas/química , Precursores de Proteínas/uso terapêutico , Receptores de Glutamato/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/uso terapêutico , Coluna Vertebral/efeitos dos fármacos , Coluna Vertebral/fisiologia
3.
Mol Pharmacol ; 98(2): 96-108, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32487735

RESUMO

In the mid-1970s, an intense race to identify endogenous substances that activated the same receptors as opiates resulted in the identification of the first endogenous opioid peptides. Since then, >20 peptides with opioid receptor activity have been discovered, all of which are generated from three precursors, proenkephalin, prodynorphin, and proopiomelanocortin, by sequential proteolytic processing by prohormone convertases and carboxypeptidase E. Each of these peptides binds to all three of the opioid receptor types (µ, δ, or κ), albeit with differing affinities. Peptides derived from proenkephalin and prodynorphin are broadly distributed in the brain, and mRNA encoding all three precursors are highly expressed in some peripheral tissues. Various approaches have been used to explore the functions of the opioid peptides in specific behaviors and brain circuits. These methods include directly administering the peptides ex vivo (i.e., to excised tissue) or in vivo (in animals), using antagonists of opioid receptors to infer endogenous peptide activity, and genetic knockout of opioid peptide precursors. Collectively, these studies add to our current understanding of the function of endogenous opioids, especially when similar results are found using different approaches. We briefly review the history of identification of opioid peptides, highlight the major findings, address several myths that are widely accepted but not supported by recent data, and discuss unanswered questions and future directions for research. SIGNIFICANCE STATEMENT: Activation of the opioid receptors by opiates and synthetic drugs leads to central and peripheral biological effects, including analgesia and respiratory depression, but these may not be the primary functions of the endogenous opioid peptides. Instead, the opioid peptides play complex and overlapping roles in a variety of systems, including reward pathways, and an important direction for research is the delineation of the role of individual peptides.


Assuntos
Peptídeos Opioides/genética , Peptídeos Opioides/metabolismo , Receptores Opioides/metabolismo , Animais , Encéfalo/metabolismo , Carboxipeptidase H/metabolismo , Encefalinas/química , Encefalinas/genética , Humanos , Pró-Opiomelanocortina/química , Pró-Opiomelanocortina/genética , Pró-Proteína Convertases/metabolismo , Precursores de Proteínas/química , Precursores de Proteínas/genética
4.
Nucleic Acids Res ; 48(14): e83, 2020 08 20.
Artigo em Inglês | MEDLINE | ID: mdl-32526036

RESUMO

Mass spectrometry (MS)-based quantitative proteomics experiments frequently generate data with missing values, which may profoundly affect downstream analyses. A wide variety of imputation methods have been established to deal with the missing-value issue. To date, however, there is a scarcity of efficient, systematic, and easy-to-handle tools that are tailored for proteomics community. Herein, we developed a user-friendly and powerful stand-alone software, NAguideR, to enable implementation and evaluation of different missing value methods offered by 23 widely used missing-value imputation algorithms. NAguideR further evaluates data imputation results through classic computational criteria and, unprecedentedly, proteomic empirical criteria, such as quantitative consistency between different charge-states of the same peptide, different peptides belonging to the same proteins, and individual proteins participating protein complexes and functional interactions. We applied NAguideR into three label-free proteomic datasets featuring peptide-level, protein-level, and phosphoproteomic variables respectively, all generated by data independent acquisition mass spectrometry (DIA-MS) with substantial biological replicates. The results indicate that NAguideR is able to discriminate the optimal imputation methods that are facilitating DIA-MS experiments over those sub-optimal and low-performance algorithms. NAguideR further provides downloadable tables and figures supporting flexible data analysis and interpretation. NAguideR is freely available at http://www.omicsolution.org/wukong/NAguideR/ and the source code: https://github.com/wangshisheng/NAguideR/.


Assuntos
Proteômica/métodos , Software , Células/efeitos dos fármacos , Simulação por Computador , Conjuntos de Dados como Assunto , Formaldeído/farmacologia , Humanos , Espectrometria de Massas , Microtúbulos/efeitos dos fármacos , Nocodazol/farmacologia , Precursores de Proteínas/química
5.
Comput Biol Chem ; 84: 107168, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31791808

RESUMO

The cyclotides are the largest known family of cyclic proteins, which are found in several plant families including Violaceae. They are circular bioactive peptides consisting of 28-37 amino acids, which possess a cyclic cystine knot (CCK) motif and could be useful in biotechnology and drug design as scaffolds for peptide-based drugs. This study describes our finding of a potentially novel gene transcript from the petals of the Iranian Viola tricolor (V. tricolor) flowers. This study is based on the cDNA screening method employed for isolation of cyclotide precursor genes and in silico analysis. Our study resulted in the finding of a novel cyclotide-like precursor from V. tricolor, which is documented in the NCBI by GenBank accession number: KP065812. The in silico analysis revealed that there are lots of similar sequences in many other plant families and they all exhibit some different features from previously discovered cyclotide precursors. The differences occur particularly in the main cyclotide domain that exists without the usual CCK structure. All of these hypothetical precursors have a conserved ER-signal sequence, a Cysteine (C)-rich sequence forming two zinc finger motifs and a cyclotide-like region containing several conserved elements including two highly conserved C residues. In conclusion, using the cDNA screening method we found a potentially new cyclotide-like precursor gene and in silico studies revealed its significant characteristics that may open up a new research line on the distribution and evolution of cyclotides.


Assuntos
Ciclotídeos/análise , Proteínas de Plantas/análise , Precursores de Proteínas/análise , Viola/química , Sequência de Aminoácidos , Ciclotídeos/química , Ciclotídeos/genética , Flores/química , Genes de Plantas , Irã (Geográfico) , Folhas de Planta/química , Proteínas de Plantas/química , Proteínas de Plantas/genética , Precursores de Proteínas/química , Precursores de Proteínas/genética , Alinhamento de Sequência , Análise de Sequência de DNA
6.
PLoS One ; 14(9): e0221550, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31504041

RESUMO

HIV envelope protein (Env) is the sole target of broadly neutralizing antibodies (BNAbs) that are capable of neutralizing diverse strains of HIV. While BNAbs develop spontaneously in a subset of HIV-infected patients, efforts to design an envelope protein-based immunogen to elicit broadly neutralizing antibody responses have so far been unsuccessful. It is hypothesized that a primary barrier to eliciting BNAbs is the fact that HIV envelope proteins bind poorly to the germline-encoded unmutated common ancestor (UCA) precursors to BNAbs. To identify variant forms of Env with increased affinities for the UCA forms of BNAbs 4E10 and 10E8, which target the Membrane Proximal External Region (MPER) of Env, libraries of randomly mutated Env variants were expressed in a yeast surface display system and screened using fluorescence activated cell sorting for cells displaying variants with enhanced abilities to bind the UCA antibodies. Based on analyses of individual clones obtained from the screen and on next-generation sequencing of sorted libraries, distinct but partially overlapping sets of amino acid substitutions conferring enhanced UCA antibody binding were identified. These were particularly enriched in substitutions of arginine for highly conserved tryptophan residues. The UCA-binding variants also generally exhibited enhanced binding to the mature forms of anti-MPER antibodies. Mapping of the identified substitutions into available structures of Env suggest that they may act by destabilizing both the initial pre-fusion conformation and the six-helix bundle involved in fusion of the viral and cell membranes, as well as providing new or expanded epitopes with increased accessibility for the UCA antibodies.


Assuntos
Anticorpos Neutralizantes/imunologia , Afinidade de Anticorpos , Proteína gp41 do Envelope de HIV/imunologia , Mutação , Precursores de Proteínas/imunologia , Anticorpos Antivirais/imunologia , Epitopos/química , Epitopos/genética , Epitopos/imunologia , Proteína gp41 do Envelope de HIV/química , Proteína gp41 do Envelope de HIV/genética , Ligação Proteica , Precursores de Proteínas/química , Precursores de Proteínas/genética , Estabilidade Proteica
7.
Mater Sci Eng C Mater Biol Appl ; 103: 109762, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31349478

RESUMO

Early detection is the most effective mean of improving prognosis for many fatal diseases such as cancer. In this context, the Surface Enhanced Resonance Raman Scattering (SERRS) technique is being proposed as alternative to fluorescent methods in detection of biomarkers, because SERRS nanostructures are bright as fluorescent tags but more stable and clearly detectable using the narrow Raman "fingerprints" of a suitable reporter. Here we show that biocompatible SERRS active gold nanostructures, functionalized with an engineered PreS1 peptide (AuNP@PEG-PreS1), detect the presence of the SerpinB3 antigen overexpressed on liver tumor cells, a biomarker of the onset of liver cell carcinomatous transformation. A proper engineering of the targeting unit, linked to the nanostructure by a polymer chain, affords a sensitivity and specificity larger than 80%, at subnanomolar concentrations. Taking into account the high sensitivity of SERRS and that SB3 overexpression is an early event in liver cell carcinomatous transformation, AuNP@PEG-PreS1 nanostructures could be used in routine diagnostic activities, to improve the accuracy of HCC detection in particular in patients with chronic liver diseases.


Assuntos
Carcinoma Hepatocelular/tratamento farmacológico , Sistemas de Liberação de Medicamentos , Ouro , Antígenos de Superfície da Hepatite B , Neoplasias Hepáticas/tratamento farmacológico , Nanopartículas Metálicas , Peptídeos , Precursores de Proteínas , Animais , Antígenos de Neoplasias/metabolismo , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Ouro/química , Ouro/farmacologia , Células Hep G2 , Antígenos de Superfície da Hepatite B/química , Antígenos de Superfície da Hepatite B/farmacologia , Humanos , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Nanopartículas Metálicas/química , Nanopartículas Metálicas/uso terapêutico , Camundongos , Camundongos Transgênicos , Proteínas de Neoplasias/metabolismo , Peptídeos/química , Peptídeos/farmacologia , Precursores de Proteínas/química , Precursores de Proteínas/farmacologia , Serpinas/metabolismo , Análise Espectral Raman
8.
J Synchrotron Radiat ; 26(Pt 4): 967-979, 2019 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-31274418

RESUMO

Dissociation of transforming growth factor beta-1 (TGFß-1) from the inhibitory protein latency-associated peptide (LAP) can occur from low doses of X-ray irradiation of the LAP-TGFß-1 complex, resulting in the activation of TGFß-1, and can have health-related consequences. Using the tools and knowledge developed in the study of radiation damage in the crystallographic setting, small-angle X-ray scattering (SAXS) and complementary techniques suggest an activation process that is initiated but not driven by the initial X-ray exposure. LAP is revealed to be extended when not bound to TGFß-1 and has a different structural conformation compared to the bound state. These studies pave the way for the structural understanding of systems impacted at therapeutic X-ray doses and show the potential impact of radiation damage studies beyond their original intent.


Assuntos
Peptídeos/química , Precursores de Proteínas/química , Fator de Crescimento Transformador beta1/química , Fator de Crescimento Transformador beta/química , Raios X , Relação Dose-Resposta à Radiação , Humanos , Conformação Proteica , Espalhamento a Baixo Ângulo , Difração de Raios X
9.
Biochim Biophys Acta Proteins Proteom ; 1867(11): 140252, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31325636

RESUMO

Intrinsically disordered proteins (IDPs) explore diverse conformations in their free states and, a few of them, also in their molecular complexes. This functional plasticity is essential for the function of IDPs, although their dynamics in both free and bound states is poorly understood. NUPR1 is a protumoral multifunctional IDP, activated during the acute phases of pancreatitis. It interacts with DNA and other IDPs, such as prothymosin α (ProTα), with dissociation constants of ~0.5 µM, and a 1:1 stoichiometry. We studied the structure and picosecond-to-nanosecond (ps-ns) dynamics by using both NMR and SAXS in: (i) isolated NUPR1; (ii) the NUPR1/ProTα complex; and (iii) the NUPR1/double stranded (ds) GGGCGCGCCC complex. Our SAXS findings show that NUPR1 remained disordered when bound to either partner, adopting a worm-like conformation; the fuzziness of bound NUPR1 was also pinpointed by NMR. Residues with the largest values of the relaxation rates (R1, R1ρ, R2 and ηxy), in the free and bound species, were mainly clustered around the 30s region of the sequence, which agree with one of the protein hot-spots already identified by site-directed mutagenesis. Not only residues in this region had larger relaxation rates, but they also moved slower than the rest of the molecule, as indicated by the reduced spectral density approach (RSDA). Upon binding, the energy landscape of NUPR1 was not funneled down to a specific, well-folded conformation, but rather its backbone flexibility was kept, with distinct motions occurring at the hot-spot region.


Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/química , DNA/química , Complexos Multiproteicos/química , Proteínas de Neoplasias/química , Precursores de Proteínas/química , Timosina/análogos & derivados , Humanos , Domínios Proteicos , Espalhamento a Baixo Ângulo , Timosina/química , Difração de Raios X
10.
Sci Adv ; 5(6): eaav9404, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31206019

RESUMO

Escherichia coli exports proteins via a translocase comprising SecA and the translocon, SecYEG. Structural changes of active translocases underlie general secretory system function, yet directly visualizing dynamics has been challenging. We imaged active translocases in lipid bilayers as a function of precursor protein species, nucleotide species, and stage of translocation using atomic force microscopy (AFM). Starting from nearly identical initial states, SecA more readily dissociated from SecYEG when engaged with the precursor of outer membrane protein A as compared to the precursor of galactose-binding protein. For the SecA that remained bound to the translocon, the quaternary structure varied with nucleotide, populating SecA2 primarily with adenosine diphosphate (ADP) and adenosine triphosphate, and the SecA monomer with the transition state analog ADP-AlF3. Conformations of translocases exhibited precursor-dependent differences on the AFM imaging time scale. The data, acquired under near-native conditions, suggest that the translocation process varies with precursor species.


Assuntos
Proteínas da Membrana Bacteriana Externa/química , Proteínas de Ligação ao Cálcio/química , Proteínas de Escherichia coli/química , Escherichia coli/genética , Bicamadas Lipídicas/química , Proteínas de Transporte de Monossacarídeos/química , Proteínas Periplásmicas de Ligação/química , Precursores de Proteínas/química , Proteínas SecA/química , Difosfato de Adenosina/química , Difosfato de Adenosina/metabolismo , Trifosfato de Adenosina/química , Trifosfato de Adenosina/metabolismo , Proteínas da Membrana Bacteriana Externa/genética , Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas de Ligação ao Cálcio/genética , Proteínas de Ligação ao Cálcio/metabolismo , Escherichia coli/química , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Expressão Gênica , Bicamadas Lipídicas/metabolismo , Microscopia de Força Atômica , Proteínas de Transporte de Monossacarídeos/genética , Proteínas de Transporte de Monossacarídeos/metabolismo , Proteínas Periplásmicas de Ligação/genética , Proteínas Periplásmicas de Ligação/metabolismo , Ligação Proteica , Multimerização Proteica , Precursores de Proteínas/genética , Precursores de Proteínas/metabolismo , Estrutura Quaternária de Proteína , Transporte Proteico , Proteolipídeos/química , Proteolipídeos/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Canais de Translocação SEC/química , Canais de Translocação SEC/genética , Canais de Translocação SEC/metabolismo , Proteínas SecA/genética , Proteínas SecA/metabolismo
11.
Structure ; 27(7): 1103-1113.e3, 2019 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-31104815

RESUMO

Sortilin is a multifunctional neuronal receptor involved in sorting of neurotrophic factors and apoptosis signaling. So far, structural characterization of sortilin and its endogenous ligands has been limited to crystallographic studies of sortilin in complex with the neuropeptide neurotensin. Here, we use hydrogen/deuterium exchange mass spectrometry to investigate the conformational response of sortilin to binding biological ligands including the peptides neurotensin and the sortilin propeptide and the proteins progranulin and pro-nerve growth factor-ß. The results show that the ligands use two binding sites inside the cavity of the ß-propeller of sortilin. However, ligands have distinct differences in their conformational impact on the receptor. Interestingly, the protein ligands induce conformational stabilization in a remote membrane-proximal domain, hinting at an unknown conformational link between the ligand binding region and this membrane-proximal region of sortilin. Our findings improve our structural understanding of sortilin and how it mediates diverse ligand-dependent functions important in neurobiology.


Assuntos
Proteínas Adaptadoras de Transporte Vesicular/química , Fator de Crescimento Neural/química , Neurotensina/química , Progranulinas/química , Precursores de Proteínas/química , Proteínas Recombinantes de Fusão/química , Proteínas Adaptadoras de Transporte Vesicular/genética , Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Clonagem Molecular , Cristalografia por Raios X , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Glutationa Transferase/química , Glutationa Transferase/genética , Glutationa Transferase/metabolismo , Células HEK293 , Humanos , Espectrometria de Massa com Troca Hidrogênio-Deutério , Ligantes , Modelos Moleculares , Fator de Crescimento Neural/genética , Fator de Crescimento Neural/metabolismo , Neurotensina/genética , Neurotensina/metabolismo , Progranulinas/genética , Progranulinas/metabolismo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Precursores de Proteínas/genética , Precursores de Proteínas/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo
12.
Biopolymers ; 110(6): e23277, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30972750

RESUMO

Surfactant protein C (SP-C) is an important constituent of lung surfactant (LS) and, along with SP-B, is included in exogenous surfactant replacement therapies for treating respiratory distress syndrome (RDS). SP-C's biophysical activity depends upon the presence of a rigid C-terminal helix, of which the secondary structure is more crucial to functionality than precise side-chain chemistry. SP-C is highly sequence-conserved, suggesting that the ß-branched, aliphatic side chains of the helix are also important. Nonnatural mimics of SP-C were created using a poly-N-substituted glycine, or "peptoid," backbone. The mimics included varying amounts of α-chiral, aliphatic side chains and α-chiral, aromatic side chains in the helical region, imparting either biomimicry or structural rigidity. Biophysical studies confirmed that the peptoids mimicked SP-C's secondary structure and replicated many of its surface-active characteristics. Surface activity was optimized by incorporating both structurally rigid and biomimetic side chain chemistries in the helical region indicating that both characteristics are important for activity. By balancing these features in one mimic, a novel analogue was created that emulates SP-C's in vitro surface activity while overcoming many of the challenges related to natural SP-C. Peptoid-based analogues hold great potential for use in a synthetic, biomimetic LS formulation for treating RDS.


Assuntos
Materiais Biomiméticos/química , Peptoides/química , Materiais Biomiméticos/síntese química , Materiais Biomiméticos/metabolismo , Dicroísmo Circular , Desenho de Fármacos , Bicamadas Lipídicas/química , Bicamadas Lipídicas/metabolismo , Microscopia de Fluorescência , Peptoides/síntese química , Peptoides/metabolismo , Conformação Proteica em alfa-Hélice , Precursores de Proteínas/química , Proteolipídeos/química , Propriedades de Superfície
13.
FEBS Lett ; 593(6): 565-572, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30775779

RESUMO

Protein import into chloroplasts is carried out by the protein translocons at the outer and inner envelope membranes (TOC and TIC). Detailed structures for these translocons are lacking, with only a low-resolution TOC complex structure available. Recently, we showed that the TOC/TIC translocons can import folded proteins, a rather unique feat for a coupled double membrane system. We also determined the maximum functional TOC/TIC pore size to be 30-35 Å. Here, we discuss how such large pores could form and compare the structural dynamics of the pore-forming Toc75 subunit to its bacterial/mitochondrial Omp85 family homologs. We put forward structural models that can be empirically tested and also briefly review the pore dynamics of other protein translocons with known structures.


Assuntos
Arabidopsis/metabolismo , Cloroplastos/metabolismo , Ervilhas/metabolismo , Proteínas de Plantas/química , Precursores de Proteínas/química , Arabidopsis/ultraestrutura , Proteínas da Membrana Bacteriana Externa/química , Proteínas da Membrana Bacteriana Externa/metabolismo , Cloroplastos/ultraestrutura , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Proteínas Hemolisinas/química , Proteínas Hemolisinas/metabolismo , Canais Iônicos/química , Canais Iônicos/metabolismo , Ervilhas/ultraestrutura , Proteínas de Plantas/metabolismo , Dobramento de Proteína , Precursores de Proteínas/metabolismo , Estrutura Secundária de Proteína , Transporte Proteico , Homologia Estrutural de Proteína
14.
Proteins ; 87(1): 81-90, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30367523

RESUMO

The translocase of the outer membrane (TOM) mediates the membrane permeation of mitochondrial matrix proteins. Tom20 is a subunit of the TOM complex and binds to the N-terminal region (ie, presequence) in mitochondrial matrix precursor proteins. Previous experimental studies indicated that the presequence recognition by Tom20 was achieved in a dynamic-equilibrium among multiple bound states of the α-helical presequence. Accordingly, the co-crystallization of Tom20 and a presequence peptide required a disulfide-bond cross-linking. A 3-residue spacer sequence (XAG) was inserted between the presequence and the anchoring Cys residue at the C-terminus to not disturb the movement of the presequence peptide in the binding site of Tom20. Two crystalline forms were obtained according to Ala or Tyr at the X position of the spacer sequence, which may reflect the dynamic-equilibrium of the presequence. Here, we have performed replica-exchange molecular dynamics (REMD) simulations to study the effect of disulfide-bond linker and single amino acid difference in the spacer region of the linker on the conformational dynamics of Tom20-presequence complex. Free energy and network analyses of the REMD simulations were compared against previous simulations of non-tethered system. We concluded that the disulfide-bond tethering did not strongly affect the conformational ensemble of the presequence peptide in the complex. Further investigation showed that the choice of Ala or Tyr at the X position did not affect the most distributions of the conformational ensemble of the presequence. The present study provides a rational basis for the disulfide-bond tethering to study the dynamics of weakly binding complexes.


Assuntos
Aldeído Desidrogenase 1/química , Biologia Computacional/métodos , Proteínas de Membrana Transportadoras/química , Fragmentos de Peptídeos/química , Precursores de Proteínas/química , Receptores de Superfície Celular/química , Aldeído Desidrogenase 1/metabolismo , Animais , Cristalização , Proteínas de Membrana Transportadoras/metabolismo , Mitocôndrias/metabolismo , Modelos Moleculares , Simulação de Dinâmica Molecular , Fragmentos de Peptídeos/metabolismo , Ligação Proteica , Precursores de Proteínas/metabolismo , Estrutura Terciária de Proteína , Ratos , Receptores de Superfície Celular/metabolismo
15.
Structure ; 27(1): 78-89.e3, 2019 01 02.
Artigo em Inglês | MEDLINE | ID: mdl-30393051

RESUMO

Nerve growth factor (NGF) is an important neurotrophic factor involved in the regulation of cell differentiation and survival of target neurons. Expressed as a proNGF precursor, NGF is matured by furin-mediated protease cleavage. Increasing evidence suggests that NGF and proNGF have distinct functional roles. While the structure of mature NGF is available, little is known about that of the pro-domain because of its dynamical structural features. We exploited an ad hoc hybrid strategy based on nuclear magnetic resonance and modeling validated by small-angle X-ray scattering to gain novel insights on the pro-domain, both in isolation and in the context of proNGF. We show that the isolated pro-domain is intrinsically unstructured but forms transient intramolecular contacts with mature NGF and has per se the ability to induce growth cone collapse, indicating functional independence. Our data represent an important step toward the structural and functional characterization of the properties of proNGF.


Assuntos
Fator de Crescimento Neural/química , Precursores de Proteínas/química , Animais , Células Cultivadas , Cones de Crescimento/metabolismo , Espectroscopia de Ressonância Magnética , Camundongos , Simulação de Dinâmica Molecular , Fator de Crescimento Neural/metabolismo , Domínios Proteicos , Precursores de Proteínas/metabolismo , Processamento de Proteína Pós-Traducional , Proteólise , Espalhamento a Baixo Ângulo , Difração de Raios X
16.
Methods ; 157: 80-87, 2019 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-30419336

RESUMO

Protein-protein interactions are essential for cellular structure and function. To delineate how the intricate assembly of protein interactions contribute to cellular processes in health and disease, new methodologies that are both highly sensitive and can be applied at large scale are needed. Here, we develop HiPLA (high-throughput imaging proximity ligation assay), a method that employs the well-established antibody-based proximity ligation assay in a high-throughput imaging screening format as a novel means to systematically visualize protein interactomes. Using HiPLA with a library of antibodies targeting nuclear proteins, we probe the interaction of 60 proteins and associated post-translational modifications (PTMs) with the nuclear lamina in a model of the premature aging disorder Hutchinson-Gilford progeria syndrome (HGPS). We identify a subset of proteins that differentially interact with the nuclear lamina in HGPS. Using HiPLA in combination with quantitative indirect immunofluorescence, we find that the majority of differential interactions are accompanied by corresponding changes in expression of the interacting protein. Taken together, HiPLA offers a novel approach to probe cellular protein-protein interaction at a large scale and reveals mechanistic insights into the assembly of protein complexes.


Assuntos
Núcleo Celular/genética , Lamina Tipo A/genética , Progéria/genética , Mapeamento de Interação de Proteínas/métodos , Núcleo Celular/patologia , Humanos , Lamina Tipo A/química , Mutação , Progéria/patologia , Precursores de Proteínas/química , Precursores de Proteínas/genética
17.
Elife ; 72018 12 18.
Artigo em Inglês | MEDLINE | ID: mdl-30558715

RESUMO

New strategies are urgently required to develop antibiotics. The siderophore uptake system has attracted considerable attention, but rational design of siderophore antibiotic conjugates requires knowledge of recognition by the cognate outer-membrane transporter. Acinetobacter baumannii is a serious pathogen, which utilizes (pre)acinetobactin to scavenge iron from the host. We report the structure of the (pre)acinetobactin transporter BauA bound to the siderophore, identifying the structural determinants of recognition. Detailed biophysical analysis confirms that BauA recognises preacinetobactin. We show that acinetobactin is not recognised by the protein, thus preacinetobactin is essential for iron uptake. The structure shows and NMR confirms that under physiological conditions, a molecule of acinetobactin will bind to two free coordination sites on the iron preacinetobactin complex. The ability to recognise a heterotrimeric iron-preacinetobactin-acinetobactin complex may rationalize contradictory reports in the literature. These results open new avenues for the design of novel antibiotic conjugates (trojan horse) antibiotics.


Assuntos
Acinetobacter baumannii/metabolismo , Imidazóis/metabolismo , Ferro/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Oxazóis/metabolismo , Precursores de Proteínas/metabolismo , Cristalografia por Raios X , Imidazóis/química , Espectroscopia de Ressonância Magnética , Proteínas de Membrana Transportadoras/química , Oxazóis/química , Ligação Proteica , Multimerização Proteica , Precursores de Proteínas/química , Oligoelementos/metabolismo
18.
Biophys J ; 115(12): 2368-2385, 2018 12 18.
Artigo em Inglês | MEDLINE | ID: mdl-30527337

RESUMO

The cadherin superfamily of proteins is defined by the presence of extracellular cadherin (EC) "repeats" that engage in protein-protein interactions to mediate cell-cell adhesion, cell signaling, and mechanotransduction. The extracellular domains of nonclassical cadherins often have a large number of EC repeats along with other subdomains of various folds. Protocadherin-15 (PCDH15), a protein component of the inner-ear tip link filament essential for mechanotransduction, has 11 EC repeats and a membrane adjacent domain (MAD12) of atypical fold. Here we report the crystal structure of a pig PCDH15 fragment including EC10, EC11, and MAD12 in a parallel dimeric arrangement. MAD12 has a unique molecular architecture and folds as a ferredoxin-like domain similar to that found in the nucleoporin protein Nup54. Analytical ultracentrifugation experiments along with size-exclusion chromatography coupled to multiangle laser light scattering and small-angle x-ray scattering corroborate the crystallographic dimer and show that MAD12 induces parallel dimerization of PCDH15 near its membrane insertion point. In addition, steered molecular dynamics simulations suggest that MAD12 is mechanically weak and may unfold before tip-link rupture. Sequence analyses and structural modeling predict the existence of similar domains in cadherin-23, protocadherin-24, and the "giant" FAT and CELSR cadherins, indicating that some of them may also exhibit MAD-induced parallel dimerization.


Assuntos
Caderinas/química , Espaço Extracelular/metabolismo , Fenômenos Mecânicos , Multimerização Proteica , Precursores de Proteínas/química , Animais , Fenômenos Biomecânicos , Caderinas/metabolismo , Camundongos , Simulação de Dinâmica Molecular , Domínios Proteicos , Dobramento de Proteína , Precursores de Proteínas/metabolismo , Estrutura Quaternária de Proteína , Suínos
19.
J Am Chem Soc ; 140(47): 16213-16221, 2018 11 28.
Artigo em Inglês | MEDLINE | ID: mdl-30387998

RESUMO

Ribosomally synthesized and post-translationally modified peptides (RiPPs) are ubiquitous natural products. Bioactive RiPPs are produced from a precursor peptide, which is modified by enzymes. Usually, a single product is encoded in a precursor peptide. However, in cyanobactins and several other RiPP pathways, a single precursor peptide encodes multiple bioactive products flanking with recognition sequences known as "cassettes". The role of multiple cassettes in one peptide is mysterious, but in general their presence is a marker of biosynthetic plasticity. Here, we show that in cyanobactin biosynthesis the presence of multiple cassettes confers distributive enzyme processing to multiple steps of the pathway, a feature we propose to be a hallmark of multicassette RiPPs. TruD heterocyclase is stochastic and distributive. Although a canonical biosynthetic route is favored with certain substrates, every conceivable biosynthetic route is accepted. Together, these factors afford greater plasticity to the biosynthetic pathway by equalizing the processing of each cassette, enabling access to chemical diversity.


Assuntos
Peptídeos Cíclicos/biossíntese , Precursores de Proteínas/metabolismo , Alquilação , Sequência de Aminoácidos , Ciclização , Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Transferases Intramoleculares/química , Transferases Intramoleculares/metabolismo , Peptídeos Cíclicos/química , Peptídeos Cíclicos/metabolismo , Domínios Proteicos , Precursores de Proteínas/química , Processamento de Proteína Pós-Traducional , Processos Estocásticos , Especificidade por Substrato
20.
Cell ; 175(5): 1365-1379.e25, 2018 11 15.
Artigo em Inglês | MEDLINE | ID: mdl-30445040

RESUMO

The exchange of metabolites between the mitochondrial matrix and the cytosol depends on ß-barrel channels in the outer membrane and α-helical carrier proteins in the inner membrane. The essential translocase of the inner membrane (TIM) chaperones escort these proteins through the intermembrane space, but the structural and mechanistic details remain elusive. We have used an integrated structural biology approach to reveal the functional principle of TIM chaperones. Multiple clamp-like binding sites hold the mitochondrial membrane proteins in a translocation-competent elongated form, thus mimicking characteristics of co-translational membrane insertion. The bound preprotein undergoes conformational dynamics within the chaperone binding clefts, pointing to a multitude of dynamic local binding events. Mutations in these binding sites cause cell death or growth defects associated with impairment of carrier and ß-barrel protein biogenesis. Our work reveals how a single mitochondrial "transfer-chaperone" system is able to guide α-helical and ß-barrel membrane proteins in a "nascent chain-like" conformation through a ribosome-free compartment.


Assuntos
Mitocôndrias/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Chaperonas Moleculares/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Membranas Intracelulares/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/química , Proteínas de Transporte da Membrana Mitocondrial/genética , Simulação de Dinâmica Molecular , Mutagênese Sítio-Dirigida , Ligação Proteica , Domínios Proteicos , Precursores de Proteínas/química , Precursores de Proteínas/metabolismo , Estrutura Secundária de Proteína , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Alinhamento de Sequência
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