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1.
bioRxiv ; 2023 Oct 27.
Artigo em Inglês | MEDLINE | ID: mdl-37961422

RESUMO

The Polycomb Group (PcG) complex PRC1 represses transcription, forms condensates in cells, and modifies chromatin architecture. These processes are connected through the essential, polymerizing Sterile Alpha Motif (SAM) present in the PRC1 subunit Polyhomeotic (Ph). In vitro, Ph SAM drives formation of short oligomers and phase separation with DNA or chromatin in the context of a Ph truncation ("mini-Ph"). Oligomer length is controlled by the long disordered linker (L) that connects the SAM to the rest of Ph--replacing Drosophila PhL with the evolutionarily diverged human PHC3L strongly increases oligomerization. How the linker controls SAM polymerization, and how polymerization and the linker affect condensate formation are not know. We analyzed PhL and PHC3L using biochemical assays and molecular dynamics (MD) simulations. PHC3L promotes mini-Ph phase separation and makes it relatively independent of DNA. In MD simulations, basic amino acids in PHC3L form contacts with acidic amino acids in the SAM. Engineering the SAM to make analogous charge-based contacts with PhL increased polymerization and phase separation, partially recapitulating the effects of the PHC3L. Ph to PHC3 linker swaps and SAM surface mutations alter Ph condensate formation in cells, and Ph function in Drosophila imaginal discs. Thus, SAM-driven phase separation and polymerization are conserved between flies and mammals, but the underlying mechanisms have diverged through changes to the disordered linker.

2.
Biochemistry ; 59(30): 2764-2775, 2020 08 04.
Artigo em Inglês | MEDLINE | ID: mdl-32627541

RESUMO

Co-translational folding studies of membrane proteins lag behind cytosolic protein investigations largely due to the technical difficulty in maintaining membrane lipid environments for correct protein folding. Stalled ribosome-bound nascent chain complexes (RNCs) can give snapshots of a nascent protein chain as it emerges from the ribosome during biosynthesis. Here, we demonstrate how SecM-facilitated nascent chain stalling and native nanodisc technologies can be exploited to capture in vivo-generated membrane protein RNCs within their native lipid compositions. We reveal that a polytopic membrane protein can be successfully stalled at various stages during its synthesis and the resulting RNC extracted within either detergent micelles or diisobutylene-maleic acid co-polymer native nanodiscs. Our approaches offer tractable solutions for the structural and biophysical interrogation of nascent membrane proteins of specified lengths, as the elongating nascent chain emerges from the ribosome and inserts into its native lipid milieu.


Assuntos
Proteínas de Membrana/metabolismo , Biossíntese de Proteínas , Ribossomos/metabolismo , Alcenos/química , Sequência de Aminoácidos , Maleatos/química , Micelas , Nanopartículas/química , Estabilidade Proteica , Estrutura Secundária de Proteína , Proteínas/química , Canais de Translocação SEC/metabolismo
3.
Elife ; 72018 09 11.
Artigo em Inglês | MEDLINE | ID: mdl-30204083

RESUMO

Locomotion circuits developed in simple animals, and circuit motifs further evolved in higher animals. To understand locomotion circuit motifs, they must be characterized in many models. The nematode Caenorhabditis elegans possesses one of the best-studied circuits for undulatory movement. Yet, for 1/6th of the cholinergic motor neurons (MNs), the AS MNs, functional information is unavailable. Ventral nerve cord (VNC) MNs coordinate undulations, in small circuits of complementary neurons innervating opposing muscles. AS MNs differ, as they innervate muscles and other MNs asymmetrically, without complementary partners. We characterized AS MNs by optogenetic, behavioral and imaging analyses. They generate asymmetric muscle activation, enabling navigation, and contribute to coordination of dorso-ventral undulation as well as anterio-posterior bending wave propagation. AS MN activity correlated with forward and backward locomotion, and they functionally connect to premotor interneurons (PINs) for both locomotion regimes. Electrical feedback from AS MNs via gap junctions may affect only backward PINs.


Assuntos
Neurônios Colinérgicos/fisiologia , Locomoção/fisiologia , Neurônios Motores/fisiologia , Optogenética , Animais , Animais Geneticamente Modificados , Caenorhabditis elegans/genética , Caenorhabditis elegans/crescimento & desenvolvimento , Interneurônios/fisiologia , Músculos/inervação , Músculos/fisiologia
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