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1.
Arch Virol ; 166(11): 3143-3150, 2021 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-34533641

RESUMO

Chandipura virus (CHPV), belonging to the genus Vesiculovirus of the family Rhabdoviridae, has been identified as one of the causes of pediatric encephalitis in India. Currently, neither vaccines nor therapeutic drugs are available against this agent. Considering that the disease progresses very fast with a high mortality rate, working towards the development of potential therapeutics against it will have a public health impact. Although the use of viral inhibitors as antiviral agents is the most common way to curb virus replication, the mutation-prone nature of viruses results in the development of resistance to antiviral agents. The recent development of proteomic platforms for analysis of purified viral agents has allowed certain upregulated host proteins that are involved in the morphogenesis and replication of viruses to be identified. Thus, the alternative approach of inhibition of host proteins involved in the regulation of virus replication could be explored for their therapeutic effectiveness. In the current study, we have evaluated the effect of inhibition of cyclophilin A (CypA), an immunophilin with peptidyl-prolyl cis/trans-isomerase activity, on the replication of CHPV. Treatment with cyclosporin A, used in vitro for the inhibition of CypA, resulted in a 3-log reduction in CHPV titer and an undetectable level of CypA in comparison to an untreated control. An in silico analysis of the interaction of the CHPV nucleoprotein with the human CypA protein showed stable interaction in molecular docking and molecular dynamics simulations. Overall, the results of this study suggest a possible role of CypA in facilitating CHPV replication, thus making it one of the potential host factors to be explored in future antiviral studies.


Assuntos
Ciclofilina A/metabolismo , Interações Hospedeiro-Patógeno/fisiologia , Infecções por Rhabdoviridae/virologia , Vesiculovirus/patogenicidade , Ciclofilina A/antagonistas & inibidores , Ciclofilina A/química , Ciclosporina/farmacologia , Interações Hospedeiro-Patógeno/efeitos dos fármacos , Humanos , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Proteínas do Nucleocapsídeo/química , Proteínas do Nucleocapsídeo/metabolismo , Vesiculovirus/efeitos dos fármacos , Vesiculovirus/fisiologia , Replicação Viral/efeitos dos fármacos
2.
J Med Chem ; 64(15): 11445-11459, 2021 08 12.
Artigo em Inglês | MEDLINE | ID: mdl-34338510

RESUMO

Blocking the interaction between the apoptosis-inducing factor (AIF) and cyclophilin A (CypA) by the AIF fragment AIF(370-394) is protective against glutamate-induced neuronal cell death and brain injury in mice. Starting from AIF(370-394), we report the generation of the disulfide-bridged and shorter variant AIF(381-389) and its structural characterization by nuclear magnetic resonance (NMR) in the free and CypA-bound state. AIF(381-389) in both the free and bound states assumes a ß-hairpin conformation similar to that of the fragment in the AIF protein and shows a highly reduced conformational flexibility. This peptide displays a similar in vitro affinity for CypA, an improved antiapoptotic activity in cells and an enhanced proteolytic stability compared to the parent peptide. The NMR-based 3D model of the AIF(381-389)/CypA complex provides a better understanding of the binding hot spots on both the peptide and the protein and can be exploited to design AIF/CypA inhibitors with improved pharmacokinetic and pharmacodynamics features.


Assuntos
Fator de Indução de Apoptose/farmacologia , Apoptose/efeitos dos fármacos , Lesões Encefálicas/tratamento farmacológico , Morte Celular/efeitos dos fármacos , Ciclofilina A/antagonistas & inibidores , Desenho de Fármacos , Animais , Fator de Indução de Apoptose/síntese química , Fator de Indução de Apoptose/química , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Ciclofilina A/metabolismo , Relação Dose-Resposta a Droga , Ácido Glutâmico/metabolismo , Humanos , Camundongos , Estrutura Molecular , Relação Estrutura-Atividade
3.
Int J Mol Sci ; 22(15)2021 Jul 23.
Artigo em Inglês | MEDLINE | ID: mdl-34360654

RESUMO

CD147, a transmembrane glycoprotein that belongs to the immunoglobulin superfamily, and cyclophilin A (CypA), one of the binding partners of CD147, are overexpressed in tumor cells and associated with the progression of several malignancies, including both solid and hematological malignancies. However, CD147 and CypA involvement in cutaneous T-cell lymphoma (CTCL) has not been reported. In this study, we examined CD147 and CypA expression and function using clinical samples of mycosis fungoides (MF) and Sézary syndrome (SS) and CTCL cell lines. CD147 and CypA were overexpressed by tumor cells of MF/SS, and CypA was also expressed by epidermal keratinocytes in MF/SS lesional skin. Serum CypA levels were increased and correlated with disease severity markers in MF/SS patients. Anti-CD147 antibody and/or anti-CypA antibody suppressed the proliferation of CTCL cell lines, both in vitro and in vivo, via downregulation of phosphorylated extracellular-regulated kinase 1/2 and Akt. These results suggest that CD147-CypA interactions can contribute to the proliferation of MF/SS tumor cells in both a autocrine and paracrine manner, and that the disruption of CD147-CypA interactions could be a new therapeutic strategy for the treatment of MF/SS.


Assuntos
Basigina/metabolismo , Proliferação de Células , Ciclofilina A/metabolismo , Linfoma Cutâneo de Células T/patologia , Micose Fungoide/patologia , Síndrome de Sézary/patologia , Neoplasias Cutâneas/patologia , Basigina/genética , Estudos de Casos e Controles , Ciclofilina A/genética , Feminino , Humanos , Linfoma Cutâneo de Células T/genética , Linfoma Cutâneo de Células T/metabolismo , Masculino , Pessoa de Meia-Idade , Micose Fungoide/genética , Micose Fungoide/metabolismo , Índice de Gravidade de Doença , Síndrome de Sézary/genética , Síndrome de Sézary/metabolismo , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/metabolismo
4.
Sci Rep ; 11(1): 12001, 2021 06 07.
Artigo em Inglês | MEDLINE | ID: mdl-34099817

RESUMO

Staphylococcus epidermidis (S. epidermidis) ATCC 12228 was incubated with 2% polyethylene glycol (PEG)-8 Laurate to yield electricity which was measured by a voltage difference between electrodes. Production of electron was validated by a Ferrozine assay. The anti-Cutibacterium acnes (C. acnes) activity of electrogenic S. epidermidis was assessed in vitro and in vivo. The voltage change (~ 4.4 mV) reached a peak 60 min after pipetting S. epidermidis plus 2% PEG-8 Laurate onto anodes. The electricity produced by S. epidermidis caused significant growth attenuation and cell lysis of C. acnes. Intradermal injection of C. acnes and S. epidermidis plus PEG-8 Laurate into the mouse ear considerably suppressed the growth of C. acnes. This suppressive effect was noticeably reversed when cyclophilin A of S. epidermidis was inhibited, indicating the essential role of cyclophilin A in electricity production of S. epidermidis against C. acnes. In summary, we demonstrate for the first time that skin S. epidermidis, in the presence of PEG-8 Laurate, can mediate cyclophilin A to elicit an electrical current that has anti-C. acnes effects. Electricity generated by S. epidermidis may confer immediate innate immunity in acne lesions to rein in the overgrowth of C. acnes at the onset of acne vulgaris.


Assuntos
Acne Vulgar/terapia , Antibiose/genética , Proteínas de Bactérias/genética , Ciclofilina A/genética , Propionibacteriaceae/patogenicidade , Staphylococcus epidermidis/efeitos dos fármacos , Acne Vulgar/microbiologia , Animais , Proteínas de Bactérias/metabolismo , Técnicas de Cocultura , Meios de Cultura/química , Meios de Cultura/farmacologia , Ciclofilina A/metabolismo , Modelos Animais de Doenças , Orelha/microbiologia , Eletricidade , Eletrodos , Feminino , Expressão Gênica , Lauratos/farmacologia , Camundongos , Camundongos Endogâmicos ICR , Polietilenoglicóis/farmacologia , Propionibacteriaceae/crescimento & desenvolvimento , Pele/microbiologia , Staphylococcus epidermidis/fisiologia , Tensoativos/farmacologia
5.
J Med Chem ; 64(12): 8272-8286, 2021 06 24.
Artigo em Inglês | MEDLINE | ID: mdl-34096287

RESUMO

A macrocyclic peptide scaffold with well-established structure-property relationship is desirable for tackling undruggable targets. Here, we adopted a natural macrocycle, cyclosporin O (CsO) and its derivatives (CP1-3), and evaluated the impact of conformation on membrane permeability, cyclophilin A (CypA) binding, and the pharmacokinetic (PK) profile. In nonpolar media, CsO showed a similar conformation to cyclosporin A (CsA), a well-known chameleonic macrocycle, but less chameleonic behavior in a polar environment. The weak chameleonicity of CsO resulted in decreased membrane permeability; however, the more rigid conformation of CsO was not detrimental to its PK profile. CsO exhibited a higher plasma concentration than CsA, which resulted from minimal CypA binding and lower accumulation in red blood cells and moderate oral bioavailability (F = 12%). Our study aids understanding of CsO, a macrocyclic peptide that is less explored than CsA but with greater potential for diversity generation and rational design.


Assuntos
Ciclofilina A/metabolismo , Ciclosporinas/metabolismo , Animais , Células CACO-2 , Permeabilidade da Membrana Celular/efeitos dos fármacos , Ciclização , Ciclofilina A/química , Ciclosporina/síntese química , Ciclosporina/metabolismo , Ciclosporina/farmacocinética , Ciclosporinas/síntese química , Ciclosporinas/farmacocinética , Desenho de Fármacos , Humanos , Ligação de Hidrogênio , Masculino , Camundongos Endogâmicos ICR , Conformação Molecular , Ligação Proteica
6.
Int J Mol Sci ; 22(9)2021 May 06.
Artigo em Inglês | MEDLINE | ID: mdl-34066541

RESUMO

Breast cancer is one of the major causes of deaths due to cancer, especially in women. The crucial barrier for breast cancer treatment is resistance to radiation therapy, one of the important local regional therapies. We previously established and characterized radio-resistant MDA-MB-231 breast cancer cells (RT-R-MDA-MB-231 cells) that harbor a high expression of cancer stem cells (CSCs) and the EMT phenotype. In this study, we performed antibody array analysis to identify the hub signaling mechanism for the radiation resistance of RT-R-MDA-MB-231 cells by comparing parental MDA-MB-231 (p-MDA-MB-231) and RT-R-MDA-MB-231 cells. Antibody array analysis unveiled that the MAPK1 protein was the most upregulated protein in RT-R-MDA-MB-231 cells compared to in p-MDA-MB-231 cells. The pathway enrichment analysis also revealed the presence of MAPK1 in almost all enriched pathways. Thus, we used an MEK/ERK inhibitor, PD98059, to block the MEK/ERK pathway and to identify the role of MAPK1 in the radio-resistance of RT-R-MDA-MB-231 cells. MEK/ERK inhibition induced cell death in both p-MDA-MB-231 and RT-R-MDA-MB-231 cells, but the death mechanism for each cell was different; p-MDA-MB-231 cells underwent apoptosis, showing cell shrinkage and PARP-1 cleavage, while RT-R-MDA-MB-231 cells underwent necroptosis, showing mitochondrial dissipation, nuclear swelling, and an increase in the expressions of CypA and AIF. In addition, MEK/ERK inhibition reversed the radio-resistance of RT-R-MDA-MB-231 cells and suppressed the increased expression of CSC markers (CD44 and OCT3/4) and the EMT phenotype (ß-catenin and N-cadherin/E-cadherin). Taken together, this study suggests that activated ERK signaling is one of the major hub signals related to the radio-resistance of MDA-MB-231 breast cancer cells.


Assuntos
Neoplasias da Mama/enzimologia , Neoplasias da Mama/radioterapia , Sistema de Sinalização das MAP Quinases , Tolerância a Radiação , Apoptose/efeitos dos fármacos , Fator de Indução de Apoptose/metabolismo , Biomarcadores Tumorais/metabolismo , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Forma Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Clonais , Ciclofilina A/metabolismo , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Feminino , Humanos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Necroptose/efeitos dos fármacos , Células-Tronco Neoplásicas/efeitos dos fármacos , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Fenótipo , Poli(ADP-Ribose) Polimerases/metabolismo , Mapas de Interação de Proteínas/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Proteômica , Tolerância a Radiação/efeitos dos fármacos
7.
J Virol ; 95(15): e0056321, 2021 07 12.
Artigo em Inglês | MEDLINE | ID: mdl-34011546

RESUMO

Human respiratory syncytial virus (hRSV) is the most common pathogen which causes acute lower respiratory infection (ALRI) in infants. Recently, virus-host interaction has become a hot spot of virus-related research, and it needs to be further elaborated for RSV infection. In this study, we found that RSV infection significantly increased the expression of cyclophilin A (cypA) in clinical patients, mice, and epithelial cells. Therefore, we evaluated the function of cypA in RSV replication and demonstrated that virus proliferation was accelerated in cypA knockdown host cells but restrained in cypA-overexpressing host cells. Furthermore, we proved that cypA limited RSV replication depending on its PPIase activity. Moreover, we performed liquid chromatography-mass spectrometry, and the results showed that cypA could interact with several viral proteins, such as RSV-N, RSV-P, and RSV-M2-1. Finally, the interaction between cypA and RSV-N was certified by coimmunoprecipitation and immunofluorescence. Those results provided strong evidence that cypA may play an inhibitory role in RSV replication through interaction with RSV-N via its PPIase activity. IMPORTANCE RSV-N, packed in the viral genome to form the ribonucleoprotein (RNP) complex, which is recognized by the RSV RNA-dependent RNA polymerase (RdRp) complex to initiate viral replication and transcription, plays an indispensable role in the viral biosynthesis process. cypA, binding to RSV-N, may impair this function by weakening the interaction between RSV-N and RSV-P, thus leading to decreased viral production. Our research provides novel insight into cypA antiviral function, including binding to viral capsid protein to inhibit viral replication, which may be helpful for new antiviral drug exploration.


Assuntos
Ciclofilina A/genética , Ciclofilina A/metabolismo , Peptidilprolil Isomerase/metabolismo , Vírus Sincicial Respiratório Humano/crescimento & desenvolvimento , Replicação Viral/fisiologia , Animais , Linhagem Celular , Chlorocebus aethiops , Feminino , Humanos , Espectrometria de Massas , Camundongos , Camundongos Endogâmicos BALB C , Interferência de RNA , RNA Interferente Pequeno/genética , Infecções por Vírus Respiratório Sincicial/patologia , Ribonucleoproteínas/metabolismo , Células Vero , Proteínas não Estruturais Virais/metabolismo , Proteínas Virais/metabolismo
8.
Int J Mol Sci ; 22(5)2021 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-33804393

RESUMO

We recently discovered a novel nargenicin A1 analog, 23-demethyl 8,13-deoxynargenicin (compound 9), with potential anti-cancer and anti-angiogenic activities against human gastric adenocarcinoma (AGS) cells. To identify the key molecular targets of compound 9, that are responsible for its biological activities, the changes in proteome expression in AGS cells following compound 9 treatment were analyzed using two-dimensional gel electrophoresis (2-DE), followed by MALDI/TOF/MS. Analyses using chemical proteomics and western blotting revealed that compound 9 treatment significantly suppressed the expression of cyclophilin A (CypA), a member of the immunophilin family. Furthermore, compound 9 downregulated CD147-mediated mitogen-activated protein kinase (MAPK) signaling pathway, including c-Jun N-terminal kinase (JNK) and extracellular signal-regulated protein kinase 1/2 (ERK1/2) by inhibiting the expression of CD147, the cellular receptor of CypA. Notably, the responses of AGS cells to CypA knockdown were significantly correlated with the anticancer and antiangiogenic effects of compound 9. CypA siRNAs reduced the expression of CD147 and phosphorylation of JNK and ERK1/2. In addition, the suppressive effects of CypA siRNAs on proliferation, migration, invasion, and angiogenesis induction of AGS cells were associated with G2/M cell cycle arrest, caspase-mediated apoptosis, inhibition of MMP-9 and MMP-2 expression, inactivation of PI3K/AKT/mTOR pathway, and inhibition of hypoxia-inducible factor-1α (HIF-1α) and vascular endothelial growth factor (VEGF) expression. The specific interaction between compound 9 and CypA was also confirmed using the drug affinity responsive target stability (DARTS) and cellular thermal shift assay (CETSA) approaches. Moreover, in silico docking analysis revealed that the structure of compound 9 was a good fit for the cyclosporin A binding cavity of CypA. Collectively, these findings provide a novel molecular basis for compound 9-mediated suppression of gastric cancer progression through the targeting of CypA.


Assuntos
Biomarcadores Tumorais/metabolismo , Ciclofilina A/metabolismo , Proteoma/análise , Proteoma/efeitos dos fármacos , Neoplasias Gástricas/tratamento farmacológico , Apoptose , Ciclo Celular , Proliferação de Células , Humanos , Lactonas/química , Lactonas/farmacologia , Nocardia/metabolismo , Proteoma/metabolismo , Transdução de Sinais , Neoplasias Gástricas/metabolismo , Neoplasias Gástricas/patologia , Células Tumorais Cultivadas
9.
mBio ; 12(2)2021 03 23.
Artigo em Inglês | MEDLINE | ID: mdl-33758083

RESUMO

Human immunodeficiency virus type 1 (HIV-1) capsid binds host proteins during infection, including cleavage and polyadenylation specificity factor 6 (CPSF6) and cyclophilin A (CypA). We observe that HIV-1 infection induces higher-order CPSF6 formation, and capsid-CPSF6 complexes cotraffic on microtubules. CPSF6-capsid complex trafficking is impacted by capsid alterations that reduce CPSF6 binding or by excess cytoplasmic CPSF6 expression, both of which are associated with decreased HIV-1 infection. Higher-order CPSF6 complexes bind and disrupt HIV-1 capsid assemblies in vitro Disruption of HIV-1 capsid binding to CypA leads to increased CPSF6 binding and altered capsid trafficking, resulting in reduced infectivity. Our data reveal an interplay between CPSF6 and CypA that is important for cytoplasmic capsid trafficking and HIV-1 infection. We propose that CypA prevents HIV-1 capsid from prematurely engaging cytoplasmic CPSF6 and that differences in CypA cellular localization and innate immunity may explain variations in HIV-1 capsid trafficking and uncoating in CD4+ T cells and macrophages.IMPORTANCE HIV is the causative agent of AIDS, which has no cure. The protein shell that encases the viral genome, the capsid, is critical for HIV replication in cells at multiple steps. HIV capsid has been shown to interact with multiple cell proteins during movement to the cell nucleus in a poorly understood process that may differ during infection of different cell types. In this study, we show that premature or too much binding of one human protein, cleavage and polyadenylation specificity factor 6 (CPSF6), disrupts the ability of the capsid to deliver the viral genome to the cell nucleus. Another human protein, cyclophilin A (CypA), can shield HIV capsid from premature binding to CPSF6, which can differ in CD4+ T cells and macrophages. Better understanding of how HIV infects cells will allow better drugs to prevent or inhibit infection and pathogenesis.


Assuntos
Proteínas do Capsídeo/genética , Capsídeo/fisiologia , Ciclofilina A/metabolismo , HIV-1/fisiologia , Interações Hospedeiro-Patógeno , Fatores de Poliadenilação e Clivagem de mRNA/genética , Linfócitos T CD4-Positivos/virologia , Proteínas do Capsídeo/metabolismo , Núcleo Celular/metabolismo , Núcleo Celular/virologia , Células HEK293 , Células HeLa , Humanos , Imunidade Inata , Macrófagos/virologia , Replicação Viral
10.
FASEB J ; 35(4): e21479, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33710680

RESUMO

Triggering receptor expressed on myeloid cells 2 (TREM2) is a cell surface receptor expressed on macrophages, microglial cells, and pre-osteoclasts, and that participates in diverse cellular function, including inflammation, bone homeostasis, neurological development, and coagulation. In spite of the indispensable role of the TREM2 protein in the maintenance of immune homeostasis and osteoclast differentiation, the exact ligand for TREM2 has not yet been identified. Here, we report a putative TREM2 ligand which is secreted from MC38 cells and identified as a cyclophilin A (CypA). A specific interaction between CypA and TREM2 was shown at both protein and cellular levels. Exogenous CypA specifically interacted and co-localized with TREM2 in RAW264.7 cells, and the physical interactions were shown to regulate TREM2 signaling transduction. The Pro144 residue in the extracellular domain of TREM2 was found to be the specific binding site of CypA. When considered together, this provides evidence that CypA interacts specifically with TREM2 as a potent ligand.


Assuntos
Ciclofilina A/metabolismo , Ligantes , Microglia/metabolismo , Células Mieloides/metabolismo , Animais , Proteínas de Transporte/metabolismo , Células Cultivadas , Humanos , Macrófagos/metabolismo , Osteoclastos/metabolismo
11.
J Exp Med ; 218(4)2021 04 05.
Artigo em Inglês | MEDLINE | ID: mdl-33533918

RESUMO

The low-density lipoprotein receptor-related protein 1 (LRP1) is an endocytic and cell signaling transmembrane protein. Endothelial LRP1 clears proteinaceous toxins at the blood-brain barrier (BBB), regulates angiogenesis, and is increasingly reduced in Alzheimer's disease associated with BBB breakdown and neurodegeneration. Whether loss of endothelial LRP1 plays a direct causative role in BBB breakdown and neurodegenerative changes remains elusive. Here, we show that LRP1 inactivation from the mouse endothelium results in progressive BBB breakdown, followed by neuron loss and cognitive deficits, which is reversible by endothelial-specific LRP1 gene therapy. LRP1 endothelial knockout led to a self-autonomous activation of the cyclophilin A-matrix metalloproteinase-9 pathway in the endothelium, causing loss of tight junctions underlying structural BBB impairment. Cyclophilin A inhibition in mice with endothelial-specific LRP1 knockout restored BBB integrity and reversed and prevented neuronal loss and behavioral deficits. Thus, endothelial LRP1 protects against neurodegeneration by inhibiting cyclophilin A, which has implications for the pathophysiology and treatment of neurodegeneration linked to vascular dysfunction.


Assuntos
Doença de Alzheimer/metabolismo , Barreira Hematoencefálica/metabolismo , Ciclofilina A/metabolismo , Células Endoteliais/metabolismo , Proteína-1 Relacionada a Receptor de Lipoproteína de Baixa Densidade/metabolismo , Transdução de Sinais/genética , Doença de Alzheimer/terapia , Animais , Células Cultivadas , Disfunção Cognitiva/genética , Disfunção Cognitiva/metabolismo , Ciclofilina A/antagonistas & inibidores , Ciclosporina/farmacologia , Modelos Animais de Doenças , Inibidores Enzimáticos/farmacologia , Feminino , Técnicas de Inativação de Genes , Terapia Genética/métodos , Proteína-1 Relacionada a Receptor de Lipoproteína de Baixa Densidade/genética , Masculino , Metaloproteinase 9 da Matriz/metabolismo , Camundongos , Camundongos Transgênicos , Neurônios/metabolismo , Transdução de Sinais/efeitos dos fármacos
12.
J Biol Chem ; 296: 100045, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33465707

RESUMO

The mammalian apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like 3 (APOBEC3 or A3) family of cytidine deaminases restrict viral infections by mutating viral DNA and impeding reverse transcription. To overcome this antiviral activity, most lentiviruses express a viral accessory protein called the virion infectivity factor (Vif), which recruits A3 proteins to cullin-RING E3 ubiquitin ligases such as cullin-5 (Cul5) for ubiquitylation and subsequent proteasomal degradation. Although Vif proteins from primate lentiviruses such as HIV-1 utilize the transcription factor core-binding factor subunit beta as a noncanonical cofactor to stabilize the complex, the maedi-visna virus (MVV) Vif hijacks cyclophilin A (CypA) instead. Because core-binding factor subunit beta and CypA are both highly conserved among mammals, the requirement for two different cellular cofactors suggests that these two A3-targeting Vif proteins have different biochemical and structural properties. To investigate this topic, we used a combination of in vitro biochemical assays and in vivo A3 degradation assays to study motifs required for the MVV Vif to bind zinc ion, Cul5, and the cofactor CypA. Our results demonstrate that although some common motifs between the HIV-1 Vif and MVV Vif are involved in recruiting Cul5, different determinants in the MVV Vif are required for cofactor binding and stabilization of the E3 ligase complex, such as the zinc-binding motif and N- and C-terminal regions of the protein. Results from this study advance our understanding of the mechanism of MVV Vif recruitment of cellular factors and the evolution of lentiviral Vif proteins.


Assuntos
Vírus Visna-Maedi/metabolismo , Produtos do Gene vif do Vírus da Imunodeficiência Humana/metabolismo , Sequência de Aminoácidos , Proteínas Culina/metabolismo , Ciclofilina A/metabolismo , Ligação Proteica , Domínios Proteicos , Proteólise , Zinco/metabolismo , Produtos do Gene vif do Vírus da Imunodeficiência Humana/química
13.
Arterioscler Thromb Vasc Biol ; 41(3): 1205-1217, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33472404

RESUMO

OBJECTIVE: Pulmonary arterial hypertension is characterized by abnormal proliferation of pulmonary artery smooth muscle cells and vascular remodeling, which leads to right ventricular (RV) failure. Bsg (Basigin) is a transmembrane glycoprotein that promotes myofibroblast differentiation, cell proliferation, and matrix metalloproteinase activation. CyPA (cyclophilin A) binds to its receptor Bsg and promotes pulmonary artery smooth muscle cell proliferation and inflammatory cell recruitment. We previously reported that Bsg promotes cardiac fibrosis and failure in the left ventricle in response to pressure-overload in mice. However, the roles of Bsg and CyPA in RV failure remain to be elucidated. Approach and Results: First, we found that protein levels of Bsg and CyPA were upregulated in the heart of hypoxia-induced pulmonary hypertension (PH) in mice and monocrotaline-induced PH in rats. Furthermore, cardiomyocyte-specific Bsg-overexpressing mice showed exacerbated RV hypertrophy, fibrosis, and dysfunction compared with their littermates under chronic hypoxia and pulmonary artery banding. Treatment with celastrol, which we identified as a suppressor of Bsg and CyPA by drug screening, decreased proliferation, reactive oxygen species, and inflammatory cytokines in pulmonary artery smooth muscle cells. Furthermore, celastrol treatment ameliorated RV systolic pressure, hypertrophy, fibrosis, and dysfunction in hypoxia-induced PH in mice and SU5416/hypoxia-induced PH in rats with reduced Bsg, CyPA, and inflammatory cytokines in the hearts and lungs. CONCLUSIONS: These results indicate that elevated Bsg in pressure-overloaded RV exacerbates RV dysfunction and that celastrol ameliorates RV dysfunction in PH model animals by suppressing Bsg and its ligand CyPA. Thus, celastrol can be a novel drug for PH and RV failure that targets Bsg and CyPA. Graphic Abstract: A graphic abstract is available for this article.


Assuntos
Basigina/antagonistas & inibidores , Ciclofilina A/antagonistas & inibidores , Hipertensão Arterial Pulmonar/tratamento farmacológico , Triterpenos/uso terapêutico , Disfunção Ventricular Direita/tratamento farmacológico , Animais , Anti-Hipertensivos/uso terapêutico , Basigina/genética , Basigina/metabolismo , Ciclofilina A/metabolismo , Modelos Animais de Doenças , Humanos , Hipóxia/complicações , Indóis/toxicidade , Camundongos , Camundongos Transgênicos , Miócitos Cardíacos/metabolismo , Hipertensão Arterial Pulmonar/patologia , Hipertensão Arterial Pulmonar/fisiopatologia , Pirróis/toxicidade , Ratos , Disfunção Ventricular Direita/patologia , Disfunção Ventricular Direita/fisiopatologia
14.
Methods Mol Biol ; 2253: 153-174, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33315223

RESUMO

PyInteraph is a software package designed for the analysis of structural communication from conformational ensembles, such as those derived from in silico simulations, under the formalism of protein structure networks. We demonstrate its usage for the calculation and analysis of intramolecular interaction networks derived from three different types of interactions, as well as with a more general protocol based on distances between centers of mass. We use the xPyder PyMOL plug-in to visualize such networks on the three-dimensional structure of the protein. We showcase our protocol on a molecular dynamics trajectory of the Cyclophilin A wild-type enzyme, a well-studied protein in which different allosteric mechanisms have been investigated.


Assuntos
Biologia Computacional/métodos , Ciclofilina A/química , Ciclofilina A/metabolismo , Algoritmos , Regulação Alostérica , Simulação de Dinâmica Molecular , Ligação Proteica , Conformação Proteica , Mapas de Interação de Proteínas , Fluxo de Trabalho
15.
J Cell Sci ; 134(3)2021 02 08.
Artigo em Inglês | MEDLINE | ID: mdl-33361281

RESUMO

Cyclophilin A (CyPA, also known as PPIA) is an abundant and ubiquitously expressed protein belonging to the immunophilin family, which has intrinsic peptidyl-prolyl-(cis/trans)-isomerase enzymatic activity. CyPA mediates immunosuppressive action of the cyclic undecapeptide cyclosporine A and is also involved in multiple cellular processes, such as protein folding, intracellular trafficking, signal transduction and transcriptional regulation. CyPA is abundantly expressed in cancer cells, and, owing to its chaperone nature, its expression is induced upon the onset of stress. In this study, we demonstrated that a significant pool of this immunophilin is primarily an intramitochondrial factor that migrates to the nucleus when cells are stimulated with stressors. CyPA shows anti-apoptotic action per se and the capability of forming ternary complexes with cytochrome c and the small acidic co-chaperone p23, the latter interaction being independent of the usual association of p23 with the heat-shock protein of 90 kDa, Hsp90. These CyPA•p23 complexes enhance the anti-apoptotic response of the cell, suggesting that both proteins form a functional unit, the high level of expression of which plays a significant role in cell survival.


Assuntos
Apoptose , Ciclofilina A , Ciclosporina , Células 3T3 , Animais , Proteínas de Transporte , Ciclofilina A/genética , Ciclofilina A/metabolismo , Células HeLa , Humanos , Camundongos , Peptidilprolil Isomerase , Dobramento de Proteína , Ratos
16.
Sci Rep ; 10(1): 18938, 2020 11 03.
Artigo em Inglês | MEDLINE | ID: mdl-33144587

RESUMO

Testicular cancer (TC) is the most common solid tumour in young men. While cisplatin-based chemotherapy is highly effective in TC patients, chemoresistance still accounts for 10% of disease-related deaths. Pre-clinical models that faithfully reflect patient tumours are needed to assist in target discovery and drug development. Tumour pieces from eight TC patients were subcutaneously implanted in NOD scid gamma (NSG) mice. Three patient-derived xenograft (PDX) models of TC, including one chemoresistant model, were established containing yolk sac tumour and teratoma components. PDX models and corresponding patient tumours were characterised by H&E, Ki-67 and cyclophilin A immunohistochemistry, showing retention of histological subtypes over several passages. Whole-exome sequencing, copy number variation analysis and RNA-sequencing was performed on these TP53 wild type PDX tumours to assess the effects of passaging, showing high concordance of molecular features between passages. Cisplatin sensitivity of PDX models corresponded with patients' response to cisplatin-based chemotherapy. MDM2 and mTORC1/2 targeted drugs showed efficacy in the cisplatin sensitive PDX models. In conclusion, we describe three PDX models faithfully reflecting chemosensitivity of TC patients. These models can be used for mechanistic studies and pre-clinical validation of novel therapeutic strategies in testicular cancer.


Assuntos
Neoplasias Testiculares/metabolismo , Ciclofilina A/genética , Ciclofilina A/metabolismo , Variações do Número de Cópias de DNA/genética , Genótipo , Humanos , Imuno-Histoquímica , Antígeno Ki-67/genética , Antígeno Ki-67/metabolismo , Masculino , Alvo Mecanístico do Complexo 1 de Rapamicina/genética , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Alvo Mecanístico do Complexo 2 de Rapamicina/genética , Alvo Mecanístico do Complexo 2 de Rapamicina/metabolismo , Mutação/genética , Neoplasias Embrionárias de Células Germinativas/genética , Neoplasias Embrionárias de Células Germinativas/metabolismo , Polimorfismo de Nucleotídeo Único/genética , Proteínas Proto-Oncogênicas c-mdm2/genética , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Análise de Sequência de RNA/métodos , Neoplasias Testiculares/genética , Sequenciamento Completo do Exoma/métodos
17.
Nucleic Acids Res ; 48(21): 12151-12168, 2020 12 02.
Artigo em Inglês | MEDLINE | ID: mdl-33231641

RESUMO

Histones are substrates of the SUMO (small ubiquitin-like modifier) conjugation pathway. Several reports suggest histone sumoylation affects transcription negatively, but paradoxically, our genome-wide analysis shows the modification concentrated at many active genes. We find that trans-tail regulation of histone-H2B ubiquitylation and H3K4 di-methylation potentiates subsequent histone sumoylation. Consistent with the known control of the Set3 histone deacetylase complex (HDAC) by H3K4 di-methylation, histone sumoylation directly recruits the Set3 complex to both protein-coding and noncoding RNA (ncRNA) genes via a SUMO-interacting motif in the HDAC Cpr1 subunit. The altered gene expression profile caused by reducing histone sumoylation matches well to the profile in cells lacking Set3. Histone H2B sumoylation and the Set3 HDAC coordinately suppress cryptic ncRNA transcription initiation internal to mRNA genes. Our results reveal an elaborate co-transcriptional histone crosstalk pathway involving the consecutive ubiquitylation, methylation, sumoylation and deacetylation of histones, which maintains transcriptional fidelity by suppressing spurious transcription.


Assuntos
Histona Desacetilases/genética , Histonas/genética , Processamento de Proteína Pós-Traducional , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Transcrição Genética , Acetilação , Ciclofilina A/genética , Ciclofilina A/metabolismo , Regulação Fúngica da Expressão Gênica , Histona Desacetilases/metabolismo , Histonas/metabolismo , Metilação , RNA/genética , RNA/metabolismo , RNA não Traduzido/genética , RNA não Traduzido/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Sumoilação , Ubiquitina/genética , Ubiquitina/metabolismo , Ubiquitinação
18.
Nat Commun ; 11(1): 6046, 2020 11 27.
Artigo em Inglês | MEDLINE | ID: mdl-33247146

RESUMO

Catalysis of cis/trans isomerization of prolines is important for the activity and misfolding of intrinsically disordered proteins. Catalysis is achieved by peptidylprolyl isomerases, a superfamily of molecular chaperones. Here, we provide atomic insight into a tug-of-war between cis/trans isomerization and molecular chaperone activity. Catalysis of proline isomerization by cyclophilin A lowers the energy barrier for α-synuclein misfolding, while isomerase-binding to a separate, disease-associated protein region opposes aggregation. We further show that cis/trans isomerization outpowers the holding activity of cyclophilin A. Removal of the proline isomerization barrier through posttranslational truncation of α-synuclein reverses the action of the proline isomerase and turns it into a potent molecular chaperone that inhibits protein misfolding. The data reveal a conserved mechanism of dual functionality in cis/trans isomerases and define its molecular determinants acting on intrinsically disordered proteins.


Assuntos
Chaperonas Moleculares/metabolismo , Prolina/química , Prolina/metabolismo , Amiloide/química , Catálise , Ciclofilina A/química , Ciclofilina A/metabolismo , Ciclosporina/farmacologia , Humanos , Isomerismo , Cinética , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Doença de Parkinson/metabolismo , Agregados Proteicos , Ligação Proteica , Domínios Proteicos , alfa-Sinucleína/química , alfa-Sinucleína/metabolismo
19.
Int Heart J ; 61(6): 1129-1134, 2020 Nov 28.
Artigo em Inglês | MEDLINE | ID: mdl-33191348

RESUMO

Patients with impaired kidney function have a high frequency of intraplaque hemorrhage (IPH) in their coronary arteries. Levels of cyclophilin A (CyPA), an indirect matrix metalloproteinase inducer, are increased in deceased patients who had impaired kidney function. In this study, we have examined the relationship between IPH and CyPA.We examined 47 samples of coronary plaque from 27 cadavers with coronary stenosis. These sections, all with > 50% coronary stenosis, were stained with an antibody against CyPA and the expression of CyPA was semi-quantified. Cadavers and plaques were classified into one of two groups depending on the presence or absence of IPH. IPH was defined as the presence of red blood cells stained with hematoxylin and eosin (HE) indicative of overt acute hemorrhage.In an individual analysis, estimation of glomerular filtration rate (eGFR) in the IPH group was significantly lower than that in the non-IPH group (P = 0.002). In a histological analysis, the percentage of stained area of CyPA in the IPH group was significantly higher than that in the non-IPH group (P < 0.0001).IPH was associated with a significantly higher expression of CyPA in this study. In addition, patients with IPH in their coronary arteries had significantly impaired kidney function.


Assuntos
Estenose Coronária/metabolismo , Ciclofilina A/metabolismo , Hemorragia/metabolismo , Placa Aterosclerótica/metabolismo , Insuficiência Renal/metabolismo , Idoso , Idoso de 80 Anos ou mais , Cadáver , Estenose Coronária/complicações , Estenose Coronária/patologia , Vasos Coronários/metabolismo , Vasos Coronários/patologia , Feminino , Taxa de Filtração Glomerular , Hemorragia/complicações , Hemorragia/patologia , Humanos , Imuno-Histoquímica , Masculino , Pessoa de Meia-Idade , Placa Aterosclerótica/complicações , Placa Aterosclerótica/patologia , Insuficiência Renal/complicações
20.
Front Immunol ; 11: 2052, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33013867

RESUMO

Mycoplasma genitalium protein of adhesion (MgPa) plays an important role in the process of adhesion and invasion of host cells by M. genitalium, and is thus significant for its pathogenic mechanisms in host cells. Our previous study has demonstrated that cyclophilin A (CypA) is the receptor for MgPa in human urothelial cells (SV-HUC-1) and can, therefore, mediate the adherence and invasion of M. genitalium into host cells by interacting with MgPa. However, the specific pathogenesis of M. genitalium to host cells and the possible pathogenic mechanism involved in the interaction of MgPa and CypA have never been clarified. The study aimed to elucidate the mechanism involved in the pathogenicity of MgPa. Recombinant MgPa (rMgPa) induced extracellular CypA (eCypA) was detected in SV-HUC-1 cells by ELISA, and the interaction between CypA and CD147 was validated using co-localization and co-immunoprecipitation assay. In addition, both extracellular signal-regulated kinases (ERK) phosphorylation and NF-κB activation evoked by rMgPa-induced eCypA were also demonstrated. The findings of this study verified that rMgPa could induce the secretion of eCypA in SV-HUC-1 cells and thus promote the protein and mRNA expression of IL-1ß, IL-6, TNF-α and MMP-9 via CypA-CD147 interaction and thus activating ERK-NF-κB pathway, which is beneficial to elucidate the pathogenesis and possible pathogenic mechanism of M. genitalium to host cells.


Assuntos
Adesinas Bacterianas/metabolismo , Basigina/metabolismo , Ciclofilina A/metabolismo , Infecções por Mycoplasma/imunologia , Mycoplasma genitalium/fisiologia , NF-kappa B/metabolismo , Urotélio/metabolismo , Linhagem Celular , Citocinas , Interações Hospedeiro-Patógeno , Humanos , Mediadores da Inflamação/metabolismo , Sistema de Sinalização das MAP Quinases , Metaloproteinase 9 da Matriz/metabolismo , Doenças Sexualmente Transmissíveis , Urotélio/citologia
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