Regulatory effects of post-translational modifications on zDHHC S-acyltransferases

The human zDHHC S-acyltransferase family comprises 23 enzymes that mediate the S-acylation of a multitude of cellular proteins, including channels, receptors, transporters, signaling molecules, scaffolds, and chaperones. This reversible post-transitional modification (PTM) involves the attachment of a fatty acyl chain, usually derived from palmitoyl-CoA, to specific cysteine residues on target proteins, which affects their stability, localization, and function. These outcomes are essential to control many processes, including synaptic transmission and plasticity, cell growth and differentiation, and infectivity of viruses and other pathogens. Given the physiological importance of S-acylation, it is unsurprising that perturbations in this process, including mutations in ZDHHC genes, have been linked to different neurological pathologies and cancers, and there is growing interest in zDHHC enzymes as novel drug targets. Although zDHHC enzymes control a diverse array of cellular processes and are associated with major disorders, our understanding of these enzymes is surprisingly incomplete, particularly with regard to the regulatory mechanisms controlling these enzymes. However, there is growing evidence highlighting the role of different PTMs in this process. In this review, we discuss how PTMs, including phosphorylation, S-acylation, and ubiquitination, affect the stability, localization, and function of zDHHC enzymes and speculate on possible effects of PTMs that have emerged from larger screening studies. Developing a better understanding of the regulatory effects of PTMs on zDHHC enzymes will provide new insight into the intracellular dynamics of S-acylation and may also highlight novel approaches to modulate S-acylation for clinical gain

O-GlcNAc transferase – an auxiliary factor or a full-blown oncogene?

The beta-linked N-acetyl-D-glucosamine (GlcNAc) is a posttranslational modification of serine and threonine residues catalyzed by the enzyme O-GlcNAc transferase (OGT). Increased OGT expression is a feature of most human cancers and inhibition of OGT decreases cancer cell proliferation. Antiproliferative effects are attributed to posttranslational modifications of known regulators of cancer cell proliferation, such as MYC, FOXM1, and EZH2. In general, OGT amplifies cell-specific phenotype, for example, OGT overexpression enhances reprogramming efficiency of mouse embryonic fibroblasts into stem cells. Genome-wide screens suggest that certain cancers are particularly dependent on OGT, and understanding these addictions is important when considering OGT as a target for cancer therapy. The O-GlcNAc modification is involved in most cellular processes, which raises concerns of ontarget undesirable effects of OGT-targeting therapy. Yet, emerging evidence suggest that, much like proteasome inhibitors, specific compounds targeting OGT elicit selective antiproliferative effects in cancer cells, and can prime malignant cells to other treatments. It is, therefore, essential to gain mechanistic insights on substrate specificity for OGT, develop reagents to more specifically enrich for O-GlcNAc-modified proteins, identify O-GlcNAc "readers," and develop OGT" small-molecule inhibitors. Here, we review the relevance of OGT in cancer progression and the potential targeting of this metabolic enzyme as a putative oncogene.Peer reviewe

DRUM: Inference of Disease-Associated m6A RNA Methylation Sites From a Multi-Layer Heterogeneous Network

Recent studies have revealed that the RNA N6-methyladenosine (m6A) modification plays a critical role in a variety of biological processes and associated with multiple diseases including cancers. Till this day, transcriptome-wide m6A RNA methylation sites have been identified by high-throughput sequencing technique combined with computational methods, and the information is publicly available in a few bioinformatics databases; however, the association between individual m6A sites and various diseases are still largely unknown. There are yet computational approaches developed for investigating potential association between individual m6A sites and diseases, which represents a major challenge in the epitranscriptome analysis. Thus, to infer the disease-related m6A sites, we implemented a novel multi-layer heterogeneous network-based approach, which incorporates the associations among diseases, genes and m6A RNA methylation sites from gene expression, RNA methylation and disease similarities data with the Random Walk with Restart (RWR) algorithm. To evaluate the performance of the proposed approach, a ten-fold cross validation is performed, in which our approach achieved a reasonable good performance (overall AUC: 0.827, average AUC 0.867), higher than a hypergeometric test-based approach (overall AUC: 0.7333 and average AUC: 0.723) and a random predictor (overall AUC: 0.550 and average AUC: 0.486). Additionally, we show that a number of predicted cancer-associated m6A sites are supported by existing literatures, suggesting that the proposed approach can effectively uncover the underlying epitranscriptome circuits of disease mechanisms. An online database DRUM, which stands for disease-associated ribonucleic acid methylation, was built to support the query of disease-associated RNA m6A methylation sites, and is freely available at: www.xjtlu.edu.cn/biologicalsciences/drum

Distinct p63 and p73 Protein Interactions Predict Specific Functions in mRNA Splicing and Polyploidy Control in Epithelia.

Epithelial organs are the first barrier against microorganisms and genotoxic stress, in which the p53 family members p63 and p73 have both overlapping and distinct functions. Intriguingly, p73 displays a very specific localization to basal epithelial cells in human tissues, while p63 is expressed in both basal and differentiated cells. Here, we analyse systematically the literature describing p63 and p73 protein-protein interactions to reveal distinct functions underlying the aforementioned distribution. We have found that p73 and p63 cooperate in the genome stability surveillance in proliferating cells; p73 specific interactors contribute to the transcriptional repression, anaphase promoting complex and spindle assembly checkpoint, whereas p63 specific interactors play roles in the regulation of mRNA processing and splicing in both proliferating and differentiated cells. Our analysis reveals the diversification of the RNA and DNA specific functions within the p53 family

Mechanism and structural requirements for formation of p62 bodies and degradation of p62 by selective autophagy

Selective autophagy is responsible for the lysosomal degradation of damaged and surplus cytoplasmic components, including misfolded proteins and dysfunctional organelles. Selective autophagy is required for protein and organelle quality control basally and upon stress. For the autophagic process to be precise, selective autophagy receptors (SARs) like SQSTM1/p62 are required. Autophagic substrates are often tagged with ubiquitin. Ubiquitinated substrates can be recognized by p62 and other p62-like SARs. SARs bind to lipidated ATG8 protein family members at the inner phagophore membrane and act as bridges that connect the substrate with the phagophore. Both SARs and their substrates are degraded after the fusion of the autophagosome with one or more lysosomes. Hence, p62 is both a substrate and a receptor for selective autophagy. p62 can polymerize into helical filaments via its N-terminal PB1 domain, bind to ATG8 proteins via its LIR (LC3 interacting region) motif and to the ubiquitin E3 ligase subunit KEAP1 via the adjacent KIR (KEAP1 interacting region) motif. The C-terminal UBA domain of p62 interacts with ubiquitinated substrates. The ability to form helical filaments and to bind to ubiquitin chains endows p62 with the property to form droplets in both the cytoplasm and nucleus of cells by liquid-liquid phase transition. The droplets have been called p62 bodies. They contain p62 and also other SARs like NBR1 and TAXBP as well as KEAP1 and ubiquitinated substrates. By recruiting ATG8 proteins and core autophagy components like FIP200 the droplets are degraded by selective autophagy. The p62 bodies can also function as signalosomes (signal transmitting, multimolecular protein complexes) which can also be degraded by selective autophagy to terminate their signaling. This thesis presents new studies of the roles of the PB1 domain, the LIR and KIR motifs and the UBA domain in the formation and degradation of p62 bodies. The first paper, a collaborative study led by the research group of Carsten Sachse, demonstrated the importance of the PB1-mediated polymerization of p62 into filaments for the formation of p62 bodies and their degradation by autophagy. In the second paper, we explored if a specific LIR-mediated binding of LC3B is required for autophagic degradation of p62. In our third paper, we focused on the UBA domain of p62 and post-translational modifications that occur and their effects on p62 droplet formation and degradation. It was clear from our findings that K435 plays a crucial role in the degradation of p62 by selective autophagy

AKT REGULATION BY PHOSPHORYLATION AND O-GLCNACYLATION AT THE C-TERMINAL TAIL

Akt is a Ser/Thr protein kinase that regulates cell growth, metabolism and is considered a therapeutic target for cancer. Regulation of Akt by membrane recruitment and post-translational modifications (PTMs) has been extensively studied. The most well-established mechanism for cellular Akt activation involves phosphorylation of its activation loop on Thr308 by PDK1 and of its C-terminal tail on Ser473 by mTORC2. In addition, dual phosphorylation on Ser477 and Thr479 has also been shown to activate Akt. Other C-terminal tail PTMs have been identified, but their functional impacts have not been well-characterized. Here we investigate the regulatory effects of phosphorylation of Tyr474 and O-GlcNAcylation of Ser473 on Akt. We use expressed protein ligation (EPL) as a tool to produce semisynthetic Akt proteins containing phosphoTyr474 and O-GlcNAcSer473 to dissect the enzymatic functions of these PTMs. We find that O-GlcNAcylation at Ser473 and phosphorylation at Tyr474 can also partially increase Akt’s kinase activity toward both peptide and protein substrates. Additionally, we performed kinase assays employing human protein microarrays to investigate global substrate specificity of Akt, comparing phosphorylated versus O-GlcNAcylated Ser473 forms. We observed a high similarity in the protein substrates phosphorylated by phosphoSer473 Akt and O-GlcNAcSer473 Akt. Two Akt substrates identified using microarrays, PPM1H, a protein phosphatase, and NEDD4L, an E3 ubiquitin ligase, were validated in solution phase assays and cell transfection experiments

Characterizing and exploiting the amyloid precursor protein-mint1 interaction as an Alzheimer’s disease therapeutic target

The generation of amyloid-β (Aβ) peptides through proteolytic processing of the amyloid precursor protein (APP) is a key pathogenic event in Alzheimer’s disease (AD). Aβ generation begins with APP endocytosis, which is mediated by the endocytic YENPTY sequence located in the cytoplasmic tail of APP. Mints, a family of cytosolic adaptor proteins, directly bind to the YENPTY motif of APP and facilitate APP endocytosis and amyloidogenic processing. In addition, loss of any one of the three Mint proteins decreases Aβ production in aging mouse models of AD, supporting the hypothesis that the APP-Mint interaction may provide a novel therapeutic target to selectively reduce Aβ production in AD. Characterizing the biochemical and cellular dynamics of the APP-Mint interaction is critical for understanding Aβ generation. Thus, we generated Mint1 mutants that bind with high affinity (Mint1Y633A) or low affinity (Mint1Y549A/F610A) to APP. These Mint1 mutants exhibited profound alterations in cellular localization, APP endocytosis, and Aβ production. Therapeutically, we generated a novel cell-permeable APP mimetic peptide (APPMP) that interferes with the APP-Mint interaction. This APPMP was designed to outcompete endogenous APP binding, with a 46-fold improved affinity to Mint. Treatment of primary neurons from an AD mouse model with several cell permeable APPMP variants reduced Aβ production with minimal cellular toxicity, supporting Mints as a promising novel therapeutic target for AD. The PTB domain of Mint1 that mediates APP binding is autoinhibited by an adjacent C-terminal α-helix. However, the molecular mechanisms underlying the relief of Mint1 autoinhibition are unclear. Since post-translational modification is one mechanism for alleviating protein autoinhibition, and Mint1 is highly regulated by phosphorylation, we performed mass spectrometry and identified several Mint1 phosphosites. In addition, we found constitutively-active Src kinase, a kinase implicated in Mint phosphorylation, enhanced APP-Mint1 binding. These results suggest that Src kinase-mediated phosphorylation of Mint1 may relieve Mint1 autoinhibition and promote APP-Mint1 interaction. Overall, this work biochemically characterized the Mint-APP interaction and how it affects amyloidogenic processing, provided a proof of concept for targeting the APP-Mint1 interaction as an AD therapeutic target, and suggested a novel mechanism for the relief of Mint1 autoinhibition

Reclassification of serine / threonine phosphorylation sites with +1 proline (S/T-P) sites as a distinct eukaryotic post-translational modification class

+1 proline is the most frequently found sequence motif around serine/threonine phosphorylation sites. While these proline-directed serine/threonine (S/T-P) phosphorylation sites accounts for about 1/3 of known human phosphorylation sites, it is less frequently studied than other types of phosphorylation sites: partly because of its unclear sequence consensus and reduced probability of generating attestable phenotypes when modified. In this study, we propose to establish this S/T-P phosphorylation sites as a distinctive subclass of protein phosphorylation by its own. We investigated sequence preferences, biophysical fingerprints & ontological associations of known human phosphorylation sites and found there is a significant difference between S/T-P sites and other serine / threonine phosphorylation sites, which would lead to difference consequences after phosphorylation. Also, we found 'horizontal’ – sequence averaged – information plays a major role in distinguishing S/T-P sites from non-phosphorylated counterparts, while other serine/threonine phosphorylation sites and tyrosine phosphorylation sites strongly rely on ‘vertical’ – sequence specific – information to differentiate those from non-phosphorylated counterparts. These behaviors were specifically associated with +1 proline: using proline residues on other locations or other residues on +1 site as criteria were not able to reproduce these pre-stated differences. Furthermore, we identified not only +1 proline is evolutionarily conserved across phosphoprotein orthologs, but also S/T-P sites were slowly enriched within mammalian level. Interestingly, +1 proline is more likely to be in the reconstructed ancestral sequences than actually phosphorylated serine/threonine residues, which might imply about the possible origin and evolutionary advantage of S/T-P phosphorylation. These results would not only provide an insight about this ‘neglected subclass’ of phosphorylation sites, but would also suggest this particular PTM is co-evolved with eukaryotic proteome to carry out roles associated with biological complexity

High-throughput profiling of sequence recognition by phosphotyrosine signaling proteins

Protein tyrosine kinase and phosphatase domains have binding specificities that depend on the amino acid sequence surrounding the target (phospho)tyrosine residue on their substrates. Although the preferred recognition motifs of many kinase and phosphatase domains have been characterized, we lack a quantitative description of sequence specificity that could guide predictions about signaling pathways or be used to design sequences for biomedical applications. Here, we present a platform that combines genetically-encoded peptide libraries and deep sequencing to profile sequence recognition by tyrosine kinases. We screened several tyrosine kinases against a million-peptide random library and used the resulting profiles to design high-activity sequences and predict phosphorylation efficiencies of substrates. We then screened several kinases against a library containing thousands of human proteome-derived peptides and their naturally-occurring variants. These screens recapitulated independently measured phosphorylation rates and revealed hundreds of phosphosite-proximal mutations that impact phosphosite recognition by tyrosine kinases. Finally, we have made progress towards extending this platform to the analysis of tyrosine phosphatase domains, by optimizing methods to produce tyrosine-phosphorylated bacterial display libraries and implementing methods to detect peptide dephosphorylation on the cell surface. Collectively, these experiments demonstrate the utility of our platform for rapid profiling of sequence specificity by tyrosine kinases and will shed new light on phosphotyrosine signaling