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1.
Zhongguo Shi Yan Xue Ye Xue Za Zhi ; 29(5): 1522-1527, 2021 Oct.
Artigo em Chinês | MEDLINE | ID: mdl-34627434

RESUMO

OBJECTIVE: To investigate the expression of cell division cycle protein 37 (Cdc37) in multiple myeloma (MM) and its effect on MM cell proliferation. METHODS: The expression of Cdc37 mRNA in CD138+ cells derived from 63 newly diagnosed MM patients and 8 healthy people were detected by real-time quantitative PCR (RT-qPCR). Cdc37 was down-regulated by lentivirus in MM cell line NCI-H929. CCK-8 assay and soft agar clone formation assay were conducted to explore the role of Cdc37 on MM proliferation in vitro. To further verify the effect of Cdc37 on MM cell proliferation in vivo, NOD/SCID mice subcutaneous tumorigenesis model was established. Flow cytometry was carried out to explore the role of Cdc37 on cell cycle. Cell cycle associated proteins and NF-κB pathway were detected by Western blot (WB). RESULTS: Cdc37 was highly expressed in newly diagnosed CD138+cells compared with healthy people. After Cdc37 suppression by shRNA lentivirus infection in NCI-H929 cells, the proliferation of MM cells were decreased in vitro and in vivo. Compared with the control group, the ratio of cells arrested in G0/G1 phase significantly increased in NCI-H929-Cdc37 shRNA cells, the expression of cyclin D1 decreased, while the expression of p21 and p53 was significantly up-regulated. Meanwhile, the activation of NF-κB signaling pathway was hampered in NCI-H929-Cdc37 shRNA cells. CONCLUSION: Cdc37 is highly expressed in newly diagnosed MM patients. Inhibition of Cdc37 results in decreased proliferation activity and G0/G1 arrest in NCI-H929 cells. The possible mechanism may be to inhibit the activation of NF-κB signaling pathway.


Assuntos
Mieloma Múltiplo , Animais , Apoptose , Proteínas de Ciclo Celular , Proliferação de Células , Chaperoninas , Humanos , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID
2.
PLoS Genet ; 17(10): e1009748, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34669693

RESUMO

Antibiotic resistance has become a major global issue. Understanding the molecular mechanisms underlying microbial adaptation to antibiotics is of keen importance to fight Antimicrobial Resistance (AMR). Aminoglycosides are a class of antibiotics that target the small subunit of the bacterial ribosome, disrupting translational fidelity and increasing the levels of misfolded proteins in the cell. In this work, we investigated the role of VchM, a DNA methyltransferase, in the response of the human pathogen Vibrio cholerae to aminoglycosides. VchM is a V. cholerae specific orphan m5C DNA methyltransferase that generates cytosine methylation at 5'-RCCGGY-3' motifs. We show that deletion of vchM, although causing a growth defect in absence of stress, allows V. cholerae cells to cope with aminoglycoside stress at both sub-lethal and lethal concentrations of these antibiotics. Through transcriptomic and genetic approaches, we show that groESL-2 (a specific set of chaperonin-encoding genes located on the second chromosome of V. cholerae), are upregulated in cells lacking vchM and are needed for the tolerance of vchM mutant to lethal aminoglycoside treatment, likely by fighting aminoglycoside-induced misfolded proteins. Interestingly, preventing VchM methylation of the four RCCGGY sites located in groESL-2 region, leads to a higher expression of these genes in WT cells, showing that the expression of these chaperonins is modulated in V. cholerae by DNA methylation.


Assuntos
Aminoglicosídeos/genética , Chaperoninas/genética , Citosina/metabolismo , Metilação de DNA/genética , DNA/genética , Vibrio cholerae/genética , Proteínas de Bactérias/genética , Regulação Bacteriana da Expressão Gênica/genética , Metiltransferases/genética
3.
Nat Commun ; 12(1): 4754, 2021 08 06.
Artigo em Inglês | MEDLINE | ID: mdl-34362932

RESUMO

Chaperonins are homo- or hetero-oligomeric complexes that use ATP binding and hydrolysis to facilitate protein folding. ATP hydrolysis exhibits both positive and negative cooperativity. The mechanism by which chaperonins coordinate ATP utilization in their multiple subunits remains unclear. Here we use cryoEM to study ATP binding in the homo-oligomeric archaeal chaperonin from Methanococcus maripaludis (MmCpn), consisting of two stacked rings composed of eight identical subunits each. Using a series of image classification steps, we obtained different structural snapshots of individual chaperonins undergoing the nucleotide binding process. We identified nucleotide-bound and free states of individual subunits in each chaperonin, allowing us to determine the ATP occupancy state of each MmCpn particle. We observe distinctive tertiary and quaternary structures reflecting variations in nucleotide occupancy and subunit conformations in each chaperonin complex. Detailed analysis of the nucleotide distribution in each MmCpn complex indicates that individual ATP binding events occur in a statistically random manner for MmCpn, both within and across the rings. Our findings illustrate the power of cryoEM to characterize a biochemical property of multi-subunit ligand binding cooperativity at the individual particle level.


Assuntos
Trifosfato de Adenosina/metabolismo , Microscopia Crioeletrônica , Chaperoninas do Grupo II/química , Chaperoninas do Grupo II/metabolismo , Chaperoninas/metabolismo , Hidrólise , Mathanococcus/metabolismo , Modelos Moleculares , Conformação Proteica , Dobramento de Proteína , Subunidades Proteicas/metabolismo
4.
Am J Physiol Lung Cell Mol Physiol ; 321(5): L803-L813, 2021 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-34431396

RESUMO

Chaperonin 60.1 (Cpn60.1) is a protein derived from Mycobacterium tuberculosis that has been shown, along with its peptide fragment IRL201104, to have beneficial effects in models of allergic inflammation. To further investigate the anti-inflammatory properties of Cpn60.1 and IRL201104, we have investigated these molecules in a model of nonallergic lung inflammation. Mice were treated with Cpn60.1 (0.5-5,000 ng/kg) or IRL201104 (0.00025-2.5 ng/kg), immediately before intranasal instillation of bacterial lipopolysaccharide (LPS). Cytokine levels and cell numbers in mouse bronchoalveolar lavage (BAL) fluid were measured 4 h after LPS administration. In some experiments, mice were depleted of lung-resident phagocytes. Cells from BAL fluid were analyzed for inflammasome function. Human umbilical vein endothelial cells (HUVECs) were analyzed for adhesion molecule expression. Human neutrophils were analyzed for integrin expression, chemotaxis, and cell polarization. Cpn60.1 and IRL201104 significantly inhibited neutrophil migration into the airways, independently of route of administration. This effect of the peptide was absent in TLR4 and annexin A1 knockout mice. Intravital microscopy revealed that IRL201104 reduced leukocyte adhesion and migration into inflamed tissues. However, IRL201104 did not significantly affect adhesion molecule expression in HUVECs or integrin expression, chemotaxis, or polarization of human neutrophils at the studied concentrations. In phagocyte-depleted animals, the anti-inflammatory effect of IRL201104 was not significant. IRL201104 significantly reduced IL-1ß and NLRP3 expression and increased A20 expression in BAL cells. This study shows that Cpn60.1 and IRL201104 potently inhibit LPS-induced neutrophil infiltration in mouse lungs by a mechanism dependent on tissue-resident phagocytes and to a much lesser extent, the proresolving factor annexin A1.


Assuntos
Anti-Inflamatórios/farmacologia , Chaperonina 60/farmacologia , Chaperoninas/farmacologia , Infiltração de Neutrófilos/efeitos dos fármacos , Fragmentos de Peptídeos/farmacologia , Pneumonia/prevenção & controle , Animais , Anexina A1/genética , Líquido da Lavagem Broncoalveolar/química , Adesão Celular/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Células Cultivadas , Citocinas/análise , Feminino , Células Endoteliais da Veia Umbilical Humana , Humanos , Integrinas/biossíntese , Interleucina-1beta/biossíntese , Lipopolissacarídeos/toxicidade , Macrófagos/imunologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteína 3 que Contém Domínio de Pirina da Família NLR/biossíntese , Neutrófilos/imunologia , Receptor 4 Toll-Like/genética
5.
Biomolecules ; 11(6)2021 06 04.
Artigo em Inglês | MEDLINE | ID: mdl-34199986

RESUMO

The natural product elaiophylin is a macrodiolide with a broad range of biological activities. However, no direct target of elaiophylin in eukaryotes has been described so far, which hinders a systematic explanation of its astonishing activity range. We recently showed that the related conglobatin A, a protein-protein interface inhibitor of the interaction between the N-terminus of Hsp90 and its cochaperone Cdc37, blocks cancer stem cell properties by selectively inhibiting K-Ras4B but not H-Ras. Here, we elaborated that elaiophylin likewise disrupts the Hsp90/ Cdc37 interaction, without affecting the ATP-pocket of Hsp90. Similarly to conglobatin A, elaiophylin decreased expression levels of the Hsp90 client HIF1α, a transcription factor with various downstream targets, including galectin-3. Galectin-3 is a nanocluster scaffold of K-Ras, which explains the K-Ras selectivity of Hsp90 inhibitors. In agreement with this K-Ras targeting and the potent effect on other Hsp90 clients, we observed with elaiophylin treatment a submicromolar IC50 for MDA-MB-231 and MIA-PaCa-2 3D spheroid formation. Finally, a strong inhibition of MDA-MB-231 cells grown in the chorioallantoic membrane (CAM) microtumor model was determined. These results suggest that several other macrodiolides may have the Hsp90/ Cdc37 interface as a target site.


Assuntos
Proteínas de Ciclo Celular/antagonistas & inibidores , Chaperoninas/antagonistas & inibidores , Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Macrolídeos/farmacologia , Nanoconjugados , Proteínas Proto-Oncogênicas p21(ras)/antagonistas & inibidores , Animais , Proteínas de Ciclo Celular/metabolismo , Chaperoninas/metabolismo , Galinhas , Membrana Corioalantoide/efeitos dos fármacos , Membrana Corioalantoide/metabolismo , Células HEK293 , Proteínas de Choque Térmico HSP90/metabolismo , Humanos , Macrolídeos/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/metabolismo
6.
Sci Rep ; 11(1): 13084, 2021 06 22.
Artigo em Inglês | MEDLINE | ID: mdl-34158536

RESUMO

The eukaryotic chaperonin TRiC/CCT is a large ATP-dependent complex essential for cellular protein folding. Its subunit arrangement into two stacked eight-membered hetero-oligomeric rings is conserved from yeast to man. A recent breakthrough enables production of functional human TRiC (hTRiC) from insect cells. Here, we apply a suite of mass spectrometry techniques to characterize recombinant hTRiC. We find all subunits CCT1-8 are N-terminally processed by combinations of methionine excision and acetylation observed in native human TRiC. Dissociation by organic solvents yields primarily monomeric subunits with a small population of CCT dimers. Notably, some dimers feature non-canonical inter-subunit contacts absent in the initial hTRiC. This indicates individual CCT monomers can promiscuously re-assemble into dimers, and lack the information to assume the specific interface pairings in the holocomplex. CCT5 is consistently the most stable subunit and engages in the greatest number of non-canonical dimer pairings. These findings confirm physiologically relevant post-translational processing and function of recombinant hTRiC and offer quantitative insight into the relative stabilities of TRiC subunits and interfaces, a key step toward reconstructing its assembly mechanism. Our results also highlight the importance of assigning contacts identified by native mass spectrometry after solution dissociation as canonical or non-canonical when investigating multimeric assemblies.


Assuntos
Chaperonina com TCP-1/química , Chaperonina com TCP-1/metabolismo , Chaperoninas/química , Chaperoninas/metabolismo , Microscopia Crioeletrônica/métodos , Humanos , Espectrometria de Massas/métodos , Conformação Proteica , Dobramento de Proteína , Subunidades Proteicas/metabolismo
7.
Biochim Biophys Acta Mol Cell Res ; 1868(10): 119081, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34147560

RESUMO

The DYRK (Dual-specificity tYrosine-phosphorylation Regulated protein Kinase) family consists of five related protein kinases (DYRK1A, DYRK1B, DYRK2, DYRK3, DYRK4). DYRKs show homology to Drosophila Minibrain, and DYRK1A in human chromosome 21 is responsible for various neuronal disorders including human Down syndrome. Here we report identification of cellular proteins that associate with specific members of DYRKs. Cellular proteins with molecular masses of 90, 70, and 50-kDa associated with DYRK1B and DYRK4. These proteins were identified as molecular chaperones Hsp90, Hsp70, and Cdc37, respectively. Microscopic analysis of GFP-DYRKs showed that DYRK1A and DYRK1B were nuclear, while DYRK2, DYRK3, and DYRK4 were mostly cytoplasmic in COS7 cells. Overexpression of DYRK1B induced nuclear re-localization of these chaperones with DYRK1B. Treatment of cells with specific Hsp90 inhibitors, geldanamycin and 17-AAG, abolished the association of Hsp90 and Cdc37 with DYRK1B and DYRK4, but not of Hsp70. Inhibition of Hsp90 chaperone activity affected intracellular dynamics of DYRK1B and DYRK4. DYRK1B and DYRK4 underwent rapid formation of cytoplasmic punctate dots after the geldanamycin treatment, suggesting that the chaperone function of Hsp90 is required for prevention of protein aggregation of the target kinases. Prolonged inhibition of Hsp90 by geldanamycin, 17-AAG, or ganetespib, decreased cellular levels of DYRK1B and DYRK4. Finally, DYRK1B and DYRK4 were ubiquitinated in cells, and ubiquitinated DYRK1B and DYRK4 further increased by Hsp90 inhibition with geldanamycin. Taken together, these results indicate that Hsp90 and Cdc37 discriminate specific members of the DYRK kinase family and play an important role in quality control of these client kinases in cells.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Chaperoninas/metabolismo , Proteínas de Choque Térmico HSP90/metabolismo , Chaperonas Moleculares/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Tirosina Quinases/metabolismo , Benzoquinonas/farmacologia , Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Humanos , Lactamas Macrocíclicas/farmacologia
8.
Sci Rep ; 11(1): 12347, 2021 06 11.
Artigo em Inglês | MEDLINE | ID: mdl-34117308

RESUMO

Protein kinases are important regulators in cellular signal transduction. As one major type of Hsp90 client, protein kinases rely on the ATP-dependent molecular chaperone Hsp90, which maintains their structure and supports their activation. Depending on client type, Hsp90 interacts with different cofactors. Here we report that besides the kinase-specific cofactor Cdc37 large PPIases of the Fkbp-type strongly bind to kinase•Hsp90•Cdc37 complexes. We evaluate the nucleotide regulation of these assemblies and identify prominent interaction sites in this quaternary complex. The synergistic interaction between the participating proteins and the conserved nature of the interaction suggests functions of the large PPIases Fkbp51/Fkbp52 and their nematode homolog FKB-6 as contributing factors to the kinase cycle of the Hsp90 machinery.


Assuntos
Proteínas de Ciclo Celular/química , Chaperoninas/química , Proteínas de Choque Térmico HSP90/química , Proteínas de Ligação a Tacrolimo/química , Animais , Sítios de Ligação , Proteínas de Ciclo Celular/metabolismo , Chaperoninas/metabolismo , Proteínas de Choque Térmico HSP90/metabolismo , Humanos , Ligação Proteica , Estabilidade Proteica , Proteínas de Ligação a Tacrolimo/metabolismo
9.
mBio ; 12(3)2021 05 04.
Artigo em Inglês | MEDLINE | ID: mdl-33947758

RESUMO

The highly conserved chaperonin GroESL performs a crucial role in protein folding; however, the essential cellular pathways that rely on this chaperone are underexplored. Loss of GroESL leads to severe septation defects in diverse bacteria, suggesting the folding function of GroESL may be integrated with the bacterial cell cycle at the point of cell division. Here, we describe new connections between GroESL and the bacterial cell cycle using the model organism Caulobacter crescentus Using a proteomics approach, we identify candidate GroESL client proteins that become insoluble or are degraded specifically when GroESL folding is insufficient, revealing several essential proteins that participate in cell division and peptidoglycan biosynthesis. We demonstrate that other cell cycle events, such as DNA replication and chromosome segregation, are able to continue when GroESL folding is insufficient. We further find that deficiency of two FtsZ-interacting proteins, the bacterial actin homologue FtsA and the constriction regulator FzlA, mediate the GroESL-dependent block in cell division. Our data show that sufficient GroESL is required to maintain normal dynamics of the FtsZ scaffold and divisome functionality in C. crescentus In addition to supporting divisome function, we show that GroESL is required to maintain the flow of peptidoglycan precursors into the growing cell wall. Linking a chaperone to cell division may be a conserved way to coordinate environmental and internal cues that signal when it is safe to divide.IMPORTANCE All organisms depend on mechanisms that protect proteins from misfolding and aggregation. GroESL is a highly conserved molecular chaperone that functions to prevent protein aggregation in organisms ranging from bacteria to humans. Despite detailed biochemical understanding of GroESL function, the in vivo pathways that strictly depend on this chaperone remain poorly defined in most species. This study provides new insights into how GroESL is linked to the bacterial cell division machinery, a crucial target of current and future antimicrobial agents. We identify a functional interaction between GroESL and the cell division proteins FzlA and FtsA, which modulate Z-ring function. FtsA is a conserved bacterial actin homologue, suggesting that as in eukaryotes, some bacteria exhibit a connection between cytoskeletal actin proteins and chaperonins. Our work further defines how GroESL is integrated with cell wall synthesis and illustrates how highly conserved folding machines ensure the functioning of fundamental cellular processes during stress.


Assuntos
Proteínas de Bactérias/metabolismo , Caulobacter crescentus/genética , Caulobacter crescentus/metabolismo , Divisão Celular/genética , Chaperoninas/genética , Chaperoninas/metabolismo , Proteínas de Bactérias/genética , Caulobacter crescentus/química , Ligação Proteica
10.
Elife ; 102021 05 24.
Artigo em Inglês | MEDLINE | ID: mdl-34029184

RESUMO

Receptor-interacting serine/threonine-protein kinase 3 (RIPK3) normally signals to necroptosis by phosphorylating MLKL. We report here that when the cellular RIPK3 chaperone Hsp90/CDC37 level is low, RIPK3 also signals to apoptosis. The apoptotic function of RIPK3 requires phosphorylation of the serine 165/threonine 166 sites on its kinase activation loop, resulting in inactivation of RIPK3 kinase activity while gaining the ability to recruit RIPK1, FADD, and caspase-8 to form a cytosolic caspase-activating complex, thereby triggering apoptosis. We found that PGF2α induces RIPK3 expression in luteal granulosa cells in the ovary to cause luteal regression through this RIPK3-mediated apoptosis pathway. Mice carrying homozygous phosphorylation-resistant RIPK3 S165A/T166A knockin mutations failed to respond to PGF2α but retained pro-necroptotic function, whereas mice with phospho-mimicking S165D/T166E homozygous knock-in mutation underwent spontaneous apoptosis in multiple RIPK3-expressing tissues and died shortly after birth. Thus, RIPK3 signals to either necroptosis or apoptosis depending on its serine 165/threonine 166 phosphorylation status.


Assuntos
Apoptose , Corpo Lúteo/enzimologia , Dinoprosta/metabolismo , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Animais , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Chaperoninas/genética , Chaperoninas/metabolismo , Corpo Lúteo/patologia , Proteína de Domínio de Morte Associada a Fas/genética , Proteína de Domínio de Morte Associada a Fas/metabolismo , Feminino , Células HEK293 , Células HT29 , Células HeLa , Humanos , Células MCF-7 , Camundongos Endogâmicos C57BL , Camundongos Knockout , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Mutação , Fosforilação , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Proteína Serina-Treonina Quinases de Interação com Receptores/genética , Transdução de Sinais
11.
J Oral Biosci ; 63(3): 271-277, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34010688

RESUMO

OBJECTIVES: Ameloblastoma is an odontogenic neoplasm of the mandible and maxilla with various histological types and subtypes. It has been reported that some ameloblastomas could arise from dentigerous cyst walls; thus, the development of ameloblastoma from dentigerous cysts may be due to differential protein expression. Our aim was to identify a membrane protein that is differentially expressed in ameloblastomas with respect to dentigerous cysts. METHODS: We analyzed the SDS-PAGE profiles of membrane proteins from ameloblastomas and dentigerous cysts. The protein in a band present in the ameloblastoma sample, but apparently absent in the dentigerous cyst sample was identified via mass spectrometry as the chaperonin Hsp60. We used western blotting and immunohistochemistry to analyze its overexpression and localization in ameloblastoma. RESULTS: We found a differential band of 95 kDa in the membrane proteins of ameloblastoma. In this band, the chaperonin Hsp60 was identified, and its overexpression was corroborated using western blotting and immunohistochemistry. Hsp60 was localized in the plasma membrane of all ameloblastoma samples studied; in addition, it was found in the cell nucleus of the plexiform subtype of conventional ameloblastoma. CONCLUSIONS: Our results suggest that Hsp60 may be involved in ameloblastoma development, and could therefore be a potential therapeutic target for ameloblastoma treatment.


Assuntos
Ameloblastoma , Chaperonina 60/genética , Cisto Dentígero , Proteínas Mitocondriais/genética , Tumores Odontogênicos , Ameloblastoma/genética , Chaperoninas , Humanos , Imuno-Histoquímica
12.
Bioorg Chem ; 111: 104867, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-33845380

RESUMO

To enhance the disruption of Hsp90-Cdc37, we designed and synthesized a series (27) of CEL-triazole derivatives. Most of the target compounds showed enhanced anti-proliferative activity on four cancer cell lines (MDA-MB-231, MCF-7, HepG2 and A459). Among them, compound 6 showed the best anti-proliferation (IC50 = 0.34 ± 0.01 µM) on MDA-MB-231. Pharmacological studies had found that compound 6 showed a higher ability to disrupt Hsp90-Cdc37 interaction in cells and inhibited the expression of the key Hsp90-Cdc37 clients in a concentration-dependent manner. Further studies indicated that an enhanced covalent binding between compound 6 and thiols (cysteine) might be one of the reasons for the increased activity. Furthermore, compound 6 arrested cells in the G0/G1 phase and induced tumor cell apoptosis significantly. Overall, for cancer treatment, compound 6 was worth further exploring.


Assuntos
Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Proteínas de Ciclo Celular/antagonistas & inibidores , Chaperoninas/antagonistas & inibidores , Descoberta de Drogas , Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Antineoplásicos/síntese química , Antineoplásicos/química , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Estrutura Molecular , Relação Estrutura-Atividade , Células Tumorais Cultivadas
13.
FEBS Lett ; 595(11): 1559-1568, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-33786814

RESUMO

Homeodomain-interacting protein kinase 2 (HIPK2) is a highly conserved, constitutively active Ser/Thr protein kinase that is involved in various important biological processes. HIPK2 activates itself by auto-phosphorylation during its synthesis, and its activity is mainly controlled through modulation of its expression by ubiquitin-dependent degradation. By comparing the expression of wild-type and kinase-defective HIPK2, we have recently described a novel mechanism of HIPK2 regulation that is based on preferential co-translational degradation of kinase-defective versus wild-type HIPK2. Here, we have addressed this novel regulatory mechanism in more detail by focusing on the possible involvement of chaperones. Our work shows that HIPK2 is a client of the CDC37-HSP90 chaperone complex and points to a novel role of CDC37 in the co-translational degradation of a client protein.


Assuntos
Proteínas de Transporte/metabolismo , Proteínas de Ciclo Celular/metabolismo , Chaperoninas/metabolismo , Proteínas de Choque Térmico HSP90/metabolismo , Biossíntese de Proteínas , Proteínas Serina-Treonina Quinases/metabolismo , Proteólise , Animais , Proteínas de Transporte/genética , Proteínas de Ciclo Celular/antagonistas & inibidores , Proteínas de Ciclo Celular/genética , Linhagem Celular , Chaperoninas/antagonistas & inibidores , Chaperoninas/genética , Fibroblastos/citologia , Fibroblastos/metabolismo , Regulação da Expressão Gênica , Proteínas de Choque Térmico HSP90/genética , Células HeLa , Humanos , Proteínas Serina-Treonina Quinases/genética , Codorniz , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Transdução de Sinais , Ubiquitina/genética , Ubiquitina/metabolismo , Ubiquitinação
14.
Int J Mol Sci ; 22(4)2021 Feb 17.
Artigo em Inglês | MEDLINE | ID: mdl-33671465

RESUMO

To change their behaviors, cells require actin proteins to assemble together into long polymers/filaments-and so a critical goal is to understand the factors that control this actin filament (F-actin) assembly and stability. We have identified a family of unusual actin regulators, the MICALs, which are flavoprotein monooxygenase/hydroxylase enzymes that associate with flavin adenine dinucleotide (FAD) and use the co-enzyme nicotinamide adenine dinucleotide phosphate (NADPH) in Redox reactions. F-actin is a specific substrate for these MICAL Redox enzymes, which oxidize specific amino acids within actin to destabilize actin filaments. Furthermore, this MICAL-catalyzed reaction is reversed by another family of Redox enzymes (SelR/MsrB enzymes)-thereby revealing a reversible Redox signaling process and biochemical mechanism regulating actin dynamics. Interestingly, in addition to the MICALs' Redox enzymatic portion through which MICALs covalently modify and affect actin, MICALs have multiple other domains. Less is known about the roles of these other MICAL domains. Here we provide approaches for obtaining high levels of recombinant protein for the Redox only portion of Mical and demonstrate its catalytic and F-actin disassembly activity. These results provide a ground state for future work aimed at defining the role of the other domains of Mical - including characterizing their effects on Mical's Redox enzymatic and F-actin disassembly activity.


Assuntos
Actinas/metabolismo , Drosophila melanogaster/enzimologia , Ensaios Enzimáticos , Oxigenases de Função Mista/química , Oxigenases de Função Mista/metabolismo , Animais , Biocatálise , Chaperoninas/metabolismo , Temperatura Baixa , Oxirredução , Domínios Proteicos , Proteínas Recombinantes/isolamento & purificação , Solubilidade
15.
Acta Crystallogr F Struct Biol Commun ; 77(Pt 3): 79-84, 2021 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-33682792

RESUMO

Chaperonins are biomolecular complexes that assist in protein folding. Thermophilic factor 55 (TF55) is a group II chaperonin found in the archaeal genus Sulfolobus that has α, ß and γ subunits. Using cryo-electron microscopy, structures of the ß-only complex of S. solfataricus TF55 (TF55ß) were determined to 3.6-4.2 Šresolution. The structures of the TF55ß complexes formed in the presence of ADP or ATP highlighted an open state in which nucleotide exchange can occur before progressing in the refolding cycle.


Assuntos
Proteínas Arqueais/ultraestrutura , Chaperoninas/ultraestrutura , Microscopia Crioeletrônica , Sulfolobus solfataricus/ultraestrutura , Difosfato de Adenosina/metabolismo , Trifosfato de Adenosina/metabolismo , Modelos Moleculares , Conformação Proteica
16.
Int J Mol Sci ; 22(3)2021 Jan 29.
Artigo em Inglês | MEDLINE | ID: mdl-33572860

RESUMO

Bardet-Biedl syndrome (BBS) is a ciliopathy characterized by retinal dystrophy, renal cysts, obesity and polydactyly. BBS genes have been implicated in ciliogenesis, hedgehog signaling and retinal pigment epithelium maturation. BBS1 and BBS5 are members of the BBSome, implicated in cilia transport of proteins, and BBS10 is a member of the chaperonin-complex, mediating BBSome assembly. In this study, involvement of BBS1, BBS5 and BBS10 in ciliogenesis and hedgehog signaling were investigated in BBS-defective patient fibroblasts as well as in RPE-hTERT cells following siRNA-mediated knockdown of the BBS genes. Furthermore, the ability of BBS1-defective induced pluripotent stem-cells (iPSCs) to differentiate into RPE cells was assessed. We report that cells lacking functional BBS5 or BBS10 have a reduced number of primary cilia, whereas cells lacking functional BBS1 display shorter primary cilia compared to wild-type cells. Hedgehog signaling was substantially impaired and Smoothened, a component of hedgehog signaling, was trapped inside the cilia of the BBS-defective cells, even in the absence of Smoothened agonist. Preliminary results demonstrated the ability of BBS1-defective iPSC to differentiate into RPE-65 expressing RPE-like cells. The BBS1-/--defective RPE-like cells were less pigmented, compared to RPE-like cells differentiated from control iPSCs, indicating an impact of BBS1 on RPE maturation.


Assuntos
Síndrome de Bardet-Biedl/metabolismo , Chaperoninas/metabolismo , Proteínas do Citoesqueleto/metabolismo , Proteínas Hedgehog/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas de Ligação a Fosfato/metabolismo , Síndrome de Bardet-Biedl/patologia , Linhagem Celular , Cílios/metabolismo , Cílios/patologia , Fibroblastos/metabolismo , Fibroblastos/patologia , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Transdução de Sinais
17.
J Clin Invest ; 131(8)2021 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-33630762

RESUMO

Bardet-Biedl syndrome (BBS) is a rare autosomal recessive disorder caused by mutations in genes encoding components of the primary cilium and is characterized by hyperphagic obesity. To investigate the molecular basis of obesity in human BBS, we developed a cellular model of BBS using induced pluripotent stem cell-derived (iPSC-derived) hypothalamic arcuate-like neurons. BBS mutations BBS1M390R and BBS10C91fsX95 did not affect neuronal differentiation efficiency but caused morphological defects, including impaired neurite outgrowth and longer primary cilia. Single-cell RNA sequencing of BBS1M390R hypothalamic neurons identified several downregulated pathways, including insulin and cAMP signaling and axon guidance. Additional studies demonstrated that BBS1M390R and BBS10C91fsX95 mutations impaired insulin signaling in both human fibroblasts and iPSC-derived neurons. Overexpression of intact BBS10 fully restored insulin signaling by restoring insulin receptor tyrosine phosphorylation in BBS10C91fsX95 neurons. Moreover, mutations in BBS1 and BBS10 impaired leptin-mediated p-STAT3 activation in iPSC-derived hypothalamic neurons. Correction of the BBS mutation by CRISPR rescued leptin signaling. POMC expression and neuropeptide production were decreased in BBS1M390R and BBS10C91fsX95 iPSC-derived hypothalamic neurons. In the aggregate, these data provide insights into the anatomic and functional mechanisms by which components of the BBSome in CNS primary cilia mediate effects on energy homeostasis.


Assuntos
Síndrome de Bardet-Biedl/metabolismo , Chaperoninas/metabolismo , Hipotálamo/metabolismo , Células-Tronco Pluripotentes Induzidas/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Mutação de Sentido Incorreto , Neurônios/metabolismo , Sistemas do Segundo Mensageiro , Substituição de Aminoácidos , Animais , Síndrome de Bardet-Biedl/genética , Chaperoninas/genética , AMP Cíclico/genética , AMP Cíclico/metabolismo , Feminino , Células HEK293 , Humanos , Masculino , Camundongos , Camundongos Transgênicos , Proteínas Associadas aos Microtúbulos/genética
18.
BMJ Case Rep ; 14(1)2021 Jan 28.
Artigo em Inglês | MEDLINE | ID: mdl-33509858

RESUMO

Bardet-Biedl syndrome (BBS) is a rare autosomal recessive ciliopathy characterised by rod-cone dystrophy, obesity, postaxial polydactyly, cognitive impairment, hypogonadism, renal abnormalities, and rarely, laryngeal webs or bifid epiglottis. Most patients present with obesity. Multiple genes are involved in causation of BBS and there is also evidence of triallelic inheritance. We herein report an Asian boy who had weak cry and stridor since birth, and on evaluation was found to have both laryngeal web and bifid epiglottis. Mutation analysis revealed a homozygous variant in BBS10 gene.


Assuntos
Síndrome de Bardet-Biedl/diagnóstico , Epiglote/anormalidades , Hipotireoidismo/diagnóstico , Laringe/anormalidades , Síndrome de Bardet-Biedl/complicações , Síndrome de Bardet-Biedl/genética , Síndrome de Bardet-Biedl/fisiopatologia , Broncoscopia , Chaperoninas/genética , Dedos/anormalidades , Dedos/fisiopatologia , Mutação da Fase de Leitura , Humanos , Hipotireoidismo/complicações , Hipotireoidismo/tratamento farmacológico , Lactente , Laringe/cirurgia , Masculino , Obesidade Pediátrica/fisiopatologia , Polidactilia/fisiopatologia , Tiroxina/uso terapêutico , Dedos do Pé/anormalidades , Dedos do Pé/fisiopatologia
19.
Mol Ther ; 29(4): 1541-1556, 2021 04 07.
Artigo em Inglês | MEDLINE | ID: mdl-33412308

RESUMO

HER2 breast cancer (BC) remains a significant problem in patients with locally advanced or metastatic BC. We investigated the relationship between T helper 1 (Th1) immune response and the proteasomal degradation pathway (PDP), in HER2-sensitive and -resistant cells. HER2 overexpression is partially maintained because E3 ubiquitin ligase Cullin5 (CUL5), which degrades HER2, is frequently mutated or underexpressed, while the client-protective co-chaperones cell division cycle 37 (Cdc37) and heat shock protein 90 (Hsp90) are increased translating to diminished survival. The Th1 cytokine interferon (IFN)-γ caused increased CUL5 expression and marked dissociation of both Cdc37 and Hsp90 from HER2, causing significant surface loss of HER2, diminished growth, and induction of tumor senescence. In HER2-resistant mammary carcinoma, either IFN-γ or Th1-polarizing anti-HER2 vaccination, when administered with anti-HER2 antibodies, demonstrated increased intratumor CUL5 expression, decreased surface HER2, and tumor senescence with significant therapeutic activity. IFN-γ synergized with multiple HER2-targeted agents to decrease surface HER2 expression, resulting in decreased tumor growth. These data suggest a novel function of IFN-γ that regulates HER2 through the PDP pathway and provides an opportunity to impact HER2 responses through anti-tumor immunity.


Assuntos
Neoplasias da Mama/tratamento farmacológico , Proteínas Culina/genética , Interferon gama/genética , Receptor ErbB-2/imunologia , Neoplasias da Mama/genética , Neoplasias da Mama/imunologia , Neoplasias da Mama/patologia , Proteínas de Ciclo Celular/genética , Linhagem Celular Tumoral , Senescência Celular/genética , Senescência Celular/imunologia , Chaperoninas/genética , Proteínas Culina/imunologia , Citocinas/genética , Feminino , Regulação Neoplásica da Expressão Gênica/genética , Regulação Neoplásica da Expressão Gênica/imunologia , Humanos , Interferon gama/imunologia , Proteólise , Receptor ErbB-2/antagonistas & inibidores , Receptor ErbB-2/genética , Células Th1/efeitos dos fármacos , Células Th1/metabolismo , Vacinação
20.
Am J Kidney Dis ; 77(3): 410-419, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33039432

RESUMO

Primary cilia are specialized sensory organelles that protrude from the apical surface of most cell types. During the past 2 decades, they have been found to play important roles in tissue development and signal transduction, with mutations in ciliary-associated proteins resulting in a group of diseases collectively known as ciliopathies. Many of these mutations manifest as renal ciliopathies, characterized by kidney dysfunction resulting from aberrant cilia or ciliary functions. This group of overlapping and genetically heterogeneous diseases includes polycystic kidney disease, nephronophthisis, and Bardet-Biedl syndrome as the main focus of this review. Renal ciliopathies are characterized by the presence of kidney cysts that develop due to uncontrolled epithelial cell proliferation, growth, and polarity, downstream of dysregulated ciliary-dependent signaling. Due to cystic-associated kidney injury and systemic inflammation, cases result in kidney failure requiring dialysis and transplantation. Of the handful of pharmacologic treatments available, none are curative. It is important to determine the molecular mechanisms that underlie the involvement of the primary cilium in cyst initiation, expansion, and progression for the development of novel and efficacious treatments. This review updates research progress in defining key genes and molecules central to ciliogenesis and renal ciliopathies.


Assuntos
Síndrome de Bardet-Biedl/genética , Cílios/metabolismo , Ciliopatias/genética , Doenças Renais Policísticas/genética , Anormalidades Múltiplas/genética , Anormalidades Múltiplas/metabolismo , Anormalidades Múltiplas/fisiopatologia , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transporte Vesicular/genética , Síndrome de Bardet-Biedl/metabolismo , Síndrome de Bardet-Biedl/fisiopatologia , Cerebelo/anormalidades , Cerebelo/metabolismo , Cerebelo/fisiopatologia , Chaperoninas/genética , Cílios/fisiologia , Transtornos da Motilidade Ciliar/genética , Transtornos da Motilidade Ciliar/metabolismo , Transtornos da Motilidade Ciliar/fisiopatologia , Ciliopatias/metabolismo , Ciliopatias/fisiopatologia , Proteínas do Citoesqueleto/genética , Encefalocele/genética , Encefalocele/metabolismo , Encefalocele/fisiopatologia , Anormalidades do Olho/genética , Anormalidades do Olho/metabolismo , Anormalidades do Olho/fisiopatologia , Humanos , Doenças Renais Císticas/genética , Doenças Renais Císticas/metabolismo , Doenças Renais Císticas/fisiopatologia , Amaurose Congênita de Leber/genética , Amaurose Congênita de Leber/metabolismo , Amaurose Congênita de Leber/fisiopatologia , Proteínas de Membrana/genética , Proteínas Associadas aos Microtúbulos/genética , Atrofias Ópticas Hereditárias/genética , Atrofias Ópticas Hereditárias/metabolismo , Atrofias Ópticas Hereditárias/fisiopatologia , Doenças Renais Policísticas/metabolismo , Doenças Renais Policísticas/fisiopatologia , Proteínas/genética , Retina/anormalidades , Retina/metabolismo , Retina/fisiopatologia , Retinite Pigmentosa/genética , Retinite Pigmentosa/metabolismo , Retinite Pigmentosa/fisiopatologia , Canais de Cátion TRPP/genética
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