Structure and mechanism of the human CTDNEP1-NEP1R1 membrane protein phosphatase complex necessary to maintain ER membrane morphology.

in Proceedings of the National Academy of Sciences of the United States of America by Shujuan Gao, Jake W Carrasquillo Rodríguez, Shirin Bahmanyar, Michael V Airola

TLDR

  • The study investigates how a protein called CTDNEP1 helps make a part of the cell called the ER. The study found that a protein called NEP1R1 helps CTDNEP1 do its job. The study also found that mutations in CTDNEP1 can cause a type of cancer called medulloblastoma. The study suggests that NEP1R1 could be a target for treatment of medulloblastoma.

Abstract

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.

Overview

  • The study investigates the role of C-terminal Domain Nuclear Envelope Phosphatase 1 (CTDNEP1) in regulating ER membrane biogenesis and its correlation with the development of medulloblastoma. The study uses knockdown of NEP1R1 to establish CTDNEP1-NEP1R1 as an evolutionarily conserved membrane protein phosphatase complex that restricts ER expansion. The study also identifies the mechanism by which NEP1R1 regulates CTDNEP1 function and explains how cancer-associated mutations inactivate CTDNEP1.

Comparative Analysis & Findings

  • The study compares the outcomes observed under different experimental conditions, specifically the knockdown of NEP1R1 and the loss of CTDNEP1 in mammalian cells. The study finds that both conditions generate identical phenotypes, establishing CTDNEP1-NEP1R1 as an evolutionarily conserved membrane protein phosphatase complex that restricts ER expansion. The study also identifies the mechanism by which NEP1R1 regulates CTDNEP1 function, which involves direct binding and activation of CTDNEP1's phosphatase activity.

Implications and Future Directions

  • The study's findings have significant implications for the field of research and clinical practice, as they provide insights into the molecular mechanisms underlying ER membrane biogenesis and the development of medulloblastoma. The study also identifies a potential therapeutic target for the treatment of medulloblastoma, as inactivating mutations in CTDNEP1 can be targeted by NEP1R1. Future research directions could include further investigation of the role of CTDNEP1-NEP1R1 in other diseases and the development of small molecule inhibitors of NEP1R1 to target CTDNEP1.
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