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1.
Proc Natl Acad Sci U S A ; 121(22): e2321167121, 2024 May 28.
Artigo em Inglês | MEDLINE | ID: mdl-38776370

RESUMO

C-terminal Domain Nuclear Envelope Phosphatase 1 (CTDNEP1) is a noncanonical protein serine/threonine phosphatase that has a conserved role in regulating ER membrane biogenesis. Inactivating mutations in CTDNEP1 correlate with the development of medulloblastoma, an aggressive childhood cancer. The transmembrane protein Nuclear Envelope Phosphatase 1 Regulatory Subunit 1 (NEP1R1) binds CTDNEP1, but the molecular details by which NEP1R1 regulates CTDNEP1 function are unclear. Here, we find that knockdown of NEP1R1 generates identical phenotypes to reported loss of CTDNEP1 in mammalian cells, establishing CTDNEP1-NEP1R1 as an evolutionarily conserved membrane protein phosphatase complex that restricts ER expansion. Mechanistically, NEP1R1 acts as an activating regulatory subunit that directly binds and increases the phosphatase activity of CTDNEP1. By defining a minimal NEP1R1 domain sufficient to activate CTDNEP1, we determine high-resolution crystal structures of the CTDNEP1-NEP1R1 complex bound to a peptide sequence acting as a pseudosubstrate. Structurally, NEP1R1 engages CTDNEP1 at a site distant from the active site to stabilize and allosterically activate CTDNEP1. Substrate recognition is facilitated by a conserved Arg residue in CTDNEP1 that binds and orients the substrate peptide in the active site. Together, this reveals mechanisms for how NEP1R1 regulates CTDNEP1 and explains how cancer-associated mutations inactivate CTDNEP1.


Assuntos
Retículo Endoplasmático , Humanos , Cristalografia por Raios X , Retículo Endoplasmático/metabolismo , Membranas Intracelulares/metabolismo , Proteínas de Membrana/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/química , Fosfoproteínas Fosfatases/metabolismo , Fosfoproteínas Fosfatases/genética , Fosfoproteínas Fosfatases/química , Ligação Proteica
2.
J Am Soc Mass Spectrom ; 35(6): 1330-1341, 2024 Jun 05.
Artigo em Inglês | MEDLINE | ID: mdl-38662915

RESUMO

Working in tandem with kinases via a dynamic interplay of phosphorylation and dephosphorylation of proteins, phosphatases regulate many cellular processes and thus represent compelling therapeutic targets. Here we leverage ultraviolet photodissociation to shed light on the binding characteristics of two covalent phosphatase inhibitors, T65 and rabeprazole, and their respective interactions with the human small C-terminal domain phosphatase 1 (SCP1) and its single-point mutant C181A, in which a nonreactive alanine replaces one key reactive cysteine. Top-down MS/MS analysis is used to localize the binding of T65 and rabeprazole on the two proteins and estimate the relative reactivities of each cysteine residue.


Assuntos
Espectrometria de Massas em Tandem , Raios Ultravioleta , Humanos , Espectrometria de Massas em Tandem/métodos , Cisteína/química , Cisteína/metabolismo , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Ligação Proteica , Fosfoproteínas Fosfatases/antagonistas & inibidores , Fosfoproteínas Fosfatases/química , Fosfoproteínas Fosfatases/metabolismo , Modelos Moleculares
3.
Biol. Res ; 33(3/4): 197-206, 2000. graf, ilus
Artigo em Inglês | LILACS | ID: lil-454066

RESUMO

Protein phosphatases are involved in many cellular processes. One of the most abundant and best studied members of this class is protein phosphatase type-2A (PP2A). In this study, PP2A was purified from the mussel Mytilus chilensis. Using both SDS-PAGE and size exclusion gel filtration under denaturant conditions, it was confirmed that the PP2A fraction was essentially pure. The isolated enzyme is a heterodimer and the molecular estimated masses of the subunits are 62 and 28 kDa. The isolated PP2A fraction has a notably high p-NPP phosphatase activity, which is inhibited by NaCl. The hydrolytic p-NPP phosphatase activity is independent of the MgCl2 concentration. The time courses of the inhibition of the PP2A activity of p-NPP hydrolysis by increasing concentrations of three phycotoxins that are specific inhibitors of PP2A are shown. Inhibitions caused by Okadaic acid, dinophysistoxin-1 (DTX1, 35-methylokadiac acid) and Microcystine L-R are dose-dependent with inhibition constants (Ki) of 1.68, 0.40 and 0.27 nM respectively. Microcystine L-R, the most potent phycotoxin inhibitor of PP2A isolated from Mytilus chilensis with an IC50 = 0.25 ng/ml, showed the highest specific inhibition effect an the p-NPP hydrolisis. The calculated IC50 for DTX1 and OA was 0.75 ng/ml and 1.8 ng/ml respectively.


Assuntos
Animais , Ácido Okadáico/farmacologia , Bivalves/enzimologia , Fosfoproteínas Fosfatases/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Peptídeos Cíclicos/farmacologia , Piranos/farmacologia , Cromatografia Líquida de Alta Pressão , Eletroforese em Gel de Poliacrilamida , Fosfoproteínas Fosfatases/química , Fosfoproteínas Fosfatases/isolamento & purificação , Microcistinas
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