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1.
J Cell Sci ; 135(5)2022 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-34156466

RESUMO

Store-operated Ca2+ entry is a central component of intracellular Ca2+ signaling pathways. The Ca2+ release-activated channel (CRAC) mediates store-operated Ca2+ entry in many different cell types. The CRAC channel is composed of the plasma membrane (PM)-localized Orai1 channel and endoplasmic reticulum (ER)-localized STIM1 Ca2+ sensor. Upon ER Ca2+ store depletion, Orai1 and STIM1 form complexes at ER-PM junctions, leading to the formation of activated CRAC channels. Although the importance of CRAC channels is well described, the underlying mechanisms that regulate the recruitment of Orai1 to ER-PM junctions are not fully understood. Here, we describe the rapid and transient S-acylation of Orai1. Using biochemical approaches, we show that Orai1 is rapidly S-acylated at cysteine 143 upon ER Ca2+ store depletion. Importantly, S-acylation of cysteine 143 is required for Orai1-mediated Ca2+ entry and recruitment to STIM1 puncta. We conclude that store depletion-induced S-acylation of Orai1 is necessary for recruitment to ER-PM junctions, subsequent binding to STIM1 and channel activation.


Assuntos
Canais de Cálcio , Cálcio , Acilação , Cálcio/metabolismo , Canais de Cálcio/genética , Canais de Cálcio/metabolismo , Sinalização do Cálcio , Membrana Celular/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Proteína ORAI1/genética , Proteína ORAI1/metabolismo , Molécula 1 de Interação Estromal/genética , Molécula 1 de Interação Estromal/metabolismo
2.
Medicine (Baltimore) ; 100(35): e27162, 2021 Sep 03.
Artigo em Inglês | MEDLINE | ID: mdl-34477172

RESUMO

ABSTRACT: Cancer-associated fibroblasts (CAFs) have been attracting attention in recent years, but their nature has not been fully elucidated. Although CAFs have been recognized as an important therapeutic target, therapeutic agents have not been developed to date. CAFs are characterized by their high migration rate and involvement in epithelial-to-mesenchymal transition with some displaying a dendritic morphology that is reminiscent of fascin expression.The present study was designed to immunohistochemically investigate fascin expression in lung adenocarcinoma including CAFs and compare the results with existing CAF markers.We immunohistochemically investigated fascin expression in not only cancer tissue but also CAFs from 26 autopsy cases of lung adenocarcinoma. Immunohistochemistry of α-smooth muscle actin and fibroblast activation protein was also performed.Fascin-positive staining in CAFs was observed in all cases, with a strong correlation observed with existing CAF markers α-smooth muscle actin and fibroblast activation protein (P < .001). In addition, the proportion of tumor cells showing fascin-positive staining was found to correlate with its expression in CAFs (P < .05).We propose that CAFs express fascin, and that fascin may mediate crosstalk between cancer tissue and CAFs. Fascin might be a novel therapeutic target for treatments that target the cancer stroma.


Assuntos
Adenocarcinoma/metabolismo , Fibroblastos Associados a Câncer/metabolismo , Proteínas de Transporte/metabolismo , Neoplasias Pulmonares/metabolismo , Proteínas dos Microfilamentos/metabolismo , Actinas/metabolismo , Adenocarcinoma/patologia , Idoso , Idoso de 80 Anos ou mais , Endopeptidases/metabolismo , Feminino , Humanos , Pulmão/patologia , Neoplasias Pulmonares/patologia , Masculino , Proteínas de Membrana/metabolismo , Pessoa de Meia-Idade
3.
Zool Res ; 42(5): 650-659, 2021 Sep 18.
Artigo em Inglês | MEDLINE | ID: mdl-34472226

RESUMO

Phosphatidylserine (PS) is distributed asymmetrically in the plasma membrane of eukaryotic cells. Phosphatidylserine flippase (P4-ATPase) transports PS from the outer leaflet of the lipid bilayer to the inner leaflet of the membrane to maintain PS asymmetry. The ß subunit TMEM30A is indispensable for transport and proper function of P4-ATPase. Previous studies have shown that the ATP11A and TMEM30A complex is the molecular switch for myotube formation. However, the role of Tmem30a in skeletal muscle regeneration remains elusive. In the current study, Tmem30a was highly expressed in the tibialis anterior (TA) muscles of dystrophin-null ( mdx) mice and BaCl 2-induced muscle injury model mice. We generated a satellite cell (SC)-specific Tmem30a conditional knockout (cKO) mouse model to investigate the role of Tmem30a in skeletal muscle regeneration. The regenerative ability of cKO mice was evaluated by analyzing the number and diameter of regenerated SCs after the TA muscles were injured by BaCl 2-injection. Compared to the control mice, the cKO mice showed decreased Pax7 + and MYH3 + SCs, indicating diminished SC proliferation, and decreased expression of muscular regulatory factors (MYOD and MYOG), suggesting impaired myoblast proliferation in skeletal muscle regeneration. Taken together, these results demonstrate the essential role of Tmem30a in skeletal muscle regeneration.


Assuntos
Proteínas de Membrana/metabolismo , Músculo Esquelético/fisiologia , Regeneração/fisiologia , Células Satélites de Músculo Esquelético/metabolismo , Animais , Proliferação de Células , Distrofina/genética , Distrofina/metabolismo , Antagonistas de Estrogênios/toxicidade , Regulação da Expressão Gênica/fisiologia , Genótipo , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos mdx , Camundongos Knockout , Músculo Esquelético/efeitos dos fármacos , Proteína MyoD/genética , Proteína MyoD/metabolismo , Miogenina/genética , Miogenina/metabolismo , Cadeias Pesadas de Miosina/genética , Cadeias Pesadas de Miosina/metabolismo , Fator de Transcrição PAX7/genética , Fator de Transcrição PAX7/metabolismo , Regeneração/genética , Tamoxifeno/toxicidade
4.
Nat Commun ; 12(1): 4087, 2021 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-34471112

RESUMO

We utilized forebrain organoids generated from induced pluripotent stem cells of patients with a syndromic form of Autism Spectrum Disorder (ASD) with a homozygous protein-truncating mutation in CNTNAP2, to study its effects on embryonic cortical development. Patients with this mutation present with clinical characteristics of brain overgrowth. Patient-derived forebrain organoids displayed an increase in volume and total cell number that is driven by increased neural progenitor proliferation. Single-cell RNA sequencing revealed PFC-excitatory neurons to be the key cell types expressing CNTNAP2. Gene ontology analysis of differentially expressed genes (DEgenes) corroborates aberrant cellular proliferation. Moreover, the DEgenes are enriched for ASD-associated genes. The cell-type-specific signature genes of the CNTNAP2-expressing neurons are associated with clinical phenotypes previously described in patients. The organoid overgrowth phenotypes were largely rescued after correction of the mutation using CRISPR-Cas9. This CNTNAP2-organoid model provides opportunity for further mechanistic inquiry and development of new therapeutic strategies for ASD.


Assuntos
Transtorno do Espectro Autista/metabolismo , Proteínas de Membrana/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Organoides/metabolismo , Prosencéfalo/metabolismo , Adolescente , Transtorno do Espectro Autista/genética , Diferenciação Celular , Proliferação de Células , Criança , Feminino , Predisposição Genética para Doença/genética , Humanos , Células-Tronco Pluripotentes Induzidas , Proteínas de Membrana/genética , Mutação , Proteínas do Tecido Nervoso/genética , Neurônios/metabolismo , Fenótipo , Análise de Sequência de RNA
5.
Nat Commun ; 12(1): 4718, 2021 08 05.
Artigo em Inglês | MEDLINE | ID: mdl-34354069

RESUMO

Phospholipid synthesis and fat storage as triglycerides are regulated by lipin phosphatidic acid phosphatases (PAPs), whose enzymatic PAP function requires association with cellular membranes. Using hydrogen deuterium exchange mass spectrometry, we find mouse lipin 1 binds membranes through an N-terminal amphipathic helix, the Ig-like domain and HAD phosphatase catalytic core, and a middle lipin (M-Lip) domain that is conserved in mammalian and mammalian-like lipins. Crystal structures of the M-Lip domain reveal a previously unrecognized protein fold that dimerizes. The isolated M-Lip domain binds membranes both in vitro and in cells through conserved basic and hydrophobic residues. Deletion of the M-Lip domain in lipin 1 reduces PAP activity, membrane association, and oligomerization, alters subcellular localization, diminishes acceleration of adipocyte differentiation, but does not affect transcriptional co-activation. This establishes the M-Lip domain as a dimeric protein fold that binds membranes and is critical for full functionality of mammalian lipins.


Assuntos
Fosfatidato Fosfatase/química , Células 3T3-L1 , Adipogenia , Sequência de Aminoácidos , Animais , Membrana Celular/metabolismo , Sequência Conservada , Cristalografia por Raios X , Células HEK293 , Humanos , Espectrometria de Massa com Troca Hidrogênio-Deutério , Proteínas de Membrana/química , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Modelos Moleculares , Simulação de Dinâmica Molecular , Fosfatidato Fosfatase/genética , Fosfatidato Fosfatase/metabolismo , Ligação Proteica , Domínios Proteicos , Dobramento de Proteína , Multimerização Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Deleção de Sequência , Homologia de Sequência de Aminoácidos , Transcrição Genética
6.
FEBS J ; 288(16): 4728-4729, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34398531

RESUMO

With the current issue of The FEBS Journal, we are introducing a new category of invited review article contributions on Emerging Methods and Technologies. These articles provide an overview and discussion of recent, emerging methods that significantly advance and improve research efforts in the different fields of molecular and cellular research of our The FEBS Journal authors and readers. Deputy Editorial Manager Manuel Breuer and our Emerging Methods and Technologies Commissioning Editor Eric Chevet introduce the series.


Assuntos
Doenças Autoimunes/imunologia , Proteínas de Membrana/metabolismo , Neoplasias/imunologia , Polietilenotereftalatos/metabolismo , Polissacarídeos/imunologia , Humanos , Proteínas de Membrana/análise , Neoplasias/terapia , Polissacarídeos/química
7.
Nat Immunol ; 22(9): 1152-1162, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34385712

RESUMO

The transcription factor TCF-1 is essential for the development and function of regulatory T (Treg) cells; however, its function is poorly understood. Here, we show that TCF-1 primarily suppresses transcription of genes that are co-bound by Foxp3. Single-cell RNA-sequencing analysis identified effector memory T cells and central memory Treg cells with differential expression of Klf2 and memory and activation markers. TCF-1 deficiency did not change the core Treg cell transcriptional signature, but promoted alternative signaling pathways whereby Treg cells became activated and gained gut-homing properties and characteristics of the TH17 subset of helper T cells. TCF-1-deficient Treg cells strongly suppressed T cell proliferation and cytotoxicity, but were compromised in controlling CD4+ T cell polarization and inflammation. In mice with polyposis, Treg cell-specific TCF-1 deficiency promoted tumor growth. Consistently, tumor-infiltrating Treg cells of patients with colorectal cancer showed lower TCF-1 expression and increased TH17 expression signatures compared to adjacent normal tissue and circulating T cells. Thus, Treg cell-specific TCF-1 expression differentially regulates TH17-mediated inflammation and T cell cytotoxicity, and can determine colorectal cancer outcome.


Assuntos
Neoplasias do Colo/patologia , Fator 1-alfa Nuclear de Hepatócito/metabolismo , Linfócitos T Citotóxicos/imunologia , Linfócitos T Reguladores/imunologia , Células Th17/imunologia , Polipose Adenomatosa do Colo/genética , Polipose Adenomatosa do Colo/imunologia , Animais , Proliferação de Células/fisiologia , Fatores de Transcrição Forkhead/imunologia , Regulação da Expressão Gênica/genética , Regulação da Expressão Gênica/imunologia , Fator 1-alfa Nuclear de Hepatócito/genética , Memória Imunológica/imunologia , Inflamação/imunologia , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Knockout , Transcrição Genética/genética , Proteínas Supressoras de Tumor/metabolismo
8.
Int J Mol Sci ; 22(15)2021 Jul 24.
Artigo em Inglês | MEDLINE | ID: mdl-34360678

RESUMO

Epidermal keratinocyte proteins include many with an eccentric amino acid content (compositional bias), atypical ultrastructural fate (built-in protease sensitivity), or assembly visible at the light microscope level (cytoplasmic granules). However, when considered through the looking glass of intrinsic disorder (ID), these apparent oddities seem quite expected. Keratinocyte proteins with highly repetitive motifs are of low complexity but high adaptation, providing polymers (e.g., profilaggrin) for proteolysis into bioactive derivatives, or monomers (e.g., loricrin) repeatedly cross-linked to self and other proteins to shield underlying tissue. Keratohyalin granules developing from liquid-liquid phase separation (LLPS) show that unique biomolecular condensates (BMC) and proteinaceous membraneless organelles (PMLO) occur in these highly customized cells. We conducted bioinformatic and in silico assessments of representative keratinocyte differentiation-dependent proteins. This was conducted in the context of them having demonstrated potential ID with the prospect of that characteristic driving formation of distinctive keratinocyte structures. Intriguingly, while ID is characteristic of many of these proteins, it does not appear to guarantee LLPS, nor is it required for incorporation into certain keratinocyte protein condensates. Further examination of keratinocyte-specific proteins will provide variations in the theme of PMLO, possibly recognizing new BMC for advancements in understanding intrinsically disordered proteins as reflected by keratinocyte biology.


Assuntos
Proteínas Intrinsicamente Desordenadas/metabolismo , Queratinócitos/metabolismo , Animais , Humanos , Proteínas de Filamentos Intermediários/metabolismo , Queratinócitos/fisiologia , Proteínas de Membrana/metabolismo
9.
FASEB J ; 35(9): e21813, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34390512

RESUMO

Cell adhesion is tightly controlled in multicellular organisms, for example, through proteolytic ectodomain shedding of the adhesion-mediating cell surface transmembrane proteins. In the brain, shedding of cell adhesion proteins is required for nervous system development and function, but the shedding of only a few adhesion proteins has been studied in detail in the mammalian brain. One such adhesion protein is the transmembrane protein endoglycan (PODXL2), which belongs to the CD34-family of highly glycosylated sialomucins. Here, we demonstrate that endoglycan is broadly expressed in the developing mouse brains and is proteolytically shed in vitro in mouse neurons and in vivo in mouse brains. Endoglycan shedding in primary neurons was mediated by the transmembrane protease a disintegrin and metalloprotease 10 (ADAM10), but not by its homolog ADAM17. Functionally, endoglycan deficiency reduced the branching of neurites extending from primary neurons in vitro, whereas deletion of ADAM10 had the opposite effect and increased neurite branching. Taken together, our study discovers a function for endoglycan in neurite branching, establishes endoglycan as an ADAM10 substrate and suggests that ADAM10 cleavage of endoglycan may contribute to neurite branching.


Assuntos
Proteína ADAM10/metabolismo , Secretases da Proteína Precursora do Amiloide/metabolismo , Moléculas de Adesão Celular/metabolismo , Desintegrinas/metabolismo , Proteínas de Membrana/metabolismo , Neuritos/metabolismo , Neurônios/metabolismo , Sialoglicoproteínas/metabolismo , Proteína ADAM17/metabolismo , Animais , Encéfalo/metabolismo , Adesão Celular/fisiologia , Linhagem Celular , Feminino , Células HEK293 , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neurogênese/fisiologia , Proteólise
10.
Nat Commun ; 12(1): 4999, 2021 08 17.
Artigo em Inglês | MEDLINE | ID: mdl-34404769

RESUMO

The type I interferon (IFN) signaling pathway has important functions in resistance to viral infection, with the downstream induction of interferon stimulated genes (ISG) protecting the host from virus entry, replication and spread. Listeria monocytogenes (Lm), a facultative intracellular foodborne pathogen, can exploit the type I IFN response as part of their pathogenic strategy, but the molecular mechanisms involved remain unclear. Here we show that type I IFN suppresses the antibacterial activity of phagocytes to promote systemic Lm infection. Mechanistically, type I IFN suppresses phagosome maturation and proteolysis of Lm virulence factors ActA and LLO, thereby promoting phagosome escape and cell-to-cell spread; the antiviral protein, IFN-induced transmembrane protein 3 (IFITM3), is required for this type I IFN-mediated alteration. Ifitm3-/- mice are resistant to systemic infection by Lm, displaying decreased bacterial spread in tissues, and increased immune cell recruitment and pro-inflammatory cytokine signaling. Together, our findings show how an antiviral mechanism in phagocytes can be exploited by bacterial pathogens, and implicate IFITM3 as a potential antimicrobial therapeutic target.


Assuntos
Antibacterianos/farmacologia , Listeria/efeitos dos fármacos , Listeriose/imunologia , Proteínas de Membrana/antagonistas & inibidores , Proteínas de Membrana/metabolismo , Fagócitos/imunologia , Fagócitos/microbiologia , Animais , Modelos Animais de Doenças , Interações Hospedeiro-Patógeno , Interferon Tipo I/metabolismo , Listeria monocytogenes/imunologia , Masculino , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fagossomos/imunologia , Células RAW 264.7 , Transcriptoma , Fatores de Virulência , Internalização do Vírus/efeitos dos fármacos
11.
Nat Commun ; 12(1): 4980, 2021 08 17.
Artigo em Inglês | MEDLINE | ID: mdl-34404792

RESUMO

Proximity labeling (PL) with genetically-targeted promiscuous enzymes has emerged as a powerful tool for unbiased proteome discovery. By combining the spatiotemporal specificity of PL with methods for functional protein enrichment, we show that it is possible to map specific protein subclasses within distinct compartments of living cells. In particular, we develop a method to enrich subcompartment-specific RNA binding proteins (RBPs) by combining peroxidase-catalyzed PL with organic-aqueous phase separation of crosslinked protein-RNA complexes ("APEX-PS"). We use APEX-PS to generate datasets of nuclear, nucleolar, and outer mitochondrial membrane (OMM) RBPs, which can be mined for novel functions. For example, we find that the OMM RBP SYNJ2BP retains specific nuclear-encoded mitochondrial mRNAs at the OMM during translation stress, facilitating their local translation and import of protein products into the mitochondrion during stress recovery. Functional PL in general, and APEX-PS in particular, represent versatile approaches for the discovery of proteins with novel function in specific subcellular compartments.


Assuntos
RNA Mensageiro/metabolismo , RNA Mitocondrial/metabolismo , Proteínas de Ligação a RNA/metabolismo , Técnicas de Inativação de Genes , Células HEK293 , Humanos , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Mitocôndrias/metabolismo , Membranas Mitocondriais/metabolismo , Fosforilação , Proteoma/metabolismo , Proteômica , RNA Mensageiro/genética , Proteínas de Ligação a RNA/genética
12.
Int J Mol Sci ; 22(15)2021 Jul 26.
Artigo em Inglês | MEDLINE | ID: mdl-34360754

RESUMO

Peroxisome abundance is regulated by homeostasis between the peroxisomal biogenesis and degradation processes. Peroxin 16 (PEX16) is a peroxisomal protein involved in trafficking membrane proteins for de novo peroxisome biogenesis. The present study demonstrates that PEX16 also modulates peroxisome abundance through pexophagic degradation. PEX16 knockdown in human retinal pigment epithelial-1 cells decreased peroxisome abundance and function, represented by reductions in the expression of peroxisome membrane protein ABCD3 and the levels of cholesterol and plasmalogens, respectively. The activation of pexophagy under PEX16 knockdown was shown by (i) abrogated peroxisome loss under PEX16 knockdown in autophagy-deficient ATG5 knockout cell lines, and (ii) increased autophagy flux and co-localization of p62-an autophagy adaptor protein-with ABCD3 in the presence of the autophagy inhibitor chloroquine. However, the levels of cholesterol and plasmalogens did not recover despite the restoration of peroxisome abundance following chloroquine treatment. Thus, PEX16 is indispensable for maintaining peroxisome homeostasis by regulating not only the commonly known biogenesis pathway but also the autophagic degradation of peroxisomes.


Assuntos
Autofagia , Técnicas de Silenciamento de Genes , Proteínas de Membrana/deficiência , Peroxissomos/metabolismo , Epitélio Pigmentado da Retina/metabolismo , Transportadores de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/metabolismo , Proteína 5 Relacionada à Autofagia/genética , Proteína 5 Relacionada à Autofagia/metabolismo , Linhagem Celular , Humanos , Proteínas de Membrana/metabolismo , Peroxissomos/genética
14.
J Chem Theory Comput ; 17(8): 5342-5357, 2021 Aug 10.
Artigo em Inglês | MEDLINE | ID: mdl-34339605

RESUMO

The realism and accuracy of lipid bilayer simulations through molecular dynamics (MD) are heavily dependent on the lipid composition. While the field is pushing toward implementing more heterogeneous and realistic membrane compositions, a lack of high-resolution lipidomic data prevents some membrane protein systems from being modeled with the highest level of realism. Given the additional diversity of real-world cellular membranes and protein-lipid interactions, it is still not fully understood how altering membrane complexity affects modeled membrane protein functions or if it matters over long-timescale simulations. This is especially true for organisms whose membrane environments have little to no computational study, such as the plant plasma membrane. Tackling these issues in tandem, a generalized, realistic, and asymmetric plant plasma membrane with more than 10 different lipid species is constructed herein. Classical MD simulations of pure membrane constructs were performed to evaluate how altering the compositional complexity of the membrane impacted the plant membrane properties. The apo form of a plant sugar transporter, OsSWEET2b, was inserted into membrane models where lipid diversity was calculated in either a size-dependent or size-independent manner. An adaptive sampling simulation regime validated by Markov-state models was performed to capture the gating dynamics of OsSWEET2b in each of these membrane constructs. In comparison to previous OsSWEET2b simulations performed in a pure POPC bilayer, we confirm that simulations performed within a native-like membrane composition alter the stabilization of apo OsSWEET2b conformational states by ∼1 kcal/mol. The free-energy barriers of intermediate conformational states decrease when realistic membrane complexity is simplified, albeit roughly within sampling error, suggesting that protein-specific responses to membranes differ due to altered packing caused by compositional fluctuations. This work serves as a case study where a more realistic bilayer composition makes unbiased conformational sampling easier to achieve than with simplified bilayers.


Assuntos
Membrana Celular/química , Proteínas de Membrana/química , Membrana Celular/metabolismo , Bicamadas Lipídicas/química , Bicamadas Lipídicas/metabolismo , Proteínas de Membrana/metabolismo , Simulação de Dinâmica Molecular , Proteínas de Transporte de Monossacarídeos/química , Proteínas de Transporte de Monossacarídeos/metabolismo , Oryza/metabolismo , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo
16.
Nat Commun ; 12(1): 5004, 2021 08 18.
Artigo em Inglês | MEDLINE | ID: mdl-34408154

RESUMO

The endoplasmic reticulum (ER) Hsp70 chaperone BiP is regulated by AMPylation, a reversible inactivating post-translational modification. Both BiP AMPylation and deAMPylation are catalysed by a single ER-localised enzyme, FICD. Here we present crystallographic and solution structures of a deAMPylation Michaelis complex formed between mammalian AMPylated BiP and FICD. The latter, via its tetratricopeptide repeat domain, binds a surface that is specific to ATP-state Hsp70 chaperones, explaining the exquisite selectivity of FICD for BiP's ATP-bound conformation both when AMPylating and deAMPylating Thr518. The eukaryotic deAMPylation mechanism thus revealed, rationalises the role of the conserved Fic domain Glu234 as a gatekeeper residue that both inhibits AMPylation and facilitates hydrolytic deAMPylation catalysed by dimeric FICD. These findings point to a monomerisation-induced increase in Glu234 flexibility as the basis of an oligomeric state-dependent switch between FICD's antagonistic activities, despite a similar mode of engagement of its two substrates - unmodified and AMPylated BiP.


Assuntos
Monofosfato de Adenosina/metabolismo , Proteínas de Choque Térmico/química , Proteínas de Choque Térmico/metabolismo , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Nucleotidiltransferases/química , Nucleotidiltransferases/metabolismo , Monofosfato de Adenosina/química , Trifosfato de Adenosina/química , Trifosfato de Adenosina/metabolismo , Motivos de Aminoácidos , Biocatálise , Dimerização , Proteínas de Choque Térmico/genética , Humanos , Proteínas de Membrana/genética , Nucleotidiltransferases/genética , Processamento de Proteína Pós-Traducional
17.
Int J Mol Sci ; 22(16)2021 Aug 17.
Artigo em Inglês | MEDLINE | ID: mdl-34445536

RESUMO

Chronic pain is a leading health and socioeconomic problem and an unmet need exists for long-lasting analgesics. SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptors) are required for neuropeptide release and noxious signal transducer surface trafficking, thus, selective expression of the SNARE-cleaving light-chain protease of botulinum neurotoxin A (LCA) in peripheral sensory neurons could alleviate chronic pain. However, a safety concern to this approach is the lack of a sensory neuronal promoter to prevent the expression of LCA in the central nervous system. Towards this, we exploit the unique characteristics of Pirt (phosphoinositide-interacting regulator of TRP), which is expressed in peripheral nociceptive neurons. For the first time, we identified a Pirt promoter element and cloned it into a lentiviral vector driving transgene expression selectively in peripheral sensory neurons. Pirt promoter driven-LCA expression yielded rapid and concentration-dependent cleavage of SNAP-25 in cultured sensory neurons. Moreover, the transcripts of pain-related genes (TAC1, tachykinin precursor 1; CALCB, calcitonin gene-related peptide 2; HTR3A, 5-hydroxytryptamine receptor 3A; NPY2R, neuropeptide Y receptor Y2; GPR52, G protein-coupled receptor 52; SCN9A, sodium voltage-gated channel alpha subunit 9; TRPV1 and TRPA1, transient receptor potential cation channel subfamily V member 1 and subfamily A member 1) in pro-inflammatory cytokines stimulated sensory neurons were downregulated by viral mediated expression of LCA. Furthermore, viral expression of LCA yielded long-lasting inhibition of pain mediator release. Thus, we show that the engineered Pirt-LCA virus may provide a novel means for long lasting pain relief.


Assuntos
Toxinas Botulínicas Tipo A/farmacologia , Neuropeptídeos/metabolismo , Dor/prevenção & controle , Sistema Nervoso Periférico/metabolismo , Células Receptoras Sensoriais/metabolismo , Proteína 25 Associada a Sinaptossoma/metabolismo , Animais , Animais Recém-Nascidos , Feminino , Humanos , Masculino , Fusão de Membrana , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Nociceptores/efeitos dos fármacos , Nociceptores/metabolismo , Dor/genética , Dor/metabolismo , Dor/patologia , Sistema Nervoso Periférico/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Células Receptoras Sensoriais/efeitos dos fármacos , Proteína 25 Associada a Sinaptossoma/genética
18.
Int J Mol Sci ; 22(16)2021 Aug 18.
Artigo em Inglês | MEDLINE | ID: mdl-34445578

RESUMO

The phenomenon of how oncogenes and tumor-suppressor mutations can synergize to promote tumor fitness and cancer progression can be studied in relatively simple animal model systems such as Drosophila melanogaster. Almost two decades after the landmark discovery of cooperative oncogenesis between oncogenic RasV12 and the loss of the tumor suppressor scribble in flies, this and other tumor models have provided new concepts and findings in cancer biology that has remarkable parallels and relevance to human cancer. Here we review findings using the RasV12; scrib-/- tumor model and how it has contributed to our understanding of how these initial simple genetic insults cooperate within the tumor cell to set in motion the malignant transformation program leading to tumor growth through cell growth, cell survival and proliferation, dismantling of cell-cell interactions, degradation of basement membrane and spreading to other organs. Recent findings have demonstrated that cooperativity goes beyond cell intrinsic mechanisms as the tumor interacts with the immediate cells of the microenvironment, the immune system and systemic organs to eventually facilitate malignant progression.


Assuntos
Carcinogênese , Proteínas de Membrana/metabolismo , Mutação , Neoplasias/patologia , Microambiente Tumoral , Proteínas Supressoras de Tumor/metabolismo , Proteínas ras/metabolismo , Animais , Humanos , Proteínas de Membrana/genética , Neoplasias/etiologia , Neoplasias/metabolismo , Transdução de Sinais , Proteínas Supressoras de Tumor/genética , Proteínas ras/genética
19.
Oxid Med Cell Longev ; 2021: 2989974, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34457111

RESUMO

In the present study, we used lipopolysaccharide- (LPS-) stimulated H9C2 cardiomyocytes to investigate whether irisin treatment attenuates septic cardiomyopathy via Fundc1-related mitophagy. Fundc1 levels and mitophagy were significantly reduced in LPS-stimulated H9C2 cardiomyocytes but were significantly increased by irisin treatment. Irisin significantly increased ATP production and the activities of mitochondrial complexes I and III in the LPS-stimulated cardiomyocytes. Irisin also improved glucose metabolism and significantly reduced LPS-induced levels of reactive oxygen species by increasing the activities of antioxidant enzymes, glutathione peroxidase (GPX), and superoxide dismutase (SOD), as well as levels of reduced glutathione (GSH). TUNEL assays showed that irisin significantly reduced LPS-stimulated cardiomyocyte apoptosis by suppressing the activation of caspase-3 and caspase-9. However, the beneficial effects of irisin on oxidative stress, mitochondrial metabolism, and viability of LPS-stimulated H9C2 cardiomyocytes were abolished by silencing Fundc1. These results demonstrate that irisin abrogates mitochondrial dysfunction, oxidative stress, and apoptosis through Fundc1-related mitophagy in LPS-stimulated H9C2 cardiomyocytes. This suggests irisin is a potentially useful treatment for septic cardiomyopathy, though further investigations are necessary to confirm our findings.


Assuntos
Apoptose , Cardiomiopatias/patologia , Fibronectinas/metabolismo , Proteínas de Membrana/metabolismo , Mitocôndrias/patologia , Proteínas Mitocondriais/metabolismo , Miócitos Cardíacos/patologia , Estresse Oxidativo , Sepse/patologia , Animais , Cardiomiopatias/etiologia , Cardiomiopatias/metabolismo , Células Cultivadas , Fibronectinas/genética , Proteínas de Membrana/genética , Mitocôndrias/metabolismo , Proteínas Mitocondriais/genética , Mitofagia , Miócitos Cardíacos/metabolismo , Ratos , Sepse/etiologia , Sepse/metabolismo
20.
Clin Lab ; 67(8)2021 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-34383404

RESUMO

BACKGROUND: Abnormal expression of miR 20a is reported in various types of malignancy neoplasms. However, its function is not consistent in different tumors. This study aims to explore the potential functions of miR 20a and its underlying mechanisms in bladder cancer. METHODS: Ninety-six patients diagnosed with bladder cancer were recruited into the study. The expression levels of miR-20a in bladder cancer samples and adjacent non-tumor samples were investigated by qRT-PCR. Wound healing, CCK8, and transwell migration assays were carried out for determining the functions of miR20a. Bioinformatics analysis was used for predicting the downstream gene of miR-20a. Western blot, qRT-PCR, and fluorescent reporter assays were used to verify the target gene. RESULTS: MiR-20a was significantly increased in bladder cancer tissues, and its rising level was closely correlated with histological grade, clinical stage, recurrence and metastasis in bladder cancer. Exogenous upregulation of miR-20a expression obviously enhanced the aggressive biological functions of bladder cancer in vitro. LASS2 was verified to be a target gene of miR-20a. Moreover, miR-20a can negatively regulate LASS2 at protein and mRNA levels. CONCLUSIONS: Increasing miR-20a is closely related to aggressive clinicopathological features. MiR 20a plays an oncogenic role in bladder cancer, which contributes to target LASS2 directly.


Assuntos
Proteínas de Membrana/genética , MicroRNAs , Esfingosina N-Aciltransferase/genética , Proteínas Supressoras de Tumor/genética , Neoplasias da Bexiga Urinária , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Regulação Neoplásica da Expressão Gênica , Humanos , Proteínas de Membrana/metabolismo , MicroRNAs/genética , Invasividade Neoplásica , Recidiva Local de Neoplasia , Esfingosina N-Aciltransferase/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Neoplasias da Bexiga Urinária/genética
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