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1.
FASEB J ; 35(8): e21681, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34196428

RESUMO

The sodium/iodide symporter (NIS) expresses at the basolateral plasma membrane of the thyroid follicular cell and mediates iodide accumulation required for normal thyroid hormonogenesis. Loss-of-function NIS variants cause congenital hypothyroidism due to impaired iodide accumulation in thyroid follicular cells underscoring the significance of NIS for thyroid physiology. Here we report novel findings derived from the thorough characterization of the nonsense NIS mutant p.R636* NIS-leading to a truncated protein missing the last eight amino acids-identified in twins with congenital hypothyroidism. R636* NIS is severely mislocalized into intracellular vesicular compartments due to the lack of a conserved carboxy-terminal type 1 PDZ-binding motif. As a result, R636* NIS is barely targeted to the plasma membrane and therefore iodide transport is reduced. Deletion of the PDZ-binding motif causes NIS accumulation into late endosomes and lysosomes. Using PDZ domain arrays, we revealed that the PDZ-domain containing protein SCRIB binds to the carboxy-terminus of NIS by a PDZ-PDZ interaction. Furthermore, in CRISPR/Cas9-based SCRIB deficient cells, NIS expression at the basolateral plasma membrane is compromised, leading to NIS localization into intracellular vesicular compartments. We conclude that the PDZ-binding motif is a plasma membrane retention signal that participates in the polarized expression of NIS by selectively interacting with the PDZ-domain containing protein SCRIB, thus retaining the transporter at the basolateral plasma membrane. Our data provide insights into the molecular mechanisms that regulate NIS expression at the plasma membrane, a topic of great interest in the thyroid cancer field considering the relevance of NIS-mediated radioactive iodide therapy for differentiated thyroid carcinoma.


Assuntos
Proteínas de Membrana/metabolismo , Simportadores/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Sequência de Aminoácidos , Animais , Linhagem Celular , Membrana Celular/metabolismo , Códon sem Sentido , Hipotireoidismo Congênito/genética , Hipotireoidismo Congênito/metabolismo , Sequência Conservada , Cães , Endossomos/metabolismo , Células HEK293 , Humanos , Lisossomos/metabolismo , Células Madin Darby de Rim Canino , Proteínas de Membrana/química , Proteínas de Membrana/genética , Modelos Moleculares , Mutagênese Sítio-Dirigida , Domínios PDZ/genética , Estrutura Secundária de Proteína , Ratos , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Simportadores/química , Simportadores/genética , Glândula Tireoide/metabolismo , Proteínas Supressoras de Tumor/química , Proteínas Supressoras de Tumor/genética
2.
Molecules ; 26(13)2021 Jun 24.
Artigo em Inglês | MEDLINE | ID: mdl-34202801

RESUMO

In this research, the selected drugs commonly used in diabetes and its comorbidities (gliclazide, cilazapril, atorvastatin, and acetylsalicylic acid) were studied for their interactions with bovine serum albumin-native and glycated. Two different spectroscopic methods, fluorescence quenching and circular dichroism, were utilized to elucidate the binding interactions of the investigational drugs. The glycation process was induced in BSA by glucose and was confirmed by the presence of advanced glycosylation end products (AGEs). The interaction between albumin and gliclazide, with the presence of another drug, was confirmed by calculation of association constants (0.11-1.07 × 104 M-1). The nature of changes in the secondary structure of a protein depends on the drug used and the degree of glycation. Therefore, these interactions may have an influence on pharmacokinetic parameters.


Assuntos
Diabetes Mellitus/tratamento farmacológico , Hipoglicemiantes/química , Soroalbumina Bovina/química , Animais , Bovinos , Humanos , Hipoglicemiantes/uso terapêutico , Ligação Proteica , Estrutura Secundária de Proteína
3.
Int J Mol Sci ; 22(12)2021 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-34203920

RESUMO

The negatively charged Asp325 residue has proved to be essential for iron export by human (HsFPN1) and primate Philippine tarsier (TsFpn) ferroportin, but its exact role during the iron transport cycle is still to be elucidated. It has been posited as being functionally equivalent to the metal ion-coordinating residue His261 in the C-lobe of the bacterial homolog BbFpn, but the two residues arise in different sequence motifs of the discontinuous TM7 transmembrane helix. Furthermore, BbFpn is not subject to extracellular regulation, contrary to its mammalian orthologues which are downregulated by hepcidin. To get further insight into the molecular mechanisms related to iron export in mammals in which Asp325 is involved, we investigated the behavior of the Asp325Ala, Asp325His, and Asp325Asn mutants in transiently transfected HEK293T cells, and performed a comparative structural analysis. Our biochemical studies clearly distinguished between the Asp325Ala and Asp325His mutants, which result in a dramatic decrease in plasma membrane expression of FPN1, and the Asp325Asn mutant, which alters iron egress without affecting protein localization. Analysis of the 3D structures of HsFPN1 and TsFpn in the outward-facing (OF) state indicated that Asp325 does not interact directly with metal ions but is involved in the modulation of Cys326 metal-binding capacity. Moreover, models of the architecture of mammalian proteins in the inward-facing (IF) state suggested that Asp325 may form an inter-lobe salt-bridge with Arg40 (TM1) when not interacting with Cys326. These findings allow to suggest that Asp325 may be important for fine-tuning iron recognition in the C-lobe, as well as for local structural changes during the IF-to-OF transition at the extracellular gate level. Inability to form a salt-bridge between TM1 and TM7b during iron translocation could lead to protein instability, as shown by the Asp325Ala and Asp325His mutants.


Assuntos
Ácido Aspártico/metabolismo , Proteínas de Transporte de Cátions/química , Proteínas de Transporte de Cátions/metabolismo , Sítios de Ligação , Transporte Biológico , Membrana Celular/metabolismo , Células HEK293 , Humanos , Ferro/metabolismo , Estrutura Secundária de Proteína , Relação Estrutura-Atividade
4.
Soft Matter ; 17(28): 6863-6872, 2021 Jul 21.
Artigo em Inglês | MEDLINE | ID: mdl-34227640

RESUMO

In the tissue-engineering field silk fibroin can be tailored to the target applications by modifying its secondary structure and molecular weight, and functionalizing the molecule with specific active groups linked to the amino acid side chains. To better tune the silk fibroin molecular weight and structural properties, we propose the creation of a lower molecular weight fibroin-derived material through a selective and tunable enzymatic attack on the fibroin chain. Cleavage at specific amino acid sites leads to precise silk fibroin fragmentation and, thus, lower molecular weight materials whose length and properties can be tuned with the enzyme concentration. The cleavage increased the presence of free amino groups, hence reactivity, and aqueous solutions of the resulting polymer remained stable for up to seven days. Films of fragmented fibroin were prepared and characterized, demonstrating that the fragmentation did not affect ß-sheet formation after methanol treatment, but differences were detected after the water-vapor annealing process, confirmed by structural and thermal analyses. The adopted fragmentation method is fast, controllable and precise, allowing the creation of a silk-derived material class that is stable in water, with a tunable molecular weight and secondary structure rearrangements, and is thus a versatile tool for the further tunability and modulation of bioengineered constructs.


Assuntos
Bombyx , Fibroínas , Animais , Estrutura Secundária de Proteína , Seda , Engenharia Tecidual
6.
Eur Rev Med Pharmacol Sci ; 25(12): 4405-4412, 2021 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-34227076

RESUMO

SARS-CoV-2 are enveloped RNA viruses that belong to the family Coronaviridae of genus Beta coronavirus, responsible for the COVID-19 pandemic. The mutation rate is high among RNA viruses and in particular, coronavirus replication is error prone with an estimated mutation rate of 4x10-4 nucleotide substitutions per site per year. Variants of SARS-CoV-2 have been reported from various countries like United Kingdom, South Africa, Denmark, Brazil and India. These variants evolved due to mutations in spike gene of SARS-CoV-2. The most concerning variants are Variant of Concern (VOC) 202012/01 from United Kingdom and B.1.617 variant of India. Other variants include B.1.351 lineages, cluster 5/SARS-CoV-2 variant of Denmark, 501.V2 variant/SARS-CoV-2 variant of South Africa, lineage B.1.1.248/lineage P.1 of Brazil. Mutations in S protein may result in changes in the transmissibility and virulence of SARS-CoV-2. To date, alterations in virulence or pathogenicity have been reported among the variants from many parts of the globe. In our opinion, since the S protein is significantly altered, the suitability of existing vaccine specifically targeting the S protein of SARS-CoV-2 variants is a major concern. The mutations in SARS-CoV-2 are a continuous and evolving process that may result in the transformation of naïve SARS-CoV-2 into totally new subsets of antigenically different SARS-CoV-2 viruses over a period of time.


Assuntos
COVID-19/epidemiologia , COVID-19/genética , Mutação/genética , SARS-CoV-2/genética , SARS-CoV-2/patogenicidade , COVID-19/transmissão , Humanos , Índia/epidemiologia , Estrutura Secundária de Proteína , SARS-CoV-2/química , Reino Unido/epidemiologia , Virulência/genética
7.
Appl Microbiol Biotechnol ; 105(13): 5471-5489, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-34250571

RESUMO

6-phosphogluconate dehydrogenase (6PGDH) participates in pentose phosphate pathway of glucose metabolism by catalyzing oxidative decarboxylation of 6-phsophogluconate (6PG) and its absence has been lethal for several eukaryotes. Despite being a validated drug target in many organisms like Plasmodium, the enzyme has not been explored in leishmanial parasites. In the present study, 6PGDH of Leishmania donovani (Ld6PGDH) is cloned and purified followed by its characterization using biochemical and structural approaches. Ld6PGDH lacks the glycine-serine-rich sequence at its C-terminal that is present in other eukaryotes including humans. Leishmanial 6PGDH possesses more affinity for substrate (6PG) and cofactor (NADP) in comparison to that of human. The enzymatic activity is inhibited by gentamicin and cefuroxime through competitive mode with functioning more potently towards leishmanial 6PGDH than its human counterpart. CD analysis has shown higher α-helical content in the secondary structure of Ld6PGDH, while fluorescence studies revealed that tryptophan residues are not completely accessible to solvent environment. The three-dimensional structure was generated through homology modelling and docked with substrate and cofactor. The docking studies demonstrated two separate binding pockets for 6PG and NADP with higher affinity for the cofactor binding, and Asn105 is interacting with substrate as well as the cofactor. Additionally, MD simulation has shown complexes of Ld6PGDH with 6PG and NADP to be more stable than its apo form. Altogether, the present study might provide the foundation to investigate this enzyme as potential target against leishmaniasis. KEY POINTS: • Ld6PGDH enzymatic activity is competitively inhibited by gentamicin and cefuroxime. • It displays more helical contents and all structural characteristics of 6PGDH family. • Interaction studies demonstrate higher affinity of cofactor than substrate for Ld6PGDH.


Assuntos
Leishmania donovani , Fosfogluconato Desidrogenase , Humanos , Cinética , Leishmania donovani/metabolismo , Via de Pentose Fosfato , Fosfogluconato Desidrogenase/genética , Estrutura Secundária de Proteína
8.
Biophys Chem ; 276: 106610, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34089978

RESUMO

In the new millennium, the outbreak of new coronavirus has happened three times: SARS-CoV, MERS-CoV, and SARS-CoV-2. Unfortunately, we still have no pharmaceutical weapons against the diseases caused by these viruses. The pandemic of SARS-CoV-2 reminds us the urgency to search new drugs with totally different mechanism that may target the weaknesses specific to coronaviruses. Herein, we disclose a computational evaluation of targeted oxidation strategy (TOS) for potential inhibition of SARS-CoV-2 by disulfiram, a 70-year-old anti-alcoholism drug, and predict a multiple-target mechanism. A preliminary list of promising TOS drug candidates targeting the two thiol proteases of SARS-CoV-2 are proposed upon virtual screening of 32,143 disulfides.


Assuntos
Dissuasores de Álcool/química , Antivirais/química , Proteases 3C de Coronavírus/antagonistas & inibidores , Proteases Semelhantes à Papaína de Coronavírus/antagonistas & inibidores , Dissulfiram/química , Inibidores de Proteases/química , SARS-CoV-2/química , Dissuasores de Álcool/farmacologia , Antivirais/farmacologia , COVID-19/tratamento farmacológico , Domínio Catalítico , Proteases 3C de Coronavírus/química , Proteases 3C de Coronavírus/genética , Proteases 3C de Coronavírus/metabolismo , Proteases Semelhantes à Papaína de Coronavírus/química , Proteases Semelhantes à Papaína de Coronavírus/genética , Proteases Semelhantes à Papaína de Coronavírus/metabolismo , Dissulfiram/farmacologia , Reposicionamento de Medicamentos , Expressão Gênica , Humanos , Cinética , Simulação de Acoplamento Molecular , Oxirredução , Inibidores de Proteases/farmacologia , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Estrutura Secundária de Proteína , Teoria Quântica , SARS-CoV-2/enzimologia , Especificidade por Substrato , Termodinâmica
9.
Int J Biol Macromol ; 183: 1939-1947, 2021 Jul 31.
Artigo em Inglês | MEDLINE | ID: mdl-34097957

RESUMO

Protein aggregation, such as amyloid fibril formation, is molecular hallmark of many neurodegenerative disorders including Alzheimer's, Parkinson's, and Prion disease. Indole alkaloids are well-known as the compounds having the ability to inhibit protein fibrillation. In this study, we experimentally and computationally have investigated the anti-amyloid property of a derivative of a synthesized tetracyclic indole alkaloid (TCIA), possessing capable functional groups. The fibrillation reaction of Hen White Egg Lysozyme (HEWL) was performed in absence and presence of the indole alkaloid. For quantitative analysis, we used Thioflovin T binding assay which showed ~50% reduction in fibril formation in the presence of 20 µM TCIA. Using TEM imaging, we observed a significant morphological change in our model protein in the presence of TCIA. In addition, we exploited FT-IR assay by which Amide I peak's shifting toward lower wavenumber was clearly observed. Using Molecular Docking, the interaction of the inhibitor (TCIA) with the protein's amyloidogenic region was modeled. Also, different biophysical parameters were calculated by Molecular Dynamics (MD) simulation. Various biochemical assays, conformational change, and hydrophobicity exposure of the protein during amyloid formation indicated that the compound assists HEWL to keep its native structure via destabilizing ß-sheet structure.


Assuntos
Benzotiazóis/química , Alcaloides Indólicos/farmacologia , Muramidase/química , Animais , Galinhas , Estabilidade Enzimática/efeitos dos fármacos , Interações Hidrofóbicas e Hidrofílicas , Alcaloides Indólicos/química , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Muramidase/efeitos dos fármacos , Agregados Proteicos/efeitos dos fármacos , Estrutura Secundária de Proteína/efeitos dos fármacos , Espectroscopia de Infravermelho com Transformada de Fourier
10.
J Clin Neurosci ; 89: 243-248, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-34119275

RESUMO

The sortilin-related receptor 1 (SORL1) gene has been the subject of many studies focusing on frequent polymorphisms, which is associated with increased risk for Alzheimer's Disease (AD). By whole-exome sequencing (WES), we identified two pathogenic missense mutations c.579C > G (p.F193L) and c.1397A > G (p.N466S) in SORL1. The two mutations were located in the same protein domain, and the two unrelated probands both had an onset of memory problems at less than 65 years of age, but their clinical manifestations and cranial imaging are different. The protein structure and function affected by these mutations were predicted using bioinformatics analysis, which suggested they were pathogenic. 3D protein structural analysis revealed that these amino acid substitutions might result in instability of protein structure and adverse intramolecular interactions. These findings suggest that both F193L and N466S should be thought as potential causative mutations in early-onset Alzheimer's disease (EOAD) patients. Further functional studies are warranted to evaluate their roles in the pathogenesis of AD.


Assuntos
Doença de Alzheimer/diagnóstico , Doença de Alzheimer/genética , Proteínas Relacionadas a Receptor de LDL/genética , Proteínas de Membrana Transportadoras/genética , Mutação/genética , Adulto , Sequência de Aminoácidos , Feminino , Predisposição Genética para Doença/genética , Humanos , Proteínas Relacionadas a Receptor de LDL/química , Masculino , Proteínas de Membrana Transportadoras/química , Pessoa de Meia-Idade , Linhagem , Estrutura Secundária de Proteína , Fatores de Risco , Sequenciamento Completo do Exoma/métodos
11.
Soft Matter ; 17(26): 6470-6476, 2021 Jul 07.
Artigo em Inglês | MEDLINE | ID: mdl-34137426

RESUMO

Owing to their tunable properties, hydrogels comprised of stimuli-sensitive polymers are one of the most appealing scaffolds with applications in tissue engineering, drug delivery and other biomedical fields. We previously reported a thermoresponsive hydrogel formed using a coiled-coil protein, Q. Here, we expand our studies to identify the gelation of Q protein at distinct pH conditions, creating a protein hydrogel system that is sensitive to temperature and pH. Through secondary structure analysis, transmission electron microscopy, and rheology, we observed that Q self-assembles and forms fiber-based hydrogels exhibiting upper critical solution temperature behavior with increased elastic properties at pH 7.4 and pH 10. At pH 6, however, Q forms polydisperse nanoparticles, which do not further self-assemble and undergo gelation. The high net positive charge of Q at pH 6 creates significant electrostatic repulsion, preventing its gelation. This study will potentially guide the development of novel scaffolds and functional biomaterials that are sensitive towards biologically relevant stimuli.


Assuntos
Materiais Biocompatíveis , Hidrogéis , Estrutura Secundária de Proteína , Reologia , Engenharia Tecidual
12.
Molecules ; 26(10)2021 May 12.
Artigo em Inglês | MEDLINE | ID: mdl-34066245

RESUMO

Natural backbone-cyclized proteins have an increased thermostability and resistance towards proteases, characteristics that have sparked interest in head-to-tail cyclization as a method to stability-enhance proteins used in diagnostics and therapeutic applications, for example. In this proof-of principle study, we have produced and investigated a head-to-tail cyclized and HER2-specific ZHER2:342 Affibody dimer. The sortase A-mediated cyclization reaction is highly efficient (>95%) under optimized conditions, and renders a cyclic ZHER3:342-dimer with an apparent melting temperature, Tm, of 68 °C, which is 3 °C higher than that of its linear counterpart. Circular dichroism spectra of the linear and cyclic dimers looked very similar in the far-UV range, both before and after thermal unfolding to 90 °C, which suggests that cyclization does not negatively impact the helicity or folding of the cyclic protein. The cyclic dimer had an apparent sub-nanomolar affinity (Kd ~750 pM) to the HER2-receptor, which is a ~150-fold reduction in affinity relative to the linear dimer (Kd ~5 pM), but the anti-HER2 Affibody dimer remained a high-affinity binder even after cyclization. No apparent difference in proteolytic stability was detected in an endopeptidase degradation assay for the cyclic and linear dimers. In contrast, in an exopeptidase degradation assay, the linear dimer was shown to be completely degraded after 5 min, while the cyclic dimer showed no detectable degradation even after 60 min. We further demonstrate that a site-specifically DyLight 594-labeled cyclic dimer shows specific binding to HER2-overexpressing cells. Taken together, the results presented here demonstrate that head-to-tail cyclization can be an effective strategy to increase the stability of an Affibody dimer.


Assuntos
Aminoaciltransferases/metabolismo , Proteínas de Bactérias/metabolismo , Biocatálise , Neoplasias da Mama/metabolismo , Cisteína Endopeptidases/metabolismo , Multimerização Proteica , Receptor ErbB-2/metabolismo , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Neoplasias da Mama/patologia , Dicroísmo Circular , Ciclização , Feminino , Humanos , Cinética , Células MCF-7 , Microscopia de Fluorescência , Peptídeo Hidrolases/metabolismo , Ligação Proteica , Estrutura Secundária de Proteína , Ressonância de Plasmônio de Superfície
13.
J R Soc Interface ; 18(179): 20210320, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-34129788

RESUMO

The spider major ampullate (MA) silk exhibits high tensile strength and extensibility and is typically a blend of MaSp1 and MaSp2 proteins with the latter comprising glycine-proline-glycine-glycine-X repeating motifs that promote extensibility and supercontraction. The MA silk from Darwin's bark spider (Caerostris darwini) is estimated to be two to three times tougher than the MA silk from other spider species. Previous research suggests that a unique MaSp4 protein incorporates proline into a novel glycine-proline-glycine-proline motif and may explain C. darwini MA silk's extraordinary toughness. However, no direct correlation has been made between the silk's molecular structure and its mechanical properties for C. darwini. Here, we correlate the relative protein secondary structure composition of MA silk from C. darwini and four other spider species with mechanical properties before and after supercontraction to understand the effect of the additional MaSp4 protein. Our results demonstrate that C. darwini MA silk possesses a unique protein composition with a lower ratio of helices (31%) and ß-sheets (20%) than other species. Before supercontraction, toughness, modulus and tensile strength correlate with percentages of ß-sheets, unordered or random coiled regions and ß-turns. However, after supercontraction, only modulus and strain at break correlate with percentages of ß-sheets and ß-turns. Our study highlights that additional information including crystal size and crystal and chain orientation is necessary to build a complete structure-property correlation model.


Assuntos
Seda , Aranhas , Animais , Casca de Planta , Estrutura Secundária de Proteína , Resistência à Tração
14.
Int J Mol Sci ; 22(10)2021 May 19.
Artigo em Inglês | MEDLINE | ID: mdl-34069753

RESUMO

Hydrocarbon stapling is a useful tool for stabilizing the secondary structure of peptides. Among several methods, hydrocarbon stapling at i,i + 1 positions was not extensively studied, and their secondary structures are not clarified. In this study, we investigate i,i + 1 hydrocarbon stapling between cis-4-allyloxy-l-proline and various olefin-tethered amino acids. Depending on the ring size of the stapled side chains and structure of the olefin-tethered amino acids, E- or Z-selectivities were observed during the ring-closing metathesis reaction (E/Z was up to 8.5:1 for 17-14-membered rings and up to 1:20 for 13-membered rings). We performed X-ray crystallographic analysis of hydrocarbon stapled peptide at i,i + 1 positions. The X-ray crystallographic structure suggested that the i,i + 1 staple stabilizes the peptide secondary structure to the right-handed α-helix. These findings are especially important for short oligopeptides because the employed stapling method uses two minimal amino acid residues adjacent to each other.


Assuntos
Hidrocarbonetos/química , Peptídeos/química , Estabilidade Proteica/efeitos dos fármacos , Alcenos/química , Sequência de Aminoácidos/genética , Aminoácidos/química , Dicroísmo Circular/métodos , Cristalografia por Raios X/métodos , Oligopeptídeos/química , Prolina/química , Conformação Proteica em alfa-Hélice/fisiologia , Estrutura Secundária de Proteína/fisiologia , Raios X
15.
J Phys Chem Lett ; 12(23): 5558-5563, 2021 Jun 17.
Artigo em Inglês | MEDLINE | ID: mdl-34101477

RESUMO

Plant cryptochromes are central blue light receptors in land plants and algae. Photoreduction of the flavin bound to the photolyase homology region (PHR) causes a dissociation of the C-terminal extension (CCT) as effector via an unclear pathway. We applied the recently developed in-cell infrared difference (ICIRD) spectroscopy to study the response of the full-length pCRY from Chlamydomonas reinhardtii in living bacterial cells, because the receptor degraded upon isolation. We demonstrate a stabilization of the flavin neutral radical as photoproduct and of the resulting ß-sheet reorganization by binding of cellular ATP. Comparison between light-induced structural responses of full-length pCRY and PHR reveals a downshift in frequency of the ß-sheet signal, implying an association of the CCT close to the only ß-sheet of the PHR in the dark. We provide a missing link in activation of plant cryptochromes after flavin photoreduction by indicating that ß-sheet reorganization causes the CCT release and restructuring.


Assuntos
Chlamydomonas reinhardtii/química , Chlamydomonas reinhardtii/metabolismo , Criptocromos/química , Criptocromos/metabolismo , Flavinas/química , Flavinas/metabolismo , Sítios de Ligação/fisiologia , Criptocromos/análise , Flavinas/análise , Conformação Proteica em Folha beta , Estrutura Secundária de Proteína , Espectrofotometria Infravermelho/métodos
16.
Anal Chem ; 93(26): 9094-9102, 2021 07 06.
Artigo em Inglês | MEDLINE | ID: mdl-34152129

RESUMO

Understanding the interaction of proteins at interfaces, which occurs at or within cell membranes and lipoprotein vesicles, is central to our understanding of protein function. Therefore, new experimental approaches to understand how protein structure is influenced by protein-interface interactions are important. Herein we build on our previous work exploring electrochemistry at the interface between two immiscible electrolyte solutions (ITIES) to investigate changes in protein secondary structure that are modulated by protein-interface interactions. The ITIES provides an experimental framework to drive protein adsorption at an interface, allowing subsequent spectroscopic analysis (e.g., Fourier transform infrared spectroscopy) to monitor changes in protein structure. Here, we reveal that the interaction between insulin and the interface destabilizes native insulin secondary structure, promoting formation of α helix secondary structures. These structural alterations result from protein-interface rather than protein-protein interactions at the interface. Although this is an emerging approach, our results provide a foundation highlighting the value of the ITIES as a tool to study protein structure and interactions at interfaces. Such knowledge may be useful to elucidate protein function within biological systems or to aid sensor development.


Assuntos
Insulina , Proteínas , Adsorção , Eletroquímica , Estrutura Secundária de Proteína
17.
Int J Mol Sci ; 22(11)2021 May 30.
Artigo em Inglês | MEDLINE | ID: mdl-34070827

RESUMO

Precocious dissociation of sisters 5 (PDS5) is an associate protein of cohesin that is conserved from yeast to humans. It acts as a regulator of the cohesin complex and plays important roles in various cellular processes, such as sister chromatid cohesion, DNA damage repair, gene transcription, and DNA replication. Vertebrates have two paralogs of PDS5, PDS5A and PDS5B, which have redundant and unique roles in regulating cohesin functions. Herein, we discuss the molecular characteristics and functions of PDS5, as well as the effects of its mutations in the development of diseases and their relevance for novel therapeutic strategies.


Assuntos
Proteínas de Ciclo Celular/genética , Reparo do DNA , Proteínas de Ligação a DNA/genética , Síndrome de Cornélia de Lange/genética , Neoplasias/genética , Proteínas Nucleares/genética , Proteínas de Saccharomyces cerevisiae/genética , Fatores de Transcrição/genética , Animais , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/metabolismo , Sequência Conservada , Dano ao DNA , Replicação do DNA , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Síndrome de Cornélia de Lange/metabolismo , Síndrome de Cornélia de Lange/patologia , Expressão Gênica , Humanos , Camundongos , Camundongos Knockout , Modelos Moleculares , Neoplasias/metabolismo , Neoplasias/patologia , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Estrutura Secundária de Proteína , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Troca de Cromátide Irmã , Fatores de Transcrição/química , Fatores de Transcrição/metabolismo
18.
Proc Natl Acad Sci U S A ; 118(26)2021 06 29.
Artigo em Inglês | MEDLINE | ID: mdl-34185681

RESUMO

The novel coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), invades a human cell via human angiotensin-converting enzyme 2 (hACE2) as the entry, causing the severe coronavirus disease (COVID-19). The interactions between hACE2 and the spike glycoprotein (S protein) of SARS-CoV-2 hold the key to understanding the molecular mechanism to develop treatment and vaccines, yet the dynamic nature of these interactions in fluctuating surroundings is very challenging to probe by those structure determination techniques requiring the structures of samples to be fixed. Here we demonstrate, by a proof-of-concept simulation of infrared (IR) spectra of S protein and hACE2, that time-resolved spectroscopy may monitor the real-time structural information of the protein-protein complexes of interest, with the help of machine learning. Our machine learning protocol is able to identify fine changes in IR spectra associated with variation of the secondary structures of S protein of the coronavirus. Further, it is three to four orders of magnitude faster than conventional quantum chemistry calculations. We expect our machine learning protocol would accelerate the development of real-time spectroscopy study of protein dynamics.


Assuntos
Enzima de Conversão de Angiotensina 2/metabolismo , Aprendizado de Máquina , SARS-CoV-2/metabolismo , Glicoproteína da Espícula de Coronavírus/metabolismo , Enzima de Conversão de Angiotensina 2/química , Humanos , Cinética , Ligação Proteica , Estrutura Secundária de Proteína , Espectrofotometria Infravermelho , Glicoproteína da Espícula de Coronavírus/química
19.
Science ; 372(6547): 1215-1219, 2021 06 11.
Artigo em Inglês | MEDLINE | ID: mdl-34112694

RESUMO

Hedgehog proteins govern crucial developmental steps in animals and drive certain human cancers. Before they can function as signaling molecules, Hedgehog precursor proteins must undergo amino-terminal palmitoylation by Hedgehog acyltransferase (HHAT). We present cryo-electron microscopy structures of human HHAT in complex with its palmitoyl-coenzyme A substrate and of a product complex with a palmitoylated Hedgehog peptide at resolutions of 2.7 and 3.2 angstroms, respectively. The structures reveal how HHAT overcomes the challenges of bringing together substrates that have different physiochemical properties from opposite sides of the endoplasmic reticulum membrane within a membrane-embedded active site for catalysis. These principles are relevant to related enzymes that catalyze the acylation of Wnt and of the appetite-stimulating hormone ghrelin. The structural and mechanistic insights may advance the development of inhibitors for cancer.


Assuntos
Aciltransferases/química , Aciltransferases/metabolismo , Retículo Endoplasmático/enzimologia , Proteínas Hedgehog/química , Palmitoil Coenzima A/química , Acilação , Biocatálise , Domínio Catalítico , Microscopia Crioeletrônica , Proteínas Hedgehog/metabolismo , Humanos , Membranas Intracelulares/enzimologia , Lipoilação , Modelos Moleculares , Simulação de Dinâmica Molecular , Palmitoil Coenzima A/metabolismo , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Domínios e Motivos de Interação entre Proteínas , Processamento de Proteína Pós-Traducional , Estrutura Secundária de Proteína
20.
Science ; 372(6547): 1220-1224, 2021 06 11.
Artigo em Inglês | MEDLINE | ID: mdl-34112695

RESUMO

Viruses are ubiquitous pathogens of global impact. Prompted by the hypothesis that their earliest progenitors recruited host proteins for virion formation, we have used stringent laboratory evolution to convert a bacterial enzyme that lacks affinity for nucleic acids into an artificial nucleocapsid that efficiently packages and protects multiple copies of its own encoding messenger RNA. Revealing remarkable convergence on the molecular hallmarks of natural viruses, the accompanying changes reorganized the protein building blocks into an interlaced 240-subunit icosahedral capsid that is impermeable to nucleases, and emergence of a robust RNA stem-loop packaging cassette ensured high encapsidation yields and specificity. In addition to evincing a plausible evolutionary pathway for primordial viruses, these findings highlight practical strategies for developing nonviral carriers for diverse vaccine and delivery applications.


Assuntos
Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Capsídeo/metabolismo , Evolução Molecular Direcionada , RNA Mensageiro/metabolismo , Substituição de Aminoácidos , Aquifex/enzimologia , Proteínas de Bactérias/química , Capsídeo/química , Microscopia Crioeletrônica , Complexos Multienzimáticos/química , Complexos Multienzimáticos/genética , Complexos Multienzimáticos/metabolismo , Nucleocapsídeo/química , Nucleocapsídeo/genética , Nucleocapsídeo/metabolismo , Domínios Proteicos , Estrutura Secundária de Proteína , Subunidades Proteicas , RNA Mensageiro/química , RNA Mensageiro/genética , Ribonucleases/metabolismo
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