RESUMO
The 2019 corona virus disease (COVID-19) has caused a global chaos, where a novel Omicron variant has challenged the healthcare system, followed by which it has been referred to as a variant of concern (VOC) by the World Health Organization (WHO), owing to its alarming transmission and infectivity rate. The large number of mutations in the receptor binding domain (RBD) of the spike protein is responsible for strengthening of the spike-angiotensin-converting enzyme 2 (ACE2) interaction, thereby explaining the elevated threat. This is supplemented by enhanced resistance of the variant towards pre-existing antibodies approved for the COVID-19 therapy. The manuscript brings into light failure of existing therapies to provide the desired effect, however simultaneously discussing the novel possibilities on the verge of establishing suitable treatment portfolio. The authors entail the risks associated with omicron resistance against antibodies and vaccine ineffectiveness on one side, and novel approaches and targets - kinase inhibitors, viral protease inhibitors, phytoconstituents, entry pathways - on the other. The manuscript aims to provide a holistic picture about the Omicron variant, by providing comprehensive discussions related to multiple aspects of the mutated spike variant, which might aid the global researchers and healthcare experts in finding an optimised solution to this pandemic.
Assuntos
COVID-19/fisiopatologia , SARS-CoV-2/genética , SARS-CoV-2/metabolismo , Enzima de Conversão de Angiotensina 2/metabolismo , Animais , COVID-19/imunologia , Vacinas contra COVID-19/imunologia , Catepsinas/metabolismo , Receptores ErbB/antagonistas & inibidores , Humanos , Esquemas de Imunização , Imunização Secundária , Fitoterapia/métodos , Plantas Medicinais , Ligação Proteica/fisiologia , Domínios e Motivos de Interação entre Proteínas/fisiologia , Elementos Estruturais de Proteínas/fisiologia , Glicoproteína da Espícula de Coronavírus/metabolismo , Inibidores de Protease Viral/farmacologia , Inibidores de Protease Viral/uso terapêuticoRESUMO
La obtención de información estructural tridimensional de una proteína es de suma importancia en campos tan variados como la bioquímica funcional, las ciencias de materiales o biomédicas. Siendo actualmente la difracción de rayos X de monocristal el estándar de oro para la consecución de este objetivo, la obtención de dicho monocristal sigue siendo un cuello de botella desde el punto de vista práctico, y poco entendido desde el punto de vista teórico. En este artículo se revisa desde la perspectiva estructural de la proteína la forma en que los rayos X permiten obtener la información estructural y las condiciones fisicoquímicas que permiten la formación de un cristal adecuado para estos experimentos.
Obtaining three-dimensional structural information of a protein is of utmost importance in various fields such as functional biochemistry, materials science, or biomedical sciences. Even though single crystal X-ray diffraction is currently the gold standard for this purpose, growing said single crystal is still a bottleneck from a practical viewpoint, and not fully understood from a theoretical point of view. In this article, we review, from a protein structure perspective, the way X-rays provide structural information, and the physicochemical conditions that promote the formation of an adequate crystal for these experiments.
Assuntos
Difração de Raios X/métodos , Proteínas/farmacologia , Elementos Estruturais de Proteínas , Bioquímica , Desenho de Fármacos , AminoácidosRESUMO
Dual agonists that can activate both the glucagon-like peptide-1 receptor (GLP-1R) and the gastric inhibitory polypeptide receptor (GIPR) have demonstrated high efficacy for the treatment of metabolic disease. Peptide-19 is a prototypical dual agonist that has high potency at both GLP-1R and GIPR but has a distinct signalling profile relative to the native peptides at the cognate receptors. In this study, we solved the structure of peptide-19 bound to the GLP-1R in complex with Gs protein, and compared the structure and dynamics of this complex to that of published structures of GLP-1R:Gs in complex with other receptor agonists. Unlike other peptide-bound receptor complexes, peptide-19:GLP-1R:Gs demonstrated a more open binding pocket where transmembrane domain (TM) 6, TM7 and the interconnecting extracellular loop 3 (ECL3) were located away from the peptide, with no interactions between peptide-19 and TM6/ECL3. Analysis of conformational variance of the complex revealed that peptide-19 was highly dynamic and underwent binding and unbinding motions facilitated by the more open TM binding pocket. Both the consensus structure of the GLP-1R complex with peptide-19 and the dynamics of this complex were distinct from previously described GLP-1R structures providing unique insights into the mode of GLP-1R activation by this dual agonist.
Assuntos
Receptor do Peptídeo Semelhante ao Glucagon 1/química , Receptor do Peptídeo Semelhante ao Glucagon 1/metabolismo , Peptídeos/química , Peptídeos/metabolismo , Microscopia Crioeletrônica/métodos , Receptor do Peptídeo Semelhante ao Glucagon 1/agonistas , Domínios Proteicos , Elementos Estruturais de ProteínasRESUMO
The Saccharomyces cerevisiae Rsm22 protein (Sc-Rsm22), encoded by the nuclear RSM22 (systematic name YKL155c) gene, is a distant homologue of Rsm22 from Trypanosoma brucei (Tb-Rsm22) and METTL17 from mouse (Mm-METTL17). All three proteins have been shown to be associated with mitochondrial gene expression, and Sc-Rsm22 has been documented to be essential for mitochondrial respiration. The Sc-Rsm22 protein comprises a polypeptide of molecular weight 72.2â kDa that is predicted to harbor an N-terminal mitochondrial targeting sequence. The precise physiological function of Rsm22-family proteins is unknown, and no structural information has been available for Sc-Rsm22 to date. In this study, Sc-Rsm22 was expressed and purified in monomeric and dimeric forms, their folding was confirmed by circular-dichroism analyses and their low-resolution structures were determined using a small-angle X-ray scattering (SAXS) approach. The solution structure of the monomeric form of Sc-Rsm22 revealed an elongated three-domain arrangement, which differs from the shape of Tb-Rsm22 in its complex with the mitochondrial small ribosomal subunit in T. brucei (PDB entry 6sg9). A bioinformatic analysis revealed that the core domain in the middle (Leu117-Asp462 in Sc-Rsm22) resembles the corresponding region in Tb-Rsm22, including a Rossmann-like methyltransferase fold followed by a zinc-finger-like structure. The latter structure is not present in this position in other methyltransferases and is therefore a unique structural motif for this family. The first half of the C-terminal domain is likely to form an OB-fold, which is typically found in RNA-binding proteins and is also seen in the Tb-Rsm22 structure. In contrast, the N-terminal domain of Sc-Rsm22 is predicted to be fully α-helical and shares no sequence similarity with other family members. Functional studies demonstrated that the monomeric variant of Sc-Rsm22 methylates mitochondrial tRNAs in vitro. These data suggest that Sc-Rsm22 is a new and unique member of the RNA methyltransferases that is important for mitochondrial protein synthesis.
Assuntos
Modelos Moleculares , Proteínas Ribossômicas/química , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/enzimologia , Elementos Estruturais de ProteínasRESUMO
Serum- and glucocorticoid-regulated kinase 3 (Sgk3) is a serine/threonine protein kinase activated by the phospholipid phosphatidylinositol 3-phosphate (PI3P) downstream of growth factor signaling via class I phosphatidylinositol 3-kinase (PI3K) signaling and by class III PI3K/Vps34-mediated PI3P production on endosomes. Upregulation of Sgk3 activity has recently been linked to a number of human cancers; however, the precise mechanism of activation of Sgk3 is unknown. Here, we use a wide range of cell biological, biochemical, and biophysical techniques, including hydrogen-deuterium exchange mass spectrometry, to investigate the mechanism of activation of Sgk3 by PI3P. We show that Sgk3 is regulated by a combination of phosphorylation and allosteric activation. We demonstrate that binding of Sgk3 to PI3P via its regulatory phox homology (PX) domain induces large conformational changes in Sgk3 associated with its activation and that the PI3P-binding pocket of the PX domain of Sgk3 is sequestered in its inactive conformation. Finally, we reconstitute Sgk3 activation via Vps34-mediated PI3P synthesis on phosphatidylinositol liposomes in vitro. In addition to identifying the mechanism of Sgk3 activation by PI3P, our findings open up potential therapeutic avenues in allosteric inhibitor development to target Sgk3 in cancer.
Assuntos
Classe I de Fosfatidilinositol 3-Quinases/metabolismo , Endossomos/metabolismo , Lipossomos/química , Neoplasias/patologia , Fosfatos de Fosfatidilinositol/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Classe III de Fosfatidilinositol 3-Quinases/metabolismo , Humanos , Técnicas In Vitro , Lipossomos/metabolismo , Espectrometria de Massas/métodos , Neoplasias/enzimologia , Fosfatos de Fosfatidilinositol/química , Proteínas Serina-Treonina Quinases/química , Elementos Estruturais de Proteínas , Transdução de SinaisRESUMO
Macrodomains are evolutionarily conserved structural elements. Many macrodomains feature as binding modules of ADP-ribose, thus participating in the recognition and removal of mono- and poly-ADP-ribosylation. Macrodomains are involved in the regulation of a variety of physiological processes and represent valuable therapeutic targets. Moreover, as part of the nonstructural proteins of certain viruses, macrodomains are also pivotal for viral replication and pathogenesis. Thus, targeting viral macrodomains with inhibitors is considered to be a promising antiviral intervention. In this review, we summarize our current understanding of human and viral macrodomains that are related to mono-ADP-ribosylation, with emphasis on the search for inhibitors. The advances summarized here will be helpful for the design of macrodomain-specific agents for therapeutic and diagnostic applications.
Assuntos
ADP-Ribosilação/efeitos dos fármacos , Antivirais/farmacologia , Domínios Proteicos , Relação Estrutura-Atividade , Proteínas não Estruturais Virais/antagonistas & inibidores , Adenosina Difosfato Ribose/metabolismo , Humanos , Processamento de Proteína Pós-Traducional , Elementos Estruturais de Proteínas , Replicação ViralRESUMO
Histone lysine demethylase 4D (KDM4D), also known as JMJD2D, plays an important role in cell proliferation and survival and has been associated with several tumor types. KDM4D has emerged as a potential target for the treatment of human cancer. Here, we reported crystal complex structures for two KDM4D inhibitors, OWS [2-(1H-pyrazol-3-yl)isonicotinic acid] and 10r (5-hydroxy-2-methylpyrazolo[1,5-a]pyrido[3,2-e]pyrimidine-3-carbonitrile), which were both determined to 2.0 Å. OWS is a newly discovered KDM4D inhibitor (IC50 = 4.28 µM) and the critical pharmacophores of this compound are confirmed by the complex structure. Compound 10r is a KDM4D inhibitor reported by us previously. To clarify the binding mode in more detail, the crystal structure was determined and the comparison analysis revealed unique interactions that had never been observed before. Overall, our data provide new structural insights for rational design and offer an opportunity for optimization of KDM4D inhibitors.
Assuntos
Antineoplásicos/química , Inibidores Enzimáticos/química , Ácidos Isonicotínicos/química , Histona Desmetilases com o Domínio Jumonji/antagonistas & inibidores , Histona Desmetilases com o Domínio Jumonji/química , Pirazóis/química , Antineoplásicos/farmacologia , Cristalografia por Raios X , Desenho de Fármacos , Inibidores Enzimáticos/farmacologia , Humanos , Ácidos Isonicotínicos/farmacologia , Histona Desmetilases com o Domínio Jumonji/isolamento & purificação , Histona Desmetilases com o Domínio Jumonji/metabolismo , Modelos Moleculares , Elementos Estruturais de Proteínas , Pirazóis/farmacologia , Relação Estrutura-AtividadeRESUMO
The race to identify a successful treatment for COVID19 will be defined by fundamental research into the replication cycle of the SARS-CoV-2 virus. This has identified five distinct stages from which numerous vaccination and clinical trials have emerged alongside an innumerable number of drug discovery studies currently in development for disease intervention. Informing every step of the viral replication cycle has been an unprecedented 'call-to-arms' by the global structural biology community. Of the 20 main SARS-CoV-2 proteins, 13 have been resolved structurally for SARS-CoV-2 with most having a related SARS-CoV and MERS-CoV structural homologue totalling some 300 structures currently available in public repositories. Herein, we review the contribution of structural studies to our understanding of the virus and their role in structure-based development of therapeutics.
Assuntos
Antivirais/química , Antivirais/uso terapêutico , COVID-19/terapia , Descoberta de Drogas/métodos , SARS-CoV-2 , Antivirais/síntese química , COVID-19/imunologia , Desenvolvimento de Medicamentos/métodos , Genoma Viral , Humanos , Modelos Moleculares , Elementos Estruturais de Proteínas , SARS-CoV-2/química , SARS-CoV-2/efeitos dos fármacos , SARS-CoV-2/imunologia , SARS-CoV-2/fisiologia , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/fisiologia , Relação Estrutura-Atividade , Proteínas Estruturais Virais/química , Proteínas Estruturais Virais/fisiologia , Replicação Viral/efeitos dos fármacos , Replicação Viral/fisiologia , Tratamento Farmacológico da COVID-19RESUMO
Rosmarinic acid (RA) is a natural compound that is gaining wide popularity owing to its broad-spectrum biological activities. RA is known for its wide range of medicinal properties and therapeutic applications in a vast range of neurodegenerative disorders thus making it a vital natural compound. Human transferrin (hTf) is a clinically significant protein that plays a pivotal role in maintaining iron homeostasis. The importance of studies pertaining to hTf is attributable to the pivotal role of iron deposition in CNS in neurodegenerative disorders. The study was intended to have an insight into the interaction between RA and hTf employing multispectroscopic approach, molecular docking, and molecular dynamic simulation studies. Fluorescence quenching studies revealed that RA shows an excellent binding affinity to hTf with a binding constant (K) of 107 M-1 and is guided by static mode of quenching. Isothermal titration calorimetry (ITC) further validated the spontaneous nature of binding. The estimation of enthalpy change (∆H) and entropy change (∆S) suggested that the RA-hTf complex formation is driven by hydrogen bonding, thereby making this process seemingly specific. Further, Fourier transform infrared (FTIR) and circular dichroism (CD) spectra suggested that RA induces conformational and structural changes in hTf. Additionally, molecular dynamics (MD) studies were carried out to investigate the stability of the hTf and hTf-RA system and suggested that binding of RA induces structural alteration in hTf with free hTf being more stable. This study provides a rationale to use RA in drug development against neurodegenerative disorders by designing novel functional foods containing RA.
Assuntos
Cinamatos/farmacologia , Depsídeos/farmacologia , Doenças Neurodegenerativas/tratamento farmacológico , Transferrina/metabolismo , Antioxidantes/metabolismo , Antioxidantes/farmacologia , Cinamatos/química , Cinamatos/metabolismo , Dicroísmo Circular/métodos , Depsídeos/química , Depsídeos/metabolismo , Humanos , Simulação de Acoplamento Molecular/métodos , Doenças Neurodegenerativas/metabolismo , Ligação Proteica , Elementos Estruturais de Proteínas , Espectrometria de Fluorescência/métodos , Termodinâmica , Transferrina/química , Ácido RosmarínicoRESUMO
Nipah Virus (NiV) has been designated as a priority disease with an urgent need for therapeutic development by World Health Organization. The monoclonal antibody m102.4 binds to the immunodominant NiV receptor-binding glycoprotein (GP), and potently neutralizes NiV, indicating its potential as a therapeutic agent. Although the co-crystal structure of m102.3, an m102.4 derivative, in complex with the GP of the related Hendra Virus (HeV) has been solved, the structural interaction between m102.4 and NiV is uncharacterized. Herein, we used structure-guided alanine-scanning mutagenesis to map the functional epitope and paratope residues that govern the antigen-antibody interaction. Our results revealed that the binding of m102.4 is mediated predominantly by two residues in the HCDR3 region, which is unusually small for an antibody-antigen interaction. We performed computational docking to generate a structural model of m102.4-NiV interaction. Our model indicates that m102.4 targets the common hydrophobic central cavity and a hydrophilic rim on the GP, as observed for the m102.3-HeV co-crystal, albeit with Fv orientation differences. In summary, our study provides insight into the m102.4-NiV interaction, demonstrating that structure-guided alanine-scanning and computational modeling can serve as the starting point for additional antibody reengineering (e.g. affinity maturation) to generate potential therapeutic candidates.
Assuntos
Alanina/genética , Anticorpos Monoclonais/metabolismo , Simulação por Computador , Glicoproteínas/metabolismo , Infecções por Henipavirus/virologia , Vírus Nipah/metabolismo , Proteínas do Envelope Viral/metabolismo , Alanina/química , Animais , Anticorpos Monoclonais/química , Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/química , Anticorpos Neutralizantes/imunologia , Anticorpos Neutralizantes/metabolismo , Complexo Antígeno-Anticorpo/química , Complexo Antígeno-Anticorpo/imunologia , Complexo Antígeno-Anticorpo/metabolismo , Epitopos/imunologia , Glicoproteínas/química , Glicoproteínas/genética , Infecções por Henipavirus/imunologia , Infecções por Henipavirus/metabolismo , Humanos , Mutagênese Sítio-Dirigida , Vírus Nipah/imunologia , Vírus Nipah/isolamento & purificação , Elementos Estruturais de Proteínas/imunologia , Proteínas do Envelope Viral/química , Proteínas do Envelope Viral/genéticaRESUMO
Autophagy is a key clearance process to recycle damaged cellular components. One important upstream regulator of autophagy is ULK1 kinase. Several three-dimensional structures of the ULK1 catalytic domain are available, but a comprehensive study, including molecular dynamics, is missing. Also, an exhaustive description of ULK1 alterations found in cancer samples is presently lacking. We here applied a framework which links -omics data to structural protein ensembles to study ULK1 alterations from genomics data available for more than 30 cancer types. We predicted the effects of mutations on ULK1 function and structural stability, accounting for protein dynamics, and the different layers of changes that a mutation can induce in a protein at the functional and structural level. ULK1 is down-regulated in gynecological tumors. In other cancer types, ULK2 could compensate for ULK1 downregulation and, in the majority of the cases, no marked changes in expression have been found. 36 missense mutations of ULK1, not limited to the catalytic domain, are co-occurring with mutations in a large number of ULK1 interactors or substrates, suggesting a pronounced effect of the upstream steps of autophagy in many cancer types. Moreover, our results pinpoint that more than 50% of the mutations in the kinase domain of ULK1, here investigated, are predicted to affect protein stability. Three mutations (S184F, D102N, and A28V) are predicted with only impact on kinase activity, either modifying the functional dynamics or the capability to exert effects from distal sites to the functional and catalytic regions. The framework here applied could be extended to other protein targets to aid the classification of missense mutations from cancer genomics studies, as well as to prioritize variants for experimental validation, or to select the appropriate biological readouts for experiments.
Assuntos
Proteína Homóloga à Proteína-1 Relacionada à Autofagia/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Neoplasias/enzimologia , Autofagia/fisiologia , Proteína Homóloga à Proteína-1 Relacionada à Autofagia/química , Proteína Homóloga à Proteína-1 Relacionada à Autofagia/genética , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/química , Peptídeos e Proteínas de Sinalização Intracelular/genética , Simulação de Dinâmica Molecular , Mutação , Neoplasias/genética , Neoplasias/patologia , Fosforilação , Elementos Estruturais de Proteínas , Transdução de Sinais , Relação Estrutura-AtividadeRESUMO
FSH-FSHR interaction is critical for folliculogenesis, spermatogenesis and progression of several cancers. Therefore, FSHR is an attractive target for fertility regulation and cancer therapeutics. Based on homology and structural analysis of hFSH-FSHR(ECD) complex, a minimal continuous stretch within FSHß seat-belt loop (FSHß (89-97)) was identified to be crucial for FSHR interaction. The ability of FSHß (89-97) peptide to neutralize FSHR activity was evaluated by a panel of in vitro and in vivo experiments. The synthetic peptide significantly inhibited binding of [125I]-FSH to rat Fshr as well as FSH-induced cAMP production. In immature rats, FSHß (89-97) peptide administration reduced FSH-mediated increase in ovarian weight. The peptide inhibited transition of follicles from pre-antral to antral stage and hindered the cell cycle progression of granulosa cells beyond G0/G1 phase. In adult rats, administration of the peptide inhibited estradiol synthesis and significantly perturbed folliculogenesis.
Assuntos
Subunidade beta do Hormônio Folículoestimulante/metabolismo , Células da Granulosa/efeitos dos fármacos , Oligopeptídeos/farmacologia , Folículo Ovariano/efeitos dos fármacos , Ovário/metabolismo , Receptores do FSH/antagonistas & inibidores , Animais , Cristalografia por Raios X/métodos , Estradiol/biossíntese , Feminino , Células da Granulosa/metabolismo , Oligopeptídeos/química , Folículo Ovariano/metabolismo , Ovário/efeitos dos fármacos , Elementos Estruturais de Proteínas , Ratos , Ratos Sprague-Dawley , Receptores do FSH/metabolismo , Relação Estrutura-AtividadeRESUMO
The glycosylation of small hydrophobic compounds is catalyzed by uridine diphosphate glycosyltransferases (UGTs). Because glycosylation is an invaluable tool for improving the stability and water solubility of hydrophobic compounds, UGTs have attracted attention for their application in the food, cosmetics, and pharmaceutical industries. However, the ability of UGTs to accept and glycosylate a wide range of substrates is not clearly understood due to the existence of a large number of UGTs. PaGT2, a UGT from Phytolacca americana, can regioselectively glycosylate piceatannol but has low activity toward other stilbenoids. To elucidate the substrate specificity and catalytic mechanism, we determined the crystal structures of PaGT2 with and without substrates and performed molecular docking studies. The structures have revealed key residues involved in substrate recognition and suggest the presence of a nonconserved catalytic residue (His81) in addition to the highly conserved catalytic histidine in UGTs (His18). The role of the identified residues in substrate recognition and catalysis is elucidated with the mutational assay. Additionally, the structure-guided mutation of Cys142 to other residues, Ala, Phe, and Gln, allows PaGT2 to glycosylate resveratrol with high regioselectivity, which is negligibly glycosylated by the wild-type enzyme. These results provide a basis for tailoring an efficient glycosyltransferase.
Assuntos
Cristalografia por Raios X/métodos , Glicosiltransferases/metabolismo , Simulação de Acoplamento Molecular/métodos , Phytolacca americana/enzimologia , Proteínas de Plantas/metabolismo , Polifenóis/metabolismo , Difosfato de Uridina/metabolismo , Sequência de Aminoácidos , Glicosilação , Glicosiltransferases/genética , Mutação , Filogenia , Proteínas de Plantas/genética , Elementos Estruturais de Proteínas , Especificidade por SubstratoRESUMO
The two different types of steroidal benzisoselenazolone hybrids were synthesized by incorporating benzisoselenazolone scaffold into dehydroepiandrosterone and B-norcholesterol. The antiproliferative activity of the synthesized compounds against some carcinoma cell lines were investigated. The results showed that some of these compounds have better inhibitory activity than abiraterone on the proliferation of tumor cells associated with human growth hormone, and have less cytotoxicity on normal human cells. In particular, the IC50 values of the compound 8a and 8f are 5.4 and 6.5⯵mol/L against human ovarian carcinoma (SKOV3) cell line, and possess SI values of 13.9 and 10.5, respectively. The information obtained from the studies may be useful for the design of novel chemotherapeutic drugs.
Assuntos
Antineoplásicos/farmacologia , Compostos Organosselênicos/farmacologia , Esteroides/farmacologia , Antineoplásicos/síntese química , Antineoplásicos/química , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Estrutura Molecular , Compostos Organosselênicos/química , Elementos Estruturais de Proteínas , Esteroides/química , Células Tumorais CultivadasRESUMO
Many studies about classification and the functional annotation of intrinsically disordered proteins (IDPs) are based on either the occurrence of long disordered regions or the fraction of disordered residues in the sequence. Taking into account both criteria we separate the human proteome, taken as a case study, into three variants of proteins: i) ordered proteins (ORDPs), ii) structured proteins with intrinsically disordered regions (IDPRs), and iii) intrinsically disordered proteins (IDPs). The focus of this work is on the different functional roles of IDPs and IDPRs, which up until now have been generally considered as a whole. Previous studies assigned a large set of functional roles to the general category of IDPs. We show here that IDPs and IDPRs have non-overlapping functional spectra, play different roles in human diseases, and deserve to be treated as distinct categories of proteins. IDPs enrich only a few classes, functions, and processes: nucleic acid binding proteins, chromatin binding proteins, transcription factors, and developmental processes. In contrast, IDPRs are spread over several functional protein classes and GO annotations which they partly share with ORDPs. As regards to diseases, we observe that IDPs enrich only cancer-related proteins, at variance with previous results reporting that IDPs are widespread also in cardiovascular and neurodegenerative pathologies. Overall, the operational separation of IDPRs from IDPs is relevant towards correct estimates of the occurrence of intrinsically disordered proteins in genome-wide studies and in the understanding of the functional spectra associated to different flavors of protein disorder.
Assuntos
Proteínas Intrinsicamente Desordenadas/química , Proteínas Intrinsicamente Desordenadas/metabolismo , Bases de Dados de Proteínas , Humanos , Proteínas Intrinsicamente Desordenadas/genética , Conformação Proteica , Dobramento de Proteína , Elementos Estruturais de Proteínas , Proteoma/química , Proteoma/genética , Proteoma/metabolismoRESUMO
The pineapple is a fruit that has wide acceptance worldwide both in natural form, as industrialized. Your peel is a residue generated by food industries and from this residue can obtain a protein extract which is a good source of bromelain. This study aimed to obtain a protein extract from pineapple peel, evaluate its enzyme activity and its healing properties in skin lesions in rats. Seven animal groups were used: control, treated with 5% of protein extract, 10% of protein extract and pure protein extract; 5% of commercial bromelain, 10% of commercial bromelain and pure commercial bromelain. The animals were subjected to a tissue incision and treated for 21 days. Proteolytic and specific activities of the protein extract were 1.30 U mg-1 and 45 x 10-3 U μg-1 and, for commercial bromelain, 1.04 U mg-1 and 6 x 10-3 U μg-1, respectively. In the histology of the lesion, there was no significant difference between the control and treated groups; however, macroscopically, the prepared topical formulations assisted in the recovery of skin lesions, providing a significant reduction in their width, in the groups treated with pure protein extract, 5 and 10% commercial bromelain, and pure bromelain.
Assuntos
Ananas/química , Biotecnologia , Elementos Estruturais de ProteínasRESUMO
Background:Crude venom of the banded tiger waspVespa affinis contains a variety of enzymes including hyaluronidases, commonly known as spreading factors.Methods:The cDNA cloning, sequence analysis and structural modelling of V. affinis venom hyaluronidase (VesA2) were herein described. Moreover, heterologous expression and mutagenesis of rVesA2 were performed.Results:V. affinis venom hyaluronidase full sequence is composed of 331 amino acids, with four predicted N-glycosylation sites. It was classified into the glycoside hydrolase family 56. The homology modelling exhibited a central core (α/β)7 composed of Asp107 and Glu109, acting as the catalytic residues. The recombinant protein was successfully expressed in E. coli with hyaluronidase activity. A recombinant mutant type with the double point mutation, Asp107Asn and Glu109Gln, completely lost this activity. The hyaluronidase from crude venom exhibited activity from pH 2 to 7. The recombinant wild type showed its maximal activity at pH 2 but decreased rapidly to nearly zero at pH 3 and was completely lost at pH 4.Conclusion:The recombinant wild-type protein showed its maximal activity at pH 2, more acidic pH than that found in the crude venom. The glycosylation was predicted to be responsible for the pH optimum and thermal stability of the enzymes activity.
Assuntos
Animais , Elementos Estruturais de Proteínas , Hialuronoglucosaminidase , Proteínas Recombinantes , Venenos de Vespas/químicaRESUMO
The C-terminal region of the minor structural protein of potato leafroll virus (PLRV), known as the readthrough protein (RTP), is involved in efficient virus movement, tissue tropism and symptom development. Analysis of numerous C-terminal deletions identified a five-amino acid motif that is required for RTP function. A PLRV mutant expressing RTP with these five amino acids deleted (Δ5aa-RTP) was compromised in systemic infection and symptom expression. Although the Δ5aa-RTP mutant was able to move long distance, limited infection foci were observed in systemically infected leaves suggesting that these five amino acids regulate virus phloem loading in the inoculated leaves and/or unloading into the systemically infected tissues. The 5aa deletion did not alter the efficiency of RTP translation, nor impair RTP self-interaction or its interaction with P17, the virus movement protein. However, the deletion did alter the subcellular localization of RTP. When co-expressed with a PLRV infectious clone, a GFP tagged wild-type RTP was localized to discontinuous punctate spots along the cell periphery and was associated with plasmodesmata, although localization was dependent upon the developmental stage of the plant tissue. In contrast, the Δ5aa-RTP-GFP aggregated in the cytoplasm. Structural modeling indicated that the 5aa deletion would be expected to perturb an α-helix motif. Two of 30 plants infected with Δ5aa-RTP developed a wild-type virus infection phenotype ten weeks post-inoculation. Analysis of the virus population in these plants by deep sequencing identified a duplication of sequences adjacent to the deletion that were predicted to restore the α-helix motif. The subcellular distribution of the RTP is regulated by the 5-aa motif which is under strong selection pressure and in turn contributes to the efficient long distance movement of the virus and the induction of systemic symptoms.
Assuntos
Luteoviridae/genética , Luteoviridae/metabolismo , Sequência de Aminoácidos/genética , Aminoácidos Aromáticos , Capsídeo/metabolismo , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/metabolismo , Luteovirus/genética , Mutação/genética , Doenças das Plantas/virologia , Folhas de Planta/metabolismo , Domínios Proteicos , Elementos Estruturais de Proteínas/genética , Deleção de Sequência/genética , Nicotiana/virologia , Proteínas Virais/metabolismoRESUMO
The classical crystal structure of insulin was determined in 1969 by D.C. Hodgkin et al. following a 35-year program of research. This structure depicted a hexamer remarkable for its self-assembly as a zinc-coordinated trimer of dimer. Prominent at the dimer interface was an "aromatic triplet" of conserved residues at consecutive positions in the B chain: PheB24 , PheB25 and TyrB26 . The elegance of this interface inspired the Oxford team to poetry: "A thing of beauty is a joy forever" (John Keats as quoted by Blundell, T.L., et al. Advances in Protein Chemistry 26:279-286 [1972]). Here, we revisit this aromatic triplet in light of recent advances in the structural biology of insulin bound as a monomer to fragments of the insulin receptor. Such co-crystal structures have defined how these side chains pack at the primary hormone-binding surface of the receptor ectodomain. On receptor binding, the B-chain ß-strand (residues B24-B28) containing the aromatic triplet detaches from the α-helical core of the hormone. Whereas TyrB26 lies at the periphery of the receptor interface and may functionally be replaced by a diverse set of substitutions, PheB24 and PheB25 engage invariant elements of receptor domains L1 and αCT. These critical contacts were anticipated by the discovery of diabetes-associated mutations at these positions by Donald Steiner et al. at the University of Chicago. Conservation of PheB24 , PheB25 and TyrB26 among vertebrate insulins reflects the striking confluence of structure-based evolutionary constraints: foldability, protective self-assembly and hormonal activity.