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1.
J Phys Chem Lett ; 11(3): 1141-1147, 2020 Feb 06.
Artigo em Inglês | MEDLINE | ID: mdl-31951412

RESUMO

Double-electron electron resonance (DEER) can be used to track the structural dynamics of proteins in their native environment, the cell. This method provides the distance distribution between two spin labels attached at specific, well-defined positions in a protein. For the method to be viable under in-cell conditions, the spin label and its attachment to the protein should exhibit high chemical stability in the cell. Here we present low-temperature, trityl-trityl DEER distance measurements on two model proteins, PpiB (prolyl cis-trans isomerase from E. coli) and GB1 (immunoglobulin G-binding protein), doubly labeled with the trityl spin label, CT02MA. Both proteins gave in-cell distance distributions similar to those observed in vitro, with maxima at 4.5-5 nm, and the data were further compared with in-cell Gd(III)-Gd(III) DEER obtained for PpiB labeled with BrPSPy-DO3A-Gd(III) at the same positions. These results highlight the challenges of designing trityl tags suitable for in-cell distance determination at ambient temperatures on live cells.


Assuntos
Proteínas de Bactérias/química , Proteínas de Transporte/química , Ciclofilinas/química , Espectroscopia de Ressonância de Spin Eletrônica , Compostos de Tritil/química , Gadolínio/química , Marcadores de Spin
2.
PLoS Genet ; 15(6): e1008196, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31173582

RESUMO

Covalent intermolecular cross-linking of collagen is essential for tissue stability. Recent studies have demonstrated that cyclophilin B (CypB), an endoplasmic reticulum (ER)-resident peptidyl-prolyl cis-trans isomerase, modulates lysine (Lys) hydroxylation of type I collagen impacting cross-linking chemistry. However, the extent of modulation, the molecular mechanism and the functional outcome in tissues are not well understood. Here, we report that, in CypB null (KO) mouse skin, two unusual collagen cross-links lacking Lys hydroxylation are formed while neither was detected in wild type (WT) or heterozygous (Het) mice. Mass spectrometric analysis of type I collagen showed that none of the telopeptidyl Lys was hydroxylated in KO or WT/Het mice. Hydroxylation of the helical cross-linking Lys residues was almost complete in WT/Het but was markedly diminished in KO. Lys hydroxylation at other sites was also lower in KO but to a lesser extent. A key glycosylation site, α1(I) Lys-87, was underglycosylated while other sites were mostly overglycosylated in KO. Despite these findings, lysyl hydroxylases and glycosyltransferase 25 domain 1 levels were significantly higher in KO than WT/Het. However, the components of ER chaperone complex that positively or negatively regulates lysyl hydroxylase activities were severely reduced or slightly increased, respectively, in KO. The atomic force microscopy-based nanoindentation modulus were significantly lower in KO skin than WT. These data demonstrate that CypB deficiency profoundly affects Lys post-translational modifications of collagen likely by modulating LH chaperone complexes. Together, our study underscores the critical role of CypB in Lys modifications of collagen, cross-linking and mechanical properties of skin.


Assuntos
Ciclofilinas/química , Lisina/química , Pró-Colágeno-Lisina 2-Oxoglutarato 5-Dioxigenase/química , Pele/enzimologia , Animais , Colágeno Tipo I/biossíntese , Colágeno Tipo I/genética , Ciclofilinas/genética , Ciclofilinas/ultraestrutura , Retículo Endoplasmático/química , Retículo Endoplasmático/enzimologia , Glicosilação , Heterozigoto , Hidroxilação , Lisina/genética , Espectrometria de Massas , Camundongos , Camundongos Knockout , Microscopia de Força Atômica , Pró-Colágeno-Lisina 2-Oxoglutarato 5-Dioxigenase/genética , Processamento de Proteína Pós-Traducional/genética , Pele/química
3.
Int J Mol Sci ; 20(12)2019 Jun 18.
Artigo em Inglês | MEDLINE | ID: mdl-31216716

RESUMO

Purpureocillium lilacinum has been widely used as a commercial biocontrol agent for the control of plant parasitic nematodes. Whole genome analysis promotes the identification of functional genes and the exploration of their molecular mechanisms. The Cyclophilin (CYP) gene family belongs to the immunophillin superfamily, and has a conserved cyclophilin-like domain (CLD). CYPs are widely identified in prokaryotes and eukaryotes, and can be divided into single- and multi-domain proteins. In the present study, 10 CYP genes possessing the CLD, named PlCYP1-P10, were identified from the genome of P. lilacinum strain 36-1. Those 10 PlCYPs were predicted to have different cellular localizations in P. lilacinum. Phylogenetic and gene structure analysis revealed the evolutionary differentiation of CYPs between Ascomycotina and Saccharomycotina fungi, but conservation within the Ascomycotina fungi. Motif and gene structure distributions further support the result of phylogenetic analysis. Each PlCYP gene had a specific expression pattern in different development stages of P. lilacinum and its parasitism stage on eggs of Meloidogyne incognita. In addition, the 10 PlCYP genes exhibited different expression abundances in response to abiotic stresses, among which PlCYP4 was highly expressed at a high temperature (35 °C), while PlCYP6 was up-regulated under 5 mM of H2O2 stress. Furthermore, the heterologous expression of PlCYP4 and PlCYP6 in Escherichia coli enhanced the cellular tolerance against a high temperature and H2O2. In summary, our study indicates the potential functions of PlCYPs in virulence and the stress response, and also provides a frame for further analysis of the CYP gene family in Ascomycotina fungi.


Assuntos
Ascomicetos/classificação , Ascomicetos/genética , Ciclofilinas/genética , Genoma Fúngico , Genômica , Família Multigênica , Sequência de Aminoácidos , Ascomicetos/metabolismo , Ciclofilinas/química , Regulação Fúngica da Expressão Gênica , Genômica/métodos , Fenótipo , Filogenia , Domínios e Motivos de Interação entre Proteínas , Análise de Sequência de DNA , Estresse Fisiológico
4.
Int J Mol Sci ; 20(10)2019 May 17.
Artigo em Inglês | MEDLINE | ID: mdl-31108847

RESUMO

Of the three interleukin-22 binding protein (IL-22BP) isoforms produced by the human IL22RA2 gene, IL-22BPi2 and IL-22BPi3 are capable of neutralizing IL-22. The longest isoform, IL-22BPi1, does not bind IL-22, is poorly secreted, and its retention within the endoplasmic reticulum (ER) is associated with induction of an unfolded protein response (UPR). Therapeutic modulation of IL-22BPi2 and IL-22BPi3 production may be beneficial in IL-22-dependent disorders. Recently, we identified the ER chaperones GRP94 and cyclophilin B in the interactomes of both IL-22BPi1 and IL-22BPi2. In this study, we investigated whether secretion of the IL-22BP isoforms could be modulated by pharmacological targeting of GRP94 and cyclophilin B, either by means of geldanamycin, that binds to the ADP/ATP pocket shared by HSP90 paralogs, or by cyclosporin A, which causes depletion of ER cyclophilin B levels through secretion. We found that geldanamycin and its analogs did not influence secretion of IL-22BPi2 or IL-22BPi3, but significantly enhanced intracellular and secreted levels of IL-22BPi1. The secreted protein was heterogeneously glycosylated, with both high-mannose and complex-type glycoforms present. In addition, cyclosporine A augmented the secretion of IL-22BPi1 and reduced that of IL-22BPi2 and IL-22BPi3. Our data indicate that the ATPase activity of GRP94 and cyclophilin B are instrumental in ER sequestration and degradation of IL-22BPi1, and that blocking these factors mobilizes IL-22BPi1 toward the secretory route.


Assuntos
Benzoquinonas/farmacologia , Ciclofilinas/metabolismo , Ciclosporina/farmacologia , Lactamas Macrocíclicas/farmacologia , Glicoproteínas de Membrana/metabolismo , Receptores de Interleucina/metabolismo , Sítios de Ligação/efeitos dos fármacos , Ciclofilinas/química , Retículo Endoplasmático/metabolismo , Perfilação da Expressão Gênica , Glicosilação , Células HEK293 , Humanos , Glicoproteínas de Membrana/química , Monócitos/metabolismo , Ligação Proteica/efeitos dos fármacos , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismo , Proteólise , Receptores de Interleucina/química , Receptores de Interleucina/genética
5.
Cell Mol Life Sci ; 76(19): 3899-3914, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-30993352

RESUMO

The P3H1/CRTAP/PPIB complex is essential for prolyl 3-hydroxylation and folding of procollagens in the endoplasmic reticulum (ER). Deficiency in any component of this ternary complex is associated with the misfolding of collagen and the onset of osteogenesis imperfecta. However, little structure information is available about how this ternary complex is assembled and retained in the ER. Here, we assessed the role of the KDEL sequence of P3H1 and probed the spatial interactions of PPIB in the complex. We show that the KDEL sequence is essential for retaining the P3H1 complex in the ER. Its removal resulted in co-secretion of P3H1 and CRTAP out of the cell, which was mediated by the binding of P3H1 N-terminal domain with CRTAP. The secreted P3H1/CRTAP can readily bind PPIB with their C-termini close to PPIB in the ternary complex. Cysteine modification, crosslinking, and mass spectrometry experiments identified PPIB surface residues involved in the complex formation, and showed that the surface of PPIB is extensively covered by the binding of P3H1 and CRTAP. Most importantly, we demonstrated that one disease-associated pathological PPIB mutation on the binding interface did not affect the PPIB prolyl-isomerase activity, but disrupted the formation of P3H1/CRTAP/PPIB ternary complex. This suggests that defects in the integrity of the P3H1 ternary complex are associated with pathological collagen misfolding. Taken together, these results provide novel structural information on how PPIB interacts with other components of the P3H1 complex and indicate that the integrity of P3H1 complex is required for proper collagen formation.


Assuntos
Ciclofilinas/química , Proteínas da Matriz Extracelular/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteoglicanas/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Ciclofilinas/genética , Ciclofilinas/metabolismo , Proteínas da Matriz Extracelular/genética , Humanos , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/genética , Modelos Moleculares , Mutação , Prolil Hidroxilases , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Proteoglicanas/química , Proteoglicanas/genética , Deleção de Sequência
6.
PLoS One ; 14(3): e0210771, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30925148

RESUMO

Cyclophilin (Cyp), a peptidyl-prolyl cis-trans isomerase (PPIase), acts as a virulence factor in many bacteria including Staphylococcus aureus. The enzymatic activity of Cyp is inhibited by cyclosporin A (CsA), an immunosuppressive drug. To precisely determine the unfolding mechanism and the domain structure of Cyp, we have investigated a chimeric S. aureus Cyp (rCyp) using various probes. Our limited proteolysis and the consequent analysis of the proteolytic fragments indicate that rCyp is composed of one domain with a short flexible tail at the C-terminal end. We also show that the urea-induced unfolding of both rCyp and rCyp-CsA is completely reversible and proceeds via the synthesis of at least one stable intermediate. Both the secondary structure and the tertiary structure of each intermediate appears very similar to those of the corresponding native protein. Conversely, the hydrophobic surface areas of the intermediates are comparatively less. Further analyses reveal no loss of CsA binding activity in rCyp intermediate. The thermodynamic stability of rCyp was also significantly increased in the presence of CsA, recommending that this protein could be employed to screen new CsA derivatives in the future.


Assuntos
Ciclofilinas/química , Ciclofilinas/metabolismo , Ciclosporina/farmacologia , Staphylococcus aureus/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Ciclosporina/química , Ligação Proteica , Domínios Proteicos , Dobramento de Proteína , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Proteólise , Ureia/farmacologia
7.
Chem Commun (Camb) ; 55(6): 846-849, 2019 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-30575826

RESUMO

Protein-protein interactions control all cellular functions. Presented is the first de novo designed protein-protein inhibitor that targets the C-terminus of heat shock protein 90 (Hsp90) and blocks co-chaperones from binding. Compound LB76, which was created from an Hsp90 co-chaperone, selectively pulls down Hsp90 from cell lysates, binds to Hsp90's C-terminal domain, and blocks the interactions between Hsp90 and TPR-containing co-chaperones. Through these interactions, LB76 inhibits the protein-folding function of Hsp90. Blocking these protein-protein interactions between Hsp90 and C-terminal co-chaperones regulate the cell's entire protein-folding machinery.


Assuntos
Biotina/química , Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Sequência de Aminoácidos , Sítios de Ligação , Biotina/metabolismo , Ciclofilinas/química , Ciclofilinas/metabolismo , Células HCT116 , Proteínas de Choque Térmico HSP90/metabolismo , Humanos , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas
8.
Biomolecules ; 8(4)2018 12 04.
Artigo em Inglês | MEDLINE | ID: mdl-30518120

RESUMO

The peptidyl prolyl isomerases (PPI) of the cyclophilin type are distributed throughout human cells, including eight found solely in the nucleus. Nuclear cyclophilins are involved in complexes that regulate chromatin modification, transcription, and pre-mRNA splicing. This review collects what is known about the eight human nuclear cyclophilins: peptidyl prolyl isomerase H (PPIH), peptidyl prolyl isomerase E (PPIE), peptidyl prolyl isomerase-like 1 (PPIL1), peptidyl prolyl isomerase-like 2 (PPIL2), peptidyl prolyl isomerase-like 3 (PPIL3), peptidyl prolyl isomerase G (PPIG), spliceosome-associated protein CWC27 homolog (CWC27), and peptidyl prolyl isomerase domain and WD repeat-containing protein 1 (PPWD1). Each "spliceophilin" is evaluated in relation to the spliceosomal complex in which it has been studied, and current work studying the biological roles of these cyclophilins in the nucleus are discussed. The eight human splicing complexes available in the Protein Data Bank (PDB) are analyzed from the viewpoint of the human spliceophilins. Future directions in structural and cellular biology, and the importance of developing spliceophilin-specific inhibitors, are considered.


Assuntos
Núcleo Celular/química , Ciclofilinas/química , Spliceossomos/química , Relação Estrutura-Atividade , Sequência de Aminoácidos , Ciclofilinas/classificação , Ciclofilinas/genética , Ciclofilinas/metabolismo , Humanos , Peptidilprolil Isomerase/química , Peptidilprolil Isomerase/genética , Conformação Proteica , Processamento de RNA/genética , Spliceossomos/genética
9.
Biomolecules ; 8(4)2018 10 31.
Artigo em Inglês | MEDLINE | ID: mdl-30384485

RESUMO

Trypanosoma cruzi is the etiological agent of Chagas disease. It affects eight million people worldwide and can be spread by several routes, such as vectorborne transmission in endemic areas and congenitally, and is also important in non-endemic regions such as the United States and Europe due to migration from Latin America. Cyclophilins (CyPs) are proteins with enzymatic peptidyl-prolyl isomerase activity (PPIase), essential for protein folding in vivo. Cyclosporin A (CsA) has a high binding affinity for CyPs and inhibits their PPIase activity. CsA has proved to be a parasiticidal drug on some protozoa, including T. cruzi. In this review, we describe the T. cruzi cyclophilin gene family, that comprises 15 paralogues. Among the proteins isolated by CsA-affinity chromatography, we found orthologues of mammalian CyPs. TcCyP19, as the human CyPA, is secreted to the extracellular environment by all parasite stages and could be part of a complex interplay involving the parasite and the host cell. TcCyP22, an orthologue of mitochondrial CyPD, is involved in the regulation of parasite cell death. Our findings on T. cruzi cyclophilins will allow further characterization of these processes, leading to new insights into the biology, the evolution of metabolic pathways, and novel targets for anti-T. cruzi control.


Assuntos
Ciclofilinas/metabolismo , Parasitos/fisiologia , Proteínas de Protozoários/metabolismo , Trypanosoma cruzi/fisiologia , Sequência de Aminoácidos , Animais , Antiprotozoários/farmacologia , Doença de Chagas/parasitologia , Ciclofilinas/química , Parasitos/efeitos dos fármacos , Proteínas de Protozoários/química
10.
J Med Chem ; 61(21): 9469-9472, 2018 11 08.
Artigo em Inglês | MEDLINE | ID: mdl-30359012

RESUMO

Targets that have large and groove-shaped binding sites, such as cyclophilin, are difficult to drug with small molecules. Macrocycles of natural product origin can be ideal starting points for such targets as illustrated by the transformation of sanglifehrin A into an orally bioavailable potential candidate drug. Optimization benefits from development of convergent, modular synthetic routes in combination with structure and property based methods for lead optimization.


Assuntos
Ciclofilinas/metabolismo , Descoberta de Drogas , Compostos Macrocíclicos/química , Compostos Macrocíclicos/farmacologia , Terapia de Alvo Molecular , Ciclofilinas/química , Modelos Moleculares , Conformação Proteica
11.
J Med Chem ; 61(21): 9473-9499, 2018 11 08.
Artigo em Inglês | MEDLINE | ID: mdl-30074795

RESUMO

Cyclophilins are a family of peptidyl-prolyl isomerases that are implicated in a wide range of diseases including hepatitis C. Our aim was to discover through total synthesis an orally bioavailable, non-immunosuppressive cyclophilin (Cyp) inhibitor with potent anti-hepatitis C virus (HCV) activity that could serve as part of an all oral antiviral combination therapy. An initial lead 2 derived from the sanglifehrin A macrocycle was optimized using structure based design to produce a potent and orally bioavailable inhibitor 3. The macrocycle ring size was reduced by one atom, and an internal hydrogen bond drove improved permeability and drug-like properties. 3 demonstrates potent Cyp inhibition ( Kd = 5 nM), potent anti-HCV 2a activity (EC50 = 98 nM), and high oral bioavailability in rat (100%) and dog (55%). The synthetic accessibility and properties of 3 support its potential as an anti-HCV agent and for interrogating the role of Cyp inhibition in a variety of diseases.


Assuntos
Ciclofilinas/antagonistas & inibidores , Desenho de Drogas , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/farmacocinética , Administração Oral , Antivirais/administração & dosagem , Antivirais/química , Antivirais/farmacocinética , Antivirais/farmacologia , Disponibilidade Biológica , Linhagem Celular , Ciclofilinas/química , Inibidores Enzimáticos/administração & dosagem , Inibidores Enzimáticos/química , Hepacivirus/efeitos dos fármacos , Lactonas/administração & dosagem , Lactonas/química , Lactonas/farmacocinética , Lactonas/farmacologia , Modelos Moleculares , Conformação Proteica , Compostos de Espiro/administração & dosagem , Compostos de Espiro/química , Compostos de Espiro/farmacocinética , Compostos de Espiro/farmacologia
12.
J Chem Phys ; 148(14): 145101, 2018 Apr 14.
Artigo em Inglês | MEDLINE | ID: mdl-29655319

RESUMO

Cyclophilin 40 (Cyp40) is a member of the immunophilin family that acts as a peptidyl-prolyl-isomerase enzyme and binds to the heat shock protein 90 (Hsp90). Its structure comprises an N-terminal cyclophilin domain and a C-terminal tetratricopeptide (TPR) domain. Cyp40 is overexpressed in prostate cancer and certain T-cell lymphomas. The groove for Hsp90 binding on the TPR domain includes residues Lys227 and Lys308, referred to as the carboxylate clamp, and is essential for Cyp40-Hsp90 binding. In this study, the effect of two mutations, K227A and K308A, and their combinative mutant was investigated by performing a total of 5.76 µs of all-atom molecular dynamics (MD) simulations in explicit solvent. All simulations, except the K308A mutant, were found to adopt two distinct (extended or compact) conformers defined by different cyclophilin-TPR interdomain distances. The K308A mutant was only observed in the extended form which is observed in the Cyp40 X-ray structure. The wild-type, K227A, and combined mutant also showed bimodal distributions. The experimental melting temperature, Tm, values of the mutants correlate with the degree of compactness with the K308A extended mutant having a marginally lower melting temperature. Another novel measure of compactness determined from the MD data, the "coordination shell volume," also shows a direct correlation with Tm. In addition, the MD simulations show an allosteric effect with the mutations in the remote TPR domain having a pronounced effect on the molecular motions of the enzymatic cyclophilin domain which helps rationalise the experimentally observed increase in enzyme activity measured for all three mutations.


Assuntos
Ciclofilinas/química , Mutação Puntual/genética , Ciclofilinas/genética , Humanos , Simulação de Dinâmica Molecular , Conformação Proteica , Domínios Proteicos/genética , Termodinâmica , Temperatura de Transição
13.
Sci Rep ; 8(1): 5410, 2018 04 03.
Artigo em Inglês | MEDLINE | ID: mdl-29615721

RESUMO

Cyclophilin 1 (TvCyP1), a cyclophilin type peptidyl-prolyl isomerase present in the human parasite Trichomonas vaginalis, interacts with Myb1 and assists in its nuclear translocation. Myb1 regulates the expression of ap65-1 gene that encodes for a disease causing cytoadherence enzyme. Here, we determined the crystal structures of TvCyP1 and its complex with the minimum TvCyP1-binding sequence of Myb1 (Myb1104-111), where TvCyP1 formed a homodimer, unlike other single domain cyclophilins. In the complex structure, one Myb1104-111 peptide was bound to each TvCyP1 protomer, with G106-P107 and Y105 fitting well into the active site and auxiliary S2 pocket, respectively. NMR data further showed that TvCyP1 can catalyze the cis/trans isomerization of P107 in Myb1104-111. Interestingly, in the well-folded Myb1 protein (Myb135-141), the minimum binding sequence adopted a different conformation from that of unstructured Myb1104-111 peptide, that could make P107 binding to the active site of TvCyP1 difficult. However, NMR studies showed that similar to Myb1104-111 peptide, Myb135-141 also interacted with the active site of TvCyP1 and the dynamics of the Myb135-141 residues near P107 was reduced upon interaction. Together, the structure of TvCyP1 and detailed structural insights on TvCyP1-Myb1 interaction provided here could pave the way for newer drugs to treat drug-resistant strains.


Assuntos
Ciclofilinas/química , Ciclofilinas/metabolismo , Multimerização Proteica , Proteínas de Protozoários/química , Proteínas de Protozoários/metabolismo , Fatores de Transcrição/metabolismo , Trichomonas vaginalis , Sítios de Ligação , Modelos Moleculares , Peptidilprolil Isomerase/metabolismo , Ligação Proteica , Domínios Proteicos , Estabilidade Proteica , Estrutura Quaternária de Proteína
14.
PLoS Comput Biol ; 14(2): e1006008, 2018 02.
Artigo em Inglês | MEDLINE | ID: mdl-29432415

RESUMO

The majority of the proteins encoded in the genomes of eukaryotes contain more than one domain. Reasons for high prevalence of multi-domain proteins in various organisms have been attributed to higher stability and functional and folding advantages over single-domain proteins. Despite these advantages, many proteins are composed of only one domain while their homologous domains are part of multi-domain proteins. In the study presented here, differences in the properties of protein domains in single-domain and multi-domain systems and their influence on functions are discussed. We studied 20 pairs of identical protein domains, which were crystallized in two forms (a) tethered to other proteins domains and (b) tethered to fewer protein domains than (a) or not tethered to any protein domain. Results suggest that tethering of domains in multi-domain proteins influences the structural, dynamic and energetic properties of the constituent protein domains. 50% of the protein domain pairs show significant structural deviations while 90% of the protein domain pairs show differences in dynamics and 12% of the residues show differences in the energetics. To gain further insights on the influence of tethering on the function of the domains, 4 pairs of homologous protein domains, where one of them is a full-length single-domain protein and the other protein domain is a part of a multi-domain protein, were studied. Analyses showed that identical and structurally equivalent functional residues show differential dynamics in homologous protein domains; though comparable dynamics between in-silico generated chimera protein and multi-domain proteins were observed. From these observations, the differences observed in the functions of homologous proteins could be attributed to the presence of tethered domain. Overall, we conclude that tethered domains in multi-domain proteins not only provide stability or folding advantages but also influence pathways resulting in differences in function or regulatory properties.


Assuntos
Domínios Proteicos , Proteínas/química , Animais , Simulação por Computador , Ciclofilinas/química , DNA Polimerase beta/química , Fibronectinas/química , Hexoquinase/química , Humanos , Modelos Moleculares , Simulação de Dinâmica Molecular , Neuraminidase/química , Ligação Proteica , Dobramento de Proteína , Mapeamento de Interação de Proteínas , Estrutura Terciária de Proteína , Proteoma , Ratos
15.
Biomol NMR Assign ; 12(1): 171-174, 2018 04.
Artigo em Inglês | MEDLINE | ID: mdl-29353448

RESUMO

Cyclophilins are enzymes that catalyze the isomerization of a prolyl-peptide bond and are found in both prokaryotes and eukaryotes. LRT2 (also known as OsCYP2) is a cyclophilin in rice (Oryza sativa), that has importance in lateral root development and stress tolerance. LRT2 is 172 amino acids long and has a molecular weight of 18.3 kDa. Here, we report the backbone and sidechain resonance assignments of 1H, 13C, 15N in the LRT2 protein using several 2D and 3D heteronuclear NMR experiments at pH 6.7 and 298 K. Our chemical shift data analysis predicts a secondary structure like the cytosolic wheat cyclophilin TaCypA-1 with 87.7% sequence identity. These assignments will be useful for further analysis in the NMR studies for function and structure of this enzyme.


Assuntos
Ciclofilinas/química , Ressonância Magnética Nuclear Biomolecular , Oryza , Proteínas de Plantas/química
16.
Biomol NMR Assign ; 12(1): 27-30, 2018 04.
Artigo em Inglês | MEDLINE | ID: mdl-28875299

RESUMO

Cyclophilins are peptidyl prolyl isomerases that play an important role in a wide variety of biological functions like protein folding and trafficking, intracellular and extracellular signaling pathways, nuclear translocation and in pre-mRNA splicing. Two cyclophilins have been identified in the parasitic organism Trichomonas vaginalis and were named as TvCyP1 and TvCyP2. The 2 enzymes have been found to interact with Myb transcription factors in the parasite which regulate the iron induced expression of ap65-1 gene leading to cytoadherence of the parasite to human vaginal epithelial cells to cause the disease trichomoniasis. TvCyP2 was found to interact specifically with Myb3 to regulate nuclear translocation of the transcription factor. It would be intriguing to identify the binding site of both proteins as it could pave way to newer targets for drug discovery. Here we report the 1H, 13C and 15N resonance assignments and secondary structure information of TvCyP2 that could help us investigate the interaction between Myb3 and TvCyP2 in detail using NMR.


Assuntos
Ciclofilinas/química , Ressonância Magnética Nuclear Biomolecular , Proteínas de Protozoários/química , Trichomonas vaginalis , Sequência de Aminoácidos , Estrutura Secundária de Proteína
17.
Postepy Biochem ; 64(1): 46-54, 2018 Jun 30.
Artigo em Polonês | MEDLINE | ID: mdl-30652836

RESUMO

Cyclophilins together with FK-506-binding proteins and parvulins, belong to a group of proteins that have peptidyl-prolyl cis-trans isomerase activity. They also belong to proteins that are collectively known as immunophilins. Cyclophilins are found in all cells of all organisms studied, in both prokaryotes and eukaryotes. The first member of the cyclophilins to be identified in mammals, cyclophilin A, is the major cellular target for the immunosuppressive drug cyclosporin A. This review discusses recently available data about proteins with cyclophilin domain (CBD). Recent studies have implicated a diverse array of additional cellular functions for cyclophilins, including roles as chaperones and in cell signalling as well as in several human diseases.


Assuntos
Ciclofilinas/metabolismo , Animais , Ciclofilinas/química , Doença , Humanos , Chaperonas Moleculares/metabolismo , Transdução de Sinais , Proteínas de Ligação a Tacrolimo/metabolismo
18.
J Mol Microbiol Biotechnol ; 27(4): 228-236, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28889121

RESUMO

The presence of peptidyl-prolyl cis/trans isomerases (PPIases, EC: 5.2.1.8) in all domains of life indicates their biological importance. Cyclophilin PpiA, present in the periplasm of gram-negative bacteria, possesses PPIase activity but its physiological functions are still not clearly defined. Here, we demonstrate that the ΔppiA deletion strain from Escherichia coli exhibits an increased ability for biofilm formation and enhanced swimming motility compared to the wild-type strain. To identify structural features of PpiA which are necessary for the negative modulation of biofilm formation, we constructed a series of mutant PpiA proteins using a combination of error-prone and site-directed mutagenesis approaches. We show that the negative effect of PpiA on biofilm formation is not dependent on its PPIase activity, since PpiA mutants with a reduced PPIase activity are able to complement the ΔppiA strain during biofilm growth.


Assuntos
Biofilmes/crescimento & desenvolvimento , Ciclofilinas/química , Escherichia coli/metabolismo , Peptidilprolil Isomerase/química , Proteínas Recombinantes/química , Ciclofilinas/genética , Ciclofilinas/metabolismo , Primers do DNA , Escherichia coli/genética , Perfilação da Expressão Gênica , Mutagênese Sítio-Dirigida , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Peptidilprolil Isomerase/genética , Conformação Proteica , Proteínas Recombinantes/genética
19.
Biomolecules ; 7(4)2017 09 29.
Artigo em Inglês | MEDLINE | ID: mdl-28961224

RESUMO

Analyses of sequences and structures of the cyclosporine A (CsA)-binding proteins (cyclophilins) and the immunosuppressive macrolide FK506-binding proteins (FKBPs) have revealed that they exhibit peculiar spatial distributions of charges, their overall hydrophobicity indexes vary within a considerable level whereas their points isoelectric (pIs) are contained from 4 to 11. These two families of peptidylprolyl cis/trans isomerases (PPIases) have several distinct functional attributes such as: (1) high affinity binding to some pharmacologically-useful hydrophobic macrocyclic drugs; (2) diversified binding epitopes to proteins that may induce transient manifolds with altered flexibility and functional fitness; and (3) electrostatic interactions between positively charged segments of PPIases and negatively charged intracellular entities that support their spatial integration. These three attributes enhance binding of PPIase/pharmacophore complexes to diverse intracellular entities, some of which perturb signalization pathways causing immunosuppression and other system-altering phenomena in humans.


Assuntos
Ciclofilinas/química , Portadores de Fármacos/uso terapêutico , Peptidilprolil Isomerase/química , Proteínas de Ligação a Tacrolimo/química , Ciclofilinas/imunologia , Ciclofilinas/uso terapêutico , Portadores de Fármacos/química , Epitopos/química , Epitopos/imunologia , Humanos , Interações Hidrofóbicas e Hidrofílicas , Imunossupressão , Compostos Macrocíclicos/imunologia , Compostos Macrocíclicos/uso terapêutico , Peptidilprolil Isomerase/imunologia , Peptidilprolil Isomerase/uso terapêutico , Eletricidade Estática , Proteínas de Ligação a Tacrolimo/imunologia , Proteínas de Ligação a Tacrolimo/uso terapêutico
20.
World J Microbiol Biotechnol ; 33(9): 164, 2017 Aug 08.
Artigo em Inglês | MEDLINE | ID: mdl-28791545

RESUMO

Cyclophilins belong to the superfamily of peptidyl-prolyl cis/trans isomerases (PPIases, EC: 5.2.1.8), the enzymes that catalyze the cis/trans isomerization of peptidyl-prolyl peptide bonds in unfolded and partially folded polypeptide chains and native state proteins. Cyclophilins have been extensively studied, since they are involved in multiple cellular processes related to human pathologies, such as neurodegenerative disorders, infectious diseases, and cancer. However, the presence of cyclophilins in all domains of life indicates a broader biological importance. In this mini-review, we summarize current advances in the study of microbial cyclophilins. Apart from their anticipated role in protein folding and chaperoning, cyclophilins are involved in several other biological processes, such as cellular signal transduction, adaptation to stress, control of pathogens virulence, and modulation of host immune response. Since many existing family members do not have well-defined functions and novel ones are being characterized, the requirement for further studies on their biological role and molecular mechanism of action is apparent.


Assuntos
Ciclofilinas/metabolismo , Virulência/efeitos dos fármacos , Ciclofilinas/química , Ciclofilinas/farmacologia , Humanos , Imunidade , Dobramento de Proteína , Transdução de Sinais
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