Predicting the targeting of tail-anchored proteins to subcellular compartments in mammalian cells

This is the author accepted manuscript. The final version is available from Company of Biologists via the DOI in this record.Tail-anchored (TA) proteins contain a single transmembrane domain (TMD) at the Cterminus, anchoring them to organelle membranes where they mediate a variety of critical cellular processes. Mutations in individual TA proteins cause a number of severe inherited disorders. However, the molecular mechanisms and signals facilitating proper TA protein targeting are not fully understood, in particular in mammals. Here, we identify additional TA proteins at peroxisomes or shared by multiple organelles in mammals and reveal that a combination of TMD hydrophobicity and tail charge determines targeting to distinct organelles. Specifically, an increase in tail charge can override a hydrophobic TMD signal and re-direct a protein from the ER to peroxisomes or mitochondria and vice versa. We demonstrate that subtle alterations in those physicochemical parameters can shift TA protein targeting between organelles, explaining why peroxisomes and mitochondria share many TA proteins. Our analyses enabled us to allocate characteristic physicochemical parameters to different organelle groups. This classification allows for the first time, successful prediction of the location of uncharacterized TA proteins.We thank colleagues who provided materials (see Tables S1-S4) and acknowledge support from A. C. Magalhães, M. Almeida, D. Tuerker, S. Kuehl and C. Davies. This work was supported by the Biotechnology and Biological Sciences Research Council (BB/K006231/1 to M.S.), a Wellcome Trust Institutional Strategic Support Award (WT097835MF, WT105618MA to M.S.), the Portuguese Foundation for Science and Technology and FEDER/COMPETE (PTDC/BIA-BCM/118605/2010 to M.S.; SFRH/BD/37647/2007 to N.B.; SFRH/BPD/77619/2011 and UID/BIM/04501/2013 to D.R.). M.W., E.A.G., and M.S. are supported by Marie Curie Initial Training Network (ITN) action PerFuMe (316723)

Acetylation reprograms MITF target selectivity and residence time

Abstract The ability of transcription factors to discriminate between different classes of binding sites associated with specific biological functions underpins effective gene regulation in development and homeostasis. How this is achieved is poorly understood. The microphthalmia-associated transcription factor MITF is a lineage-survival oncogene that plays a crucial role in melanocyte development and melanoma. MITF suppresses invasion, reprograms metabolism and promotes both proliferation and differentiation. How MITF distinguishes between differentiation and proliferation-associated targets is unknown. Here we show that compared to many transcription factors MITF exhibits a very long residence time which is reduced by p300/CBP-mediated MITF acetylation at K206. While K206 acetylation also decreases genome-wide MITF DNA-binding affinity, it preferentially directs DNA binding away from differentiation-associated CATGTG motifs toward CACGTG elements. The results reveal an acetylation-mediated switch that suppresses differentiation and provides a mechanistic explanation of why a human K206Q MITF mutation is associated with Waardenburg syndrome

MITF has a central role in regulating starvation-induced autophagy in melanoma.

The MITF transcription factor is a master regulator of melanocyte development and a critical factor in melanomagenesis. The related transcription factors TFEB and TFE3 regulate lysosomal activity and autophagy processes known to be important in melanoma. Here we show that MITF binds the CLEAR-box element in the promoters of lysosomal and autophagosomal genes in melanocytes and melanoma cells. The crystal structure of MITF bound to the CLEAR-box reveals how the palindromic nature of this motif induces symmetric MITF homodimer binding. In metastatic melanoma tumors and cell lines, MITF positively correlates with the expression of lysosomal and autophagosomal genes, which, interestingly, are different from the lysosomal and autophagosomal genes correlated with TFEB and TFE3. Depletion of MITF in melanoma cells and melanocytes attenuates the response to starvation-induced autophagy, whereas the overexpression of MITF in melanoma cells increases the number of autophagosomes but is not sufficient to induce autophagic flux. Our results suggest that MITF and the related factors TFEB and TFE3 have separate roles in regulating a starvation-induced autophagy response in melanoma. Understanding the normal and pathophysiological roles of MITF and related transcription factors may provide important clinical insights into melanoma therapy

Acetylation reprograms MITF target selectivity and residence time

The ability of transcription factors to discriminate between different classes of binding sites associated with specific biological functions underpins effective gene regulation in development and homeostasis. How this is achieved is poorly understood. The microphthalmia-associated transcription factor MITF is a lineage-survival oncogene that plays a crucial role in melanocyte development and melanoma. MITF suppresses invasion, reprograms metabolism and promotes both proliferation and differentiation. How MITF distinguishes between differentiation and proliferation-associated targets is unknown. Here we show that compared to many transcription factors MITF exhibits a very long residence time which is reduced by p300/CBP-mediated MITF acetylation at K206. While K206 acetylation also decreases genome-wide MITF DNA-binding affinity, it preferentially directs DNA binding away from differentiation-associated CATGTG motifs toward CACGTG elements. The results reveal an acetylation-mediated switch that suppresses differentiation and provides a mechanistic explanation of why a human K206Q MITF mutation is associated with Waardenburg syndrome

A short isoform of ATG7 fails to lipidate LC3/GABARAP

Publisher's version (útgefin grein)Autophagy is a degradation pathway important for cellular homeostasis. The E1-like enzyme ATG7 is a key component of the autophagy machinery, with the main function of mediating the lipidation of LC3/GABARAP during autophagosome formation. By analysing mRNA-sequencing data we found that in addition to the full-length ATG7 isoform, various tissues express a shorter isoform lacking an exon of 27 amino acids in the C-terminal part of the protein, termed ATG7(2). We further show that ATG7(2) does not bind LC3B and fails to mediate the lipidation of members of the LC3/GABARAP family. We have thus identified an isoform of ATG7 that is unable to carry out the best characterized function of the protein during the autophagic response. This short isoform will have to be taken into consideration when further studying the role of ATG7.This work was supported by a START Marie Curie/Icelandic Research Fund grant (M.H.O.; grant number 120457-041), Icelandic Research Fund grant (M.H.O.; grant number 184727-051), an Icelandic Cancer Society Research Fund grant (M.H.O.), Icelandic Research Fund grant (E.S.; grant number 152715) and by an Erwin Schrödinger fellowship grant from the Austrian Science Fund (V.F.; grant number: J 3864-B26).Peer Reviewe

Tuning transcription factor availability through acetylation-mediated genomic redistribution

It is widely assumed that decreasing transcription factor DNA-binding affinity reduces transcription initiation by diminishing occupancy of sequence-specific regulatory elements. However, in vivo transcription factors find their binding sites while confronted with a large excess of low-affinity degenerate motifs. Here, using the melanoma lineage survival oncogene MITF as a model, we show that low-affinity binding sites act as a competitive reservoir in vivo from which transcription factors are released by mitogen-activated protein kinase (MAPK)-stimulated acetylation to promote increased occupancy of their regulatory elements. Consequently, a low-DNA-binding-affinity acetylation-mimetic MITF mutation supports melanocyte development and drives tumorigenesis, whereas a high-affinity non-acetylatable mutant does not. The results reveal a paradoxical acetylation-mediated molecular clutch that tunes transcription factor availability via genome-wide redistribution and couples BRAF to tumorigenesis. Our results further suggest that p300/CREB-binding protein-mediated transcription factor acetylation may represent a common mechanism to control transcription factor availability

An NAD+ Phosphorylase Toxin Triggers Mycobacterium tuberculosis Cell Death.

Toxin-antitoxin (TA) systems regulate fundamental cellular processes in bacteria and represent potential therapeutic targets. We report a new RES-Xre TA system in multiple human pathogens, including Mycobacterium tuberculosis. The toxin, MbcT, is bactericidal unless neutralized by its antitoxin MbcA. To investigate the mechanism, we solved the 1.8 Å-resolution crystal structure of the MbcTA complex. We found that MbcT resembles secreted NAD+-dependent bacterial exotoxins, such as diphtheria toxin. Indeed, MbcT catalyzes NAD+ degradation in vitro and in vivo. Unexpectedly, the reaction is stimulated by inorganic phosphate, and our data reveal that MbcT is a NAD+ phosphorylase. In the absence of MbcA, MbcT triggers rapid M. tuberculosis cell death, which reduces mycobacterial survival in macrophages and prolongs the survival of infected mice. Our study expands the molecular activities employed by bacterial TA modules and uncovers a new class of enzymes that could be exploited to treat tuberculosis and other infectious diseases

The MITF family of transcription factors: Role in endolysosomal biogenesis, Wnt signaling, and oncogenesis

Canonical Wnt signaling influences cellular fate and proliferation through inhibition of Glycogen Synthase Kinase (GSK3) and the subsequent stabilization of its many substrates, most notably β-Catenin, a transcriptional co-activator. MITF, a melanoma oncogene member of the microphthalmia family of transcription factors (MiT), was recently found to contain novel GSK3 phosphorylation sites and to be stabilized by Wnt. Other MiT members, TFEB and TFE3, are known to play important roles in cellular clearance pathways by transcriptionally regulating the biogenesis of lysosomes and autophagosomes via activation of CLEAR elements in gene promoters of target genes. Recent studies suggest that MITF can also upregulate many lysosomal genes. MiT family members are dysregulated in cancer and are considered oncogenes, but the underlying oncogenic mechanisms remain unclear. Here we review the role of MiT members, including MITF, in lysosomal biogenesis, and how cancers overexpressing MITF, TFEB or TFE3 could rewire the lysosomal pathway, inhibit cellular senescence, and activate Wnt signaling by increasing sequestration of negative regulators of Wnt signaling in multivesicular bodies (MVBs). Microarray studies suggest that MITF expression inhibits macroautophagy. In melanoma the MITF-driven increase in MVBs generates a positive feedback loop between MITF, Wnt, and MVBs

A novel region within a conserved domain in ATG7 emerged in vertebrates

The E1-like enzyme ATG7 belongs to a group of ATG proteins that mediate the autophagy process. Autophagy is a highly conserved degradation pathway important for maintaining homeostasis in eukaryotic cells. Here, we study the evolution of E1 enzymes and specifically describe a region of ATG7 that emerged early in vertebrates. This vertebrate-specific region (VSR) is situated within the adenylation domain of the protein, which is the most conserved domain of E1 enzymes and is of prokaryotic origin. A comparative analysis revealed that ATG7 is unique in this respect, as in other E1 enzyme family members this domain is highly conserved from yeast to humans and has not experienced insertions of extra amino acids. The function of the VSR domain is unknown, but two residues within the region, D522 and S531 have been linked with cancer in humans. Analysis of natural selection indicates positive selection on S531 only on the mammalian clade. Notably, this was the only residue in ATG7 showing this signal. Interestingly, structural analysis of ATG7 predicted that the VSR may be intrinsically disordered and could harbor a macro-molecular binding site. Analysis of cells expressing ATG7 lacking the VSR indicated that these cells are unable to facilitate the lipidation of LC3, suggesting an important role of this region in autophagy. Abbreviations: aBSREL - an adaptive branch-site random effects likelihood; AD - adenylation domain; ATGs - autophagy-related genes; Baf-A1 - Bafilomycin-A1; EV - empty-vector; CTD - C-terminal domain; ECTD - extreme C-terminal domain; EMT - epithelial-mesenchymal transition; FEL - fixed effects likelihood; GABARAP - gamma-aminobutyric acid receptor-associated protein; LC3 - microtubule-associated protein 1A/1B-light chain 3; MEFs - mouse embryonic fibroblasts; MOCS3 - molybdenum cofactor synthesis 3; NTD - N-terminal domain; UBL ubiquitin like protein; VSR - vertebrate specific regio