Dataset Information

Structural basis of ubiquitination mediated by protein splicing in early Eukarya

ABSTRACT: This study is part of a project focused on the study of the ability of inteins to perform protein splicing. Our aim, in particular, is to characterize by mass spectrometry the splicing products of the ubl4- BIL2-ubl5 and of Ras, which strongly interacts with ubl4- BIL2-ubl5 precursor.

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ORGANISM(S): Escherichia Coli

SUBMITTER: Serena Camerini

LAB HEAD: Serena Camerini

PROVIDER: PXD020525 | Pride | 2021-01-18

REPOSITORIES: Pride

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Structural basis of ubiquitination mediated by protein splicing in early Eukarya.

Chiarini Valerio V Fiorillo Annarita A Camerini Serena S Crescenzi Marco M Nakamura Shin S Battista Theo T Guidoni Leonardo L Colotti Gianni G Ilari Andrea A

Biochimica et biophysica acta. General subjects 20210111 5

<h4>Background</h4>Inteins are intervening proteins, which are known to perform protein splicing. The reaction results in the production of an intein domain and an inteinless protein, which shows no trace of the insertion. BIL2 is part of the polyubiquitin locus of Tetrahymena thermophila (BUBL), where two bacterial-intein-like (BIL) domains lacking the C + 1 nucleophile, are flanked by two independent ubiquitin-like domains (ubl4/ubl5).<h4>Methods</h4>We solved the X-ray structures of BIL2 in b ...[more]

PMID: 33444728

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Project description:MLC1 is membrane protein highly expressed by brain perivascular astrocytes. Mutations in MLC1 account for megalencephalic leukoencephalopathy with subcortical cysts (MLC), an incurable leu-kodystrophy characterized by macrocephaly, brain edema and cysts, myelin vacuolation and as-trocyte swelling, that cause cognitive and motor dysfunctions. MLC pathological models demon-strated that in astrocytes MLC1 mutations affect the swelling-activated Cl- currents (ICl,swell) medi-ated by volume-regulated anion channel (VRAC) and the consequent regulatory volume decrease (RVD), and also induce the abnormal activation of intracellular signaling pathways linked to in-flammation/osmotic stress. Despite this knowledge, MLC1 function and MLC molecular pathogen-esis are still elusive. Following the observations that calcium regulates all the MLC1-modulated processes and that intracellular Ca2+ homeostasis is altered in MLC1-defective cells, we applied biochemistry, molecular biology, video imaging, electrophysiology and proteomic techniques on cultured human and mouse astrocytes to study MLC1/calcium signaling relationships. We revealed that MLC1 binds the Ca2+ effector proteins calmodulin (CaM) and Ca2+/CaM-dependent protein ki-nase II (CaMKII) and, as result, changes its assembly, localization and functional properties in re-sponse to Ca2+ influx or release. Noteworthy, CaM binding to the COOH terminal promotes MLC1 trafficking to the plasma membrane, while CaMKII phosphorylation of the NH2 end potentiates MLC1 activation of ICl,swell, that are critically important for RVD. Overall, our results show that MLC1 is a Ca2+-regulated protein linking VRAC function and volume regulation to Ca2+ signaling in astro-cytes, opening new avenues to clarify the defective molecular pathways of MLC and other diseas-es where astrocyte swelling and brain edema underlie the pathological process. In this contest we submit here the MS data showing MLC phosphorylation on Threonine 17.

			Action	DRS
	Band_B_ubl5_K22_K44.msf	Msf
	Band_B_ubl5_K22_K44.raw	Raw
	Band_R_Ras_K27_K166.msf	Msf
	Band_R_Ras_K27_K166.raw	Raw