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1.
J Biol Chem ; 300(1): 105541, 2024 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-38072052

RESUMO

Munc18-interacting proteins (Mints) are multidomain adaptors that regulate neuronal membrane trafficking, signaling, and neurotransmission. Mint1 and Mint2 are highly expressed in the brain with overlapping roles in the regulation of synaptic vesicle fusion required for neurotransmitter release by interacting with the essential synaptic protein Munc18-1. Here, we have used AlphaFold2 to identify and then validate the mechanisms that underpin both the specific interactions of neuronal Mint proteins with Munc18-1 as well as their wider interactome. We found that a short acidic α-helical motif within Mint1 and Mint2 is necessary and sufficient for specific binding to Munc18-1 and binds a conserved surface on Munc18-1 domain3b. In Munc18-1/2 double knockout neurosecretory cells, mutation of the Mint-binding site reduces the ability of Munc18-1 to rescue exocytosis, and although Munc18-1 can interact with Mint and Sx1a (Syntaxin1a) proteins simultaneously in vitro, we find that they have mutually reduced affinities, suggesting an allosteric coupling between the proteins. Using AlphaFold2 to then examine the entire cellular network of putative Mint interactors provides a structural model for their assembly with a variety of known and novel regulatory and cargo proteins including ADP-ribosylation factor (ARF3/ARF4) small GTPases and the AP3 clathrin adaptor complex. Validation of Mint1 interaction with a new predicted binder TJAP1 (tight junction-associated protein 1) provides experimental support that AlphaFold2 can correctly predict interactions across such large-scale datasets. Overall, our data provide insights into the diversity of interactions mediated by the Mint family and show that Mints may help facilitate a key trigger point in SNARE (soluble N-ethylmaleimide-sensitive factor attachment receptor) complex assembly and vesicle fusion.


Assuntos
Mentha , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Membrana Celular/metabolismo , Mentha/metabolismo , Proteínas Munc18/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Neurônios/metabolismo , Ligação Proteica , Proteínas SNARE/genética , Proteínas SNARE/metabolismo , Sintaxina 1/metabolismo , Humanos , Animais , Ratos , Células PC12
2.
Arthritis Res Ther ; 24(1): 273, 2022 12 14.
Artigo em Inglês | MEDLINE | ID: mdl-36517907

RESUMO

BACKGROUND: Bone marrow-derived mesenchymal stem cells (BMSCs) are general progenitor cells of osteoblasts and adipocytes and they are characterized as a fundamental mediator for bone formation. The current research studied the molecular mechanisms underlying circRNA-regulated BMSC osteogenic differentiation. METHODS: Next-generation sequencing (NGS) was employed to study abnormal circRNA and mRNA expression in BMSCs before and after osteogenic differentiation induction. Bioinformatics analysis and luciferase reporting analysis were employed to confirm correlations among miRNA, circRNA, and mRNA. RT-qPCR, ALP staining, and alizarin red staining illustrated the osteogenic differentiation ability of BMSCs. RESULTS: Data showed that circ-Iqsec1 expression increased during BMSC osteogenic differentiation. circ-Iqsec1 downregulation reduced BMSC osteogenic differentiation ability. The present investigation discovered that Satb2 played a role during BMSC osteogenic differentiation. Satb2 downregulation decreased BMSC osteogenic differentiation ability. Bioinformatics and luciferase data showed that miR-187-3p linked circ-Iqsec1 and Satb2. miR-187-3p downregulation or Satb2 overexpression restored the osteogenic differentiation capability of BMSCs post silencing circ-Iqsec1 in in vivo and in vitro experiments. Satb2 upregulation restored osteogenic differentiation capability of BMSCs post miR-187-3p overexpression. CONCLUSION: Taken together, our study found that circ-Iqsec1 induced BMSC osteogenic differentiation through the miR-187-3p/Satb2 signaling pathway.


Assuntos
Proteínas de Ligação à Região de Interação com a Matriz , Células-Tronco Mesenquimais , MicroRNAs , Osteogênese , Humanos , Medula Óssea/metabolismo , Diferenciação Celular/genética , Diferenciação Celular/fisiologia , Células Cultivadas , Proteínas de Ligação à Região de Interação com a Matriz/genética , Proteínas de Ligação à Região de Interação com a Matriz/metabolismo , Células-Tronco Mesenquimais/metabolismo , Células-Tronco Mesenquimais/fisiologia , MicroRNAs/genética , MicroRNAs/metabolismo , Osteogênese/genética , Osteogênese/fisiologia , RNA Circular/genética , RNA Circular/metabolismo , RNA Mensageiro/metabolismo , Transdução de Sinais , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
3.
Cell Calcium ; 89: 102226, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32505782

RESUMO

Contact sites between the endoplasmic reticulum (ER) and plasma membrane (PM) regulate both non-vesicular lipid transfer as well as Ca2+ signaling with multiple interactions between the two pathways. Here I discuss recent findings that offer exciting insights into the role of store-operated Ca2+ entry (SOCE), Oxysterol-binding protein (OSBP)-related proteins ORP3, Arf5 and the Arf GEF IQSec1 in this crosstalk and how they regulate cell migration and focal adhesion disassembly.


Assuntos
Sinalização do Cálcio , Membrana Celular/metabolismo , Movimento Celular , Retículo Endoplasmático/metabolismo , Lipídeos/química , Animais , Adesões Focais/metabolismo , Humanos
4.
Elife ; 92020 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-32234213

RESUMO

Coordinated assembly and disassembly of integrin-mediated focal adhesions (FAs) is essential for cell migration. Many studies have shown that FA disassembly requires Ca2+ influx, however our understanding of this process remains incomplete. Here, we show that Ca2+ influx via STIM1/Orai1 calcium channels, which cluster near FAs, leads to activation of the GTPase Arf5 via the Ca2+-activated GEF IQSec1, and that both IQSec1 and Arf5 activation are essential for adhesion disassembly. We further show that IQSec1 forms a complex with the lipid transfer protein ORP3, and that Ca2+ influx triggers PKC-dependent translocation of this complex to ER/plasma membrane (PM) contact sites adjacent to FAs. In addition to allosterically activating IQSec1, ORP3 also extracts PI4P from the PM, in exchange for phosphatidylcholine. ORP3-mediated lipid exchange is also important for FA turnover. Together, these findings identify a new pathway that links calcium influx to FA turnover during cell migration.


Assuntos
Cálcio/metabolismo , Proteínas de Ligação a Ácido Graxo/fisiologia , Adesões Focais/fisiologia , Fatores de Troca do Nucleotídeo Guanina/fisiologia , Fatores de Ribosilação do ADP/fisiologia , Membrana Celular/metabolismo , Células Cultivadas , Humanos , Metabolismo dos Lipídeos , Fosfatidilcolinas/metabolismo , Fosfatidilinositóis/fisiologia
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