RESUMO
Hsp60 chaperonins and their Hsp10 cofactors assist protein folding in all living cells, constituting the paradigmatic example of molecular chaperones. Despite extensive investigations of their structure and mechanism, crucial questions regarding how these chaperonins promote folding remain unsolved. Here, we report that the bacterial Hsp60 chaperonin GroEL forms a stable, functionally relevant complex with the chaperedoxin CnoX, a protein combining a chaperone and a redox function. Binding of GroES (Hsp10 cofactor) to GroEL induces CnoX release. Cryoelectron microscopy provided crucial structural information on the GroEL-CnoX complex, showing that CnoX binds GroEL outside the substrate-binding site via a highly conserved C-terminal α-helix. Furthermore, we identified complexes in which CnoX, bound to GroEL, forms mixed disulfides with GroEL substrates, indicating that CnoX likely functions as a redox quality-control plugin for GroEL. Proteins sharing structural features with CnoX exist in eukaryotes, suggesting that Hsp60 molecular plugins have been conserved through evolution.
Assuntos
Chaperonas Moleculares , Dobramento de Proteína , Microscopia Crioeletrônica , Chaperonas Moleculares/metabolismo , Oxirredução , Chaperoninas/química , Chaperoninas/metabolismo , Chaperonina 60/química , Chaperonina 10/metabolismoRESUMO
Circular RNAs (circRNAs) are upregulated during neurogenesis. Where and how circRNAs are localized and what roles they play during this process have remained elusive. Comparing the nuclear and cytoplasmic circRNAs between H9 cells and H9-derived forebrain (FB) neurons, we identify that a subset of adenosine (A)-rich circRNAs are restricted in H9 nuclei but exported to cytosols upon differentiation. Such a subcellular relocation of circRNAs is modulated by the poly(A)-binding protein PABPC1. In the H9 nucleus, newly produced (A)-rich circRNAs are bound by PABPC1 and trapped by the nuclear basket protein TPR to prevent their export. Modulating (A)-rich motifs in circRNAs alters their subcellular localization, and introducing (A)-rich circRNAs in H9 cytosols results in mRNA translation suppression. Moreover, decreased nuclear PABPC1 upon neuronal differentiation enables the export of (A)-rich circRNAs, including circRTN4(2,3), which is required for neurite outgrowth. These findings uncover subcellular localization features of circRNAs, linking their processing and function during neurogenesis.
Assuntos
Transporte Ativo do Núcleo Celular , Adenosina , Núcleo Celular , Neurogênese , Neurônios , Proteína I de Ligação a Poli(A) , RNA Circular , RNA , RNA Circular/metabolismo , RNA Circular/genética , Neurônios/metabolismo , Adenosina/metabolismo , Núcleo Celular/metabolismo , Humanos , Proteína I de Ligação a Poli(A)/metabolismo , Proteína I de Ligação a Poli(A)/genética , Animais , RNA/metabolismo , RNA/genética , Linhagem Celular , Diferenciação Celular , Citoplasma/metabolismo , Prosencéfalo/metabolismoRESUMO
O-linked N-acetylglucosamine transferase (OGT) is found in all metazoans and plays an important role in development but at the single-cell level is only essential in dividing mammalian cells. Postmitotic mammalian cells and cells of invertebrates such as Caenorhabditis elegans and Drosophila can survive without copies of OGT. Why OGT is required in dividing mammalian cells but not in other cells remains unknown. OGT has multiple biochemical activities. Beyond its well-known role in adding ß-O-GlcNAc to serine and threonine residues of nuclear and cytoplasmic proteins, OGT also acts as a protease in the maturation of the cell cycle regulator host cell factor 1 (HCF-1) and serves as an integral member of several protein complexes, many of them linked to gene expression. In this review, we summarize current understanding of the mechanisms underlying OGT's biochemical activities and address whether known functions of OGT could be related to its essential role in dividing mammalian cells.
Assuntos
Células Eucarióticas/enzimologia , Fator C1 de Célula Hospedeira/química , N-Acetilglucosaminiltransferases/química , Processamento de Proteína Pós-Traducional , Acilação , Animais , Caenorhabditis elegans/enzimologia , Caenorhabditis elegans/genética , Divisão Celular , Sobrevivência Celular , Drosophila melanogaster/enzimologia , Drosophila melanogaster/genética , Células Eucarióticas/citologia , Glicosilação , Fator C1 de Célula Hospedeira/genética , Fator C1 de Célula Hospedeira/metabolismo , Humanos , Mamíferos , Camundongos , Modelos Moleculares , N-Acetilglucosaminiltransferases/genética , N-Acetilglucosaminiltransferases/metabolismo , Especificidade da EspécieRESUMO
Folding of stringent clients requires transfer from Hsp70 to Hsp90. The co-chaperone Hop physically connects the chaperone machineries. Here, we define its role from the remodeling of Hsp70/40-client complexes to the mechanism of client transfer and the conformational switching from stalled to active client-processing states of Hsp90. We show that Hsp70 together with Hsp40 completely unfold a stringent client, the glucocorticoid receptor ligand-binding domain (GR-LBD) in large assemblies. Hop remodels these for efficient transfer onto Hsp90. As p23 enters, Hsp70 leaves the complex via switching between binding sites in Hop. Current concepts assume that to proceed to client folding, Hop dissociates and the co-chaperone p23 stabilizes the Hsp90 closed state. In contrast, we show that p23 functionally interacts with Hop, relieves the stalling Hsp90-Hop interaction, and closes Hsp90. This reaction allows folding of the client and is thus the key regulatory step for the progression of the chaperone cycle.
Assuntos
Dobramento de Proteína , Piridinolcarbamato , Proteínas de Choque Térmico HSP70/genética , Proteínas de Choque Térmico HSP70/metabolismo , Proteínas de Choque Térmico HSP90/metabolismo , Humanos , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Ligação Proteica , Receptores de Glucocorticoides/metabolismoRESUMO
O-GlcNAc transferase (OGT) is an essential mammalian enzyme that glycosylates myriad intracellular proteins and cleaves the transcriptional coregulator Host Cell Factor 1 to regulate cell cycle processes. Via these catalytic activities as well as noncatalytic protein-protein interactions, OGT maintains cell homeostasis. OGT's tetratricopeptide repeat (TPR) domain is important in substrate recognition, but there is little information on how changing the TPR domain impacts its cellular functions. Here, we investigate how altering OGT's TPR domain impacts cell growth after the endogenous enzyme is deleted. We find that disrupting the TPR residues required for OGT dimerization leads to faster cell growth, whereas truncating the TPR domain slows cell growth. We also find that OGT requires eight of its 13 TPRs to sustain cell viability. OGT-8, like the nonviable shorter OGT variants, is mislocalized and has reduced Ser/Thr glycosylation activity; moreover, its interactions with most of wild-type OGT's binding partners are broadly attenuated. Therefore, although OGT's five N-terminal TPRs are not essential for cell viability, they are required for proper subcellular localization and for mediating many of OGT's protein-protein interactions. Because the viable OGT truncation variant we have identified preserves OGT's essential functions, it may facilitate their identification.
Assuntos
N-Acetilglucosaminiltransferases , N-Acetilglucosaminiltransferases/metabolismo , N-Acetilglucosaminiltransferases/genética , Humanos , Repetições de Tetratricopeptídeos , Glicosilação , Fator C1 de Célula Hospedeira/metabolismo , Fator C1 de Célula Hospedeira/genética , Células HEK293 , Domínios Proteicos , Proliferação de Células , Sobrevivência Celular , Animais , Ligação ProteicaRESUMO
The total number of nuclear pore complexes (NPCs) per nucleus varies greatly between different cell types and is known to change during cell differentiation and cell transformation. However, the underlying mechanisms that control how many nuclear transport channels are assembled into a given nuclear envelope remain unclear. Here, we report that depletion of the NPC basket protein Tpr, but not Nup153, dramatically increases the total NPC number in various cell types. This negative regulation of Tpr occurs via a phosphorylation cascade of extracellular signal-regulated kinase (ERK), the central kinase of the mitogen-activated protein kinase (MAPK) pathway. Tpr serves as a scaffold for ERK to phosphorylate the nucleoporin (Nup) Nup153, which is critical for early stages of NPC biogenesis. Our results reveal a critical role of the Nup Tpr in coordinating signal transduction pathways during cell proliferation and the dynamic organization of the nucleus.
Assuntos
Complexo de Proteínas Formadoras de Poros Nucleares/fisiologia , Poro Nuclear/fisiologia , Proteínas Proto-Oncogênicas/fisiologia , Animais , Células Cultivadas , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Humanos , Interfase , Camundongos , Membrana Nuclear/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Proteínas Proto-Oncogênicas/metabolismoRESUMO
Protein folding, quality control, maturation, and trafficking are essential processes for proper cellular homeostasis. Around one-third of the human proteome is targeted to the endoplasmic reticulum (ER), the organelle that serves as entrance into the secretory pathway. Successful protein trafficking is paramount for proper cellular function and to that end there are many ER resident proteins that ensure efficient secretion. Here, biochemical and cell biological analysis was used to determine that TTC17 is a large, soluble, ER-localized protein that plays an important role in secretory trafficking. Transcriptional analysis identified the predominantly expressed protein isoform of TTC17 in various cell lines. Further, TTC17 localizes to the ER and interacts with a wide variety of chaperones and cochaperones normally associated with ER protein folding, quality control, and maturation processes. TTC17 was found to be significantly upregulated by ER stress and through the creation and use of TTC17-/- cell lines, quantitative mass spectrometry identified secretory pathway wide trafficking defects in the absence of TTC17. Notably, trafficking of insulin-like growth factor type 1 receptor, glycoprotein nonmetastatic melanoma protein B, clusterin, and UDP-glucose:glycoprotein glucosyltransferase 1 were significantly altered in H4 neuroglioma cells. This study defines a novel ER trafficking factor and provides insight into the protein-protein assisted trafficking in the early secretory pathway.
Assuntos
Estresse do Retículo Endoplasmático , Dobramento de Proteína , Humanos , Retículo Endoplasmático/genética , Retículo Endoplasmático/metabolismo , Glicoproteínas/metabolismo , Linhagem CelularRESUMO
Chloroplasts are not only critical photosynthesis sites in plants, but they also participate in plastidial retrograde signaling in response to developmental and environmental signals. MEcPP (2-C-Methyl-D-erythritol-2,4-cyclopyrophosphate) is an intermediary in the methylerythritol phosphate (MEP) pathway in chloroplasts. It is a critical precursor for the synthesis of isoprenoids and terpenoid derivatives, which play crucial roles in plant growth and development, photosynthesis, reproduction, and defense against environmental constraints. Accumulation of MEcPP under stressful conditions triggers the expression of IMPα-9 and TPR2, contributing to the activation of abiotic stress-responsive genes. In this correspondence, we discuss plastidial retrograde signaling in support of a recently published paper in Molecular Plant (Zeng et al. 2024). We hope that it can shed more insight on the retrograde signaling cascade.
Assuntos
Cloroplastos , Estresse Fisiológico , Cloroplastos/metabolismo , Regulação da Expressão Gênica de Plantas , Transdução de Sinais , Arabidopsis/genética , Arabidopsis/metabolismo , Eritritol/metabolismo , Eritritol/análogos & derivados , Proteínas de Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Fosfatos Açúcares/metabolismo , MAP Quinase Quinase Quinase 5/metabolismo , MAP Quinase Quinase Quinase 5/genéticaRESUMO
Nuclear pore complex (NPC) is composed of multiple nucleoporins (Nups). A plethora of studies have highlighted the significance of NPC in plant immunity. However, the specific roles of individual Nups are poorly understood. NUCLEAR PORE ANCHOR (NUA) is a component of NPC. Loss of NUA leads to an increase in SUMO conjugates and pleiotropic developmental defects in Arabidopsis thaliana. Herein, we revealed that NUA is required for plant defense against multiple pathogens. NUCLEAR PORE ANCHOR associates with the transcriptional corepressor TOPLESS-RELATED1 (TPR1) and contributes to TPR1 deSUMOylation. Significantly, NUA-interacting protein EARLY IN SHORT DAYS 4 (ESD4), a SUMO protease, specifically deSUMOylates TPR1. It has been previously established that the SUMO E3 ligase SAP AND MIZ1 DOMAIN-CONTAINING LIGASE 1 (SIZ1)-mediated SUMOylation of TPR1 represses the immune-related function of TPR1. Consistent with this notion, the hyper-SUMOylated TPR1 in nua-3 leads to upregulated expression of TPR1 target genes and compromised TPR1-mediated disease resistance. Taken together, our work uncovers a mechanism by which NUA positively regulates plant defense responses by coordination with ESD4 to deSUMOylate TPR1. Our findings, together with previous studies, reveal a regulatory module in which SIZ1 and NUA/ESD4 control the homeostasis of TPR1 SUMOylation to maintain proper immune output.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Imunidade Vegetal , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Regulação da Expressão Gênica de Plantas , Ligases/metabolismo , Poro Nuclear/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , SumoilaçãoRESUMO
The UCS (UNC-45/CRO1/She4p) family of proteins has emerged as chaperones specific for the folding, assembly, and function of myosin. UCS proteins participate in various myosin-dependent cellular processes including myofibril organization and muscle functions, cell differentiation, striated muscle development, cytokinesis, and endocytosis. Mutations in the genes that code for UCS proteins cause serious defects in myosin-dependent cellular processes. UCS proteins that contain an N-terminal tetratricopeptide repeat (TPR) domain are called UNC-45. Vertebrates usually possess two variants of UNC-45, the ubiquitous general-cell UNC-45 (UNC-45A) and the striated muscle UNC-45 (UNC-45B), which is exclusively expressed in skeletal and cardiac muscles. Except for the TPR domain in UNC-45, UCS proteins comprise of several irregular armadillo (ARM) repeats that are organized into a central domain, a neck region, and the canonical C-terminal UCS domain that functions as the chaperoning module. With or without TPR, UCS proteins form linear oligomers that serve as scaffolds that mediate myosin folding, organization into myofibrils, repair, and motility. This chapter reviews emerging functions of these proteins with a focus on UNC-45 as a dedicated chaperone for folding, assembly, and function of myosin at protein and potentially gene levels. Recent experimental evidences strongly support UNC-45 as an absolute regulator of myosin, with each domain of the chaperone playing different but complementary roles during the folding, assembly, and function of myosin, as well as recruiting Hsp90 as a co-chaperone to optimize key steps. It is becoming increasingly clear that UNC-45 also regulates the transcription of several genes involved in myosin-dependent cellular processes.
Assuntos
Proteínas de Caenorhabditis elegans , Animais , Proteínas de Caenorhabditis elegans/química , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Chaperonas Moleculares/metabolismo , Miosinas/genética , Miosinas/química , Proteínas de Choque Térmico HSP90/genética , Proteínas de Choque Térmico HSP90/metabolismoRESUMO
The Hsp90 chaperone is known to interact with a diverse array of client proteins. However, in every case examined, Hsp90 is also accompanied by a single or several co-chaperone proteins. One class of co-chaperone contains a tetratricopeptide repeat (TPR) domain that targets the co-chaperone to the C-terminal region of Hsp90. Within this class are Hsp90-binding peptidylprolyl isomerases, most of which belong to the FK506-binding protein (FKBP) family. Despite the common association of FKBP co-chaperones with Hsp90, it is abundantly clear that the client protein influences, and is often influenced by, the particular FKBP bound to Hsp90. Examples include Xap2 in aryl hydrocarbon receptor complexes and FKBP52 in steroid receptor complexes. In this chapter, we discuss the known functional roles played by FKBP co-chaperones and, where possible, relate distinctive functions to structural differences between FKBP members.
Assuntos
Proteínas de Choque Térmico HSP90 , Proteínas de Ligação a Tacrolimo , Humanos , Proteínas de Choque Térmico HSP90/química , Proteínas de Choque Térmico HSP90/genética , Proteínas de Choque Térmico HSP90/metabolismo , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Peptidilprolil Isomerase/metabolismo , Ligação Proteica , Proteínas de Ligação a Tacrolimo/genética , Proteínas de Ligação a Tacrolimo/química , Proteínas de Ligação a Tacrolimo/metabolismo , Imunofilinas/genética , Imunofilinas/metabolismoRESUMO
Study of CD4+ T cell response and T cell receptor (TCR) specificity is crucial for understanding etiology of immune-mediated diseases and developing targeted therapies. However, solubility, accessibility, and stability of synthetic antigenic peptides used in T cell assays may be a critical point in such studies. Here we present a T cell activation reporter system using recombinant proteins containing antigenic epitopes fused with bacterial thioredoxin (trx-peptides) and obtained by bacterial expression. We report that co-incubation of CD4+ HA1.7 TCR+ reporter Jurkat 76 TRP cells with CD80+ HLA-DRB1*01:01+ HeLa cells or CD4+ Ob.1A12 TCR+ Jurkat 76 TRP with CD80+ HLA-DRB1*15:01+ HeLa cells resulted in activation of reporter Jurkat 76 TPR after addition of recombinant trx-peptide fusion proteins, containing TCR-specific epitopes. Trx-peptides were comparable with corresponding synthetic peptides in their capacity to activate Jurkat 76 TPR. These data demonstrate that thioredoxin as a carrier protein (trx) for antigenic peptides exhibits minimal interference with recognition of MHC-specific peptides by TCRs and consequent T cell activation. Our findings highlight potential feasibility of trx-peptides as a reagent for assessing the immunogenicity of antigenic fragments.
Assuntos
Linfócitos T CD4-Positivos , Peptídeos , Receptores de Antígenos de Linfócitos T , Proteínas Recombinantes de Fusão , Tiorredoxinas , Humanos , Tiorredoxinas/imunologia , Tiorredoxinas/genética , Células Jurkat , Linfócitos T CD4-Positivos/imunologia , Receptores de Antígenos de Linfócitos T/imunologia , Receptores de Antígenos de Linfócitos T/metabolismo , Proteínas Recombinantes de Fusão/farmacologia , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Peptídeos/farmacologia , Peptídeos/imunologia , Peptídeos/química , Ativação Linfocitária/efeitos dos fármacos , Células HeLaRESUMO
We previously identified a cell cycle-dependent periodic subcellular distribution of cancer metastasis-associated antigen 1 (MTA1) and unraveled a novel role of MTA1 in inhibiting spindle damage-induced spindle assembly checkpoint (SAC) activation in cancer cells. However, the more detailed subcellular localization of MTA1 in mitotic cells and its copartner in SAC regulation in cancer cells are still poorly understood. Here, through immunofluorescent colocalization analysis of MTA1 and alpha-tubulin in mitotic cancer cells, we reveal that MTA1 is dynamically localized to the spindle apparatus throughout the entire mitotic process. We also demonstrated a reversible upregulation of MTA1 expression upon spindle damage-induced SAC activation, and time-lapse imaging assays indicated that MTA1 silencing delayed the mitotic metaphase-anaphase transition in cancer cells. Further investigation revealed that MTA1 interacts and colocalizes with Translocated Promoter Region (TPR) on spindle microtubules in mitotic cells, and this interaction is attenuated on SAC activation. TPR is well-implicated in SAC regulation via binding the MAD1-MAD2 complex, however, no interactions between MTA1 and MAD1 or MAD2 were detected in our coimmunoprecipitation (co-IP) assays, suggesting that the MTA1-TPR may represent a distinct SAC-associated complex separate from the previously reported TPR-MAD1/MAD2 complex. Our data provide new insights into the subcellular localization and molecular function of MTA1 in SAC regulation in cancer, and indicate that intervention of the MTA1-TPR interaction may be effective to modulate SAC and hence chromosomal instability (CIN) in tumorigenesis.
Assuntos
Proteínas de Ciclo Celular , Pontos de Checagem da Fase M do Ciclo Celular , Proteínas de Ciclo Celular/metabolismo , Proteínas Nucleares/metabolismo , Fuso Acromático/metabolismo , Pontos de Checagem do Ciclo Celular , Proteínas Mad2/metabolismo , Cinetocoros/metabolismoRESUMO
Peroxisomes are organelles with vital functions in metabolism and their dysfunction is associated with human diseases. To fulfill their multiple roles, peroxisomes import nuclear-encoded matrix proteins, most carrying a peroxisomal targeting signal (PTS) 1. The receptor Pex5p recruits PTS1-proteins for import into peroxisomes; whether and how this process is posttranslationally regulated is unknown. Here, we identify 22 phosphorylation sites of Pex5p. Yeast cells expressing phospho-mimicking Pex5p-S507/523D (Pex5p2D) show decreased import of GFP with a PTS1. We show that the binding affinity between a PTS1-protein and Pex5p2D is reduced. An in vivo analysis of the effect of the phospho-mimicking mutant on PTS1-proteins revealed that import of most, but not all, cargos is affected. The physiological effect of the phosphomimetic mutations correlates with the binding affinity of the corresponding extended PTS1-sequences. Thus, we report a novel Pex5p phosphorylation-dependent mechanism for regulating PTS1-protein import into peroxisomes. In a broader view, this suggests that posttranslational modifications can function in fine-tuning the peroxisomal protein composition and, thus, cellular metabolism.
Assuntos
Peroxissomos , Receptores Citoplasmáticos e Nucleares , Humanos , Fosforilação , Peroxissomos/metabolismo , Receptor 1 de Sinal de Orientação para Peroxissomos/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Proteínas de Transporte/metabolismo , Saccharomyces cerevisiae/metabolismo , Transporte ProteicoRESUMO
BACKGROUND: Syphilis is a chronic sexually transmitted disease caused by Treponema pallidum subspecies pallidum (T. pallidum), which is a public health problem that seriously affects human health worldwide. T. pallidum is characterized by early transmission and immune escape and is therefore termed an "invisible pathogen". METHODS: This review systematically summarizes the host's innate and adaptive immune responses to T. pallidum infection as well as the escape mechanisms of T. pallidum. PURPOSE: To lay the foundation for assessing the pathogenic mechanism and the systematic prevention and treatment of syphilis. CONCLUSION: The immune escape mechanism of T. pallidum plays an important role in its survival. Exploring the occurrence and development of these mechanisms has laid the foundation for the development of syphilis vaccine.
Assuntos
Sífilis , Treponema pallidum , Humanos , Vacinas BacterianasRESUMO
Selecting suitable promoters to drive gene overexpression can provide significant insight into the development of engineered bacteria. In this study, we analyzed the transcriptome data of Burkholderia pyrrocinia JK-SH007 and identified 54 highly expressed genes. The promoter sequences were located using genome-wide data and scored using the prokaryotic promoter prediction software BPROM to further screen out 18 promoter sequences. We also developed a promoter trap system based on two reporter proteins adapted for promoter optimization in B. pyrrocinia JK-SH007: firefly luciferase encoded by the luciferase gene set (Luc) and trimethoprim (TP)-resistant dihydrofolate reductase (TPr). Ultimately, eight constitutive promoters were successfully inserted into the probe vector and transformed into B. pyrrocinia JK-SH007. The transformants were successfully grown on Tp antibiotic plates, and firefly luciferase expression was determined by measuring the relative light unit (RLU). Five of the promoters (P4, P9, P10, P14, and P19) showed 1.01-2.51-fold higher activity than the control promoter λ phage transcriptional promoter (PRPL). The promoter activity was further validated via qPCR analysis, indicating that promoters P14 and P19 showed stable high transcription levels at all time points. Then, GFP and RFP proteins were overexpressed in JK-SH007. In addition, promoters P14 and P19 were successfully used to drive gene expression in Burkholderia multivorans WS-FJ9 and Escherichia coli S17-1. The two constitutive promoters can be used not only in B. pyrrocinia JK-SH007 itself to gene overexpression but also to expand the scope of application.
Assuntos
Complexo Burkholderia cepacia , Luciferases de Vaga-Lume , Regiões Promotoras Genéticas , Genes ReporterRESUMO
Transient receptor potential (TRP) ion channels are expressed in neuronal and some non-neuronal cells and are involved particularly in pain and thermosensation. We previously showed that TRPA1 is functionally expressed in human osteoarthritic (OA) chondrocytes and mediates inflammation, cartilage degradation, and pain in monosodium-iodoacetate-induced experimental OA. In the present study, we explored the expression of TRP-channels in primary human OA chondrocytes and investigated whether drugs used in the treatment of OA, ibuprofen and glucocorticoids, have effects on TRP-channel expression. OA cartilage was obtained from knee replacement surgery and chondrocytes were isolated with enzyme digestion. NGS analysis showed the expression of 19 TRP-genes in OA chondrocytes, with TRPM7, TRPV4, TRPC1, and TRPM8 having the highest counts in unstimulated cells. These results were verified with RT-PCR in samples from a different group of patients. Interleukin-1ß (IL-1ß) significantly increased TRPA1 expression, while TRPM8 and TRPC1 expression was decreased, and TRPM7 and TRPV4 expression remained unaffected. Furthermore, dexamethasone attenuated the effect of IL-1ß on TRPA1 and TRPM8 expression. The TRPM8 and TRPA1 agonist menthol increased the expression of the cartilage-degrading enzymes MMP-1, MMP-3, and MMP-13 and the inflammatory factors iNOS and IL-6 in OA chondrocytes. In conclusion, human OA chondrocytes express 19 different TRP-genes, of which the significant TRPM8 expression is a novel finding. Dexamethasone attenuated IL-1ß-induced TRPA1 expression. Interestingly, the TRPM8 and TRPA1 agonist menthol increased MMP expression. These results support the concept of TRPA1 and TRMP8 as potential novel drug targets in arthritis.
Assuntos
Canais de Cátion TRPM , Canais de Potencial de Receptor Transitório , Humanos , Canais de Cátion TRPM/metabolismo , Canais de Cátion TRPV/genética , Canais de Cátion TRPV/metabolismo , Mentol/farmacologia , Condrócitos/metabolismo , Canal de Cátion TRPA1/genética , Canal de Cátion TRPA1/metabolismo , Canais de Potencial de Receptor Transitório/genética , Dor/metabolismo , Dexametasona/farmacologia , Dexametasona/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas de Membrana/metabolismoRESUMO
KEY MESSAGE: This review presents the multiple ways how topless and topless-related proteins regulate defense activation in plants and help in optimizing the defense-growth tradeoff. Eukaryotic gene expression is tightly regulated at various levels by hormones, transcription regulators, post-translational modifications, and transcriptional coregulators. TOPLESS (TPL)/TOPLESS-related (TPR) corepressors regulate gene expression by interacting with other transcription factors. TPRs regulate auxin, gibberellins, jasmonic acid, strigolactone, and brassinosteroid signaling in plants. In general, except for GA, TPLs suppress these signaling pathways to prevent unwanted activation of hormone signaling. The association of TPL/TPRs in these hormonal signaling reflects a wide role of this class of corepressors in plants' normal and stress physiology. The involvement of TPL in immune responses was first demonstrated a decade ago as a repressor of DND1 and DND2 that are negative regulators of plant immune response. Over the last decade, several research groups have established a larger role of TPL/TPRs in plant immunity during both pattern- and effector-triggered immunity. Very recent research unraveled the significant involvement of TPRs in balancing the growth and defense trade-off. TPRs, along with proteasomal degradation complex, miRNA, and phasiRNA, suppress the activation of autoimmunity in plants under normal conditions and promote defense under pathogen attack.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Proteínas Correpressoras/metabolismo , Regulação da Expressão Gênica de Plantas , Imunidade Vegetal/genética , Fatores de Transcrição/metabolismoRESUMO
BACKGROUND: Long non-coding RNAs (lncRNAs) have been demonstrated to play vital roles in cancer development and progression. However, their biological roles and function mechanisms in liver cancer remain largely unknown. METHODS: RNA-seq was performed with clinical hepatoma tissues and paired adjacent normal liver tissues to identify differentially expressed lncRNAs. qPCR was utilized to examine the expression levels of lncRNAs. We studied the function of TLNC1 in cell growth and metastasis of hepatoma with both cell and mouse models. RNA-seq, RNA pull-down coupled with mass spectrometry, RNA immunoprecipitation, dual luciferase reporter assay, and surface plasmon resonance analysis were used to analyze the functional mechanism of TLNC1. RESULTS: Based on the intersection of our own RNA-seq, TCGA RNA-seq, and TCGA survival analysis data, TLNC1 was identified as a potential tumorigenic lncRNA of liver cancer. TLNC1 significantly enhanced the growth and metastasis of hepatoma cells both in vitro and in vivo. TLNC1 exerted its tumorigenic function through interaction with TPR and inducing the TPR-mediated transportation of p53 from nucleus to cytoplasm, thus repressing the transcription of p53 target genes and finally contributing to the progression of liver cancer. CONCLUSIONS: TLNC1 is a promising prognostic factor of liver cancer, and the TLNC1-TPR-p53 axis can serve as a potential therapeutic target for hepatoma treatment.
Assuntos
Carcinoma Hepatocelular , Neoplasias Hepáticas , RNA Longo não Codificante , Animais , Carcinogênese , Carcinoma Hepatocelular/genética , Humanos , Neoplasias Hepáticas/genética , Camundongos , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Transdução de Sinais , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismoRESUMO
In humans, structural or functional defects of the sperm flagellum induce asthenozoospermia, which accounts for the main sperm defect encountered in infertile men. Herein we focused on morphological abnormalities of the sperm flagellum (MMAF), a phenotype also termed "short tails," which constitutes one of the most severe sperm morphological defects resulting in asthenozoospermia. In previous work based on whole-exome sequencing of a cohort of 167 MMAF-affected individuals, we identified bi-allelic loss-of-function mutations in more than 30% of the tested subjects. In this study, we further analyzed this cohort and identified five individuals with homozygous truncating variants in TTC29, a gene preferentially and highly expressed in the testis, and encoding a tetratricopeptide repeat-containing protein related to the intraflagellar transport (IFT). One individual carried a frameshift variant, another one carried a homozygous stop-gain variant, and three carried the same splicing variant affecting a consensus donor site. The deleterious effect of this last variant was confirmed on the corresponding transcript and protein product. In addition, we produced and analyzed TTC29 loss-of-function models in the flagellated protist T. brucei and in M. musculus. Both models confirmed the importance of TTC29 for flagellar beating. We showed that in T. brucei the TPR structural motifs, highly conserved between the studied orthologs, are critical for TTC29 axonemal localization and flagellar beating. Overall our work demonstrates that TTC29 is a conserved axonemal protein required for flagellar structure and beating and that TTC29 mutations are a cause of male sterility due to MMAF.