Novel chemical proteomics approaches to study N-myristoylation and N-terminal methionine excision

N-terminal modifications may constitute the first modifications any protein acquires and can modulate protein function by altering protein stability and 3D structure, promoting or interfering with protein-protein interactions and regulating membrane targeting, localisation or secretion. Here, two interlinked N-terminal protein modifications have been studied: initiator methionine (iMet) excision and co-translational N-myristoylation. N-myristoylation involves de attachment of myristic acid, a 14-carbon saturated fatty acid, onto exposed N-terminal glycines of protein substrates. In humans, this reaction is catalysed by N-myristoyltransferases 1 and 2. The two NMT isoforms share a high degree of sequence and structural similarity, which has hindered the development of isoform-specific inhibitors to date and prevented the dissection of isoform- specific substrate pools. Using CRISPR/Cas9 in combination with a previously described metabolic labelling approach and whole proteome profiling, NMT1, and not NMT2, was defined as the main enzyme responsible for N-myristoylation of proteins in the cancer cell. In addition, a novel method was designed to accurately assess on-target activity of NMT inhibitors and the fate of N-myristoylated substrates across the whole proteome upon NMT inhibition. This new approach relies on post-lysis labelling of exposed N-terminal glycines by S. aureus sortase A (SrtA) and no longer relying on metabolic labelling, it can be applied to any type of biological sample. Methionine aminopeptidases (MetAPs) catalyse initiator methionine (iMet) removal from nascent proteins and are essential to maintain healthy proteome dynamics by priming other N-terminal modifications such as N-acetylation or N-myristoylation and modulating protein localisation and stability. MetAP2 has been explored by pharmaceutical companies for decades for the treatment of cancer and obesity. However, the links between MetAP2 inhibition and phenotypic effects are still poorly understood. Here, a novel chemical proteomics workflow is proposed to elucidate the substrates of MetAP2 systematically and uncover the missing link between MetAP2 inhibition and phenotype. This new strategy is based on metabolic labelling of cells with the methionine analogue azidohomoalanine (AHA) and in combination with specific pharmacological inhibition of MetAP2, allowed identification of >70 substrates of MetAP2, 94% of which were unknown until reported here. Together, this work provides fundamental insights into the biological role and importance of N-myristoylation and iMet excision in cancer and shapes the path for future steps in the use of NMT and MetAP2 inhibitors for the treatment of human disease.Open Acces

Study on the activation mechanism of BCL-2 related ovarian killer (BOK)

BCL-2 family proteins are key regulators of the mitochondrial apoptotic machinery, controlling the mitochondrial outer membrane (MOM) permeabilization (MOMP). BCL-2 related Ovarian Killer (BOK) is a poorly understood pro-apoptotic member of this protein family. It has been reported that BOK localizes predominantly (although not exclusively) at membranes of the endoplasmic reticulum and of the Golgi apparatus. However, it is unclear whether BOK also operates at the MOM to promote apoptosis, as other pro-apoptotic BCL-2 family members do. Basing on the fact that the other two BAX-like pro-apoptotic members have been reported to oligomerize in order to induce MOMP, site-directed mutagenesis was used to generate two point mutations that predictably eliminated BOK’s oligomerization capacity. Then, the effect of such mutations on BOK’s membrane activity was examined using fluorescence spectroscopy

FSP1 is a glutathione-independent ferroptosis suppressor

Ferroptosis is an iron-dependent form of necrotic cell death marked by oxidative damage to phospholipids1,2. To date, ferroptosis has been believed to be controlled only by the phospholipid hydroperoxide-reducing enzyme glutathione peroxidase 4 (GPX4)3,4 and radical-trapping antioxidants5,6. However, elucidation of the factors that underlie the sensitivity of a given cell type to ferroptosis7 is critical to understand the pathophysiological role of ferroptosis and how it may be exploited for the treatment of cancer. Although metabolic constraints8 and phospholipid composition9,10 contribute to ferroptosis sensitivity, no cell-autonomous mechanisms have been identified that account for the resistance of cells to ferroptosis. Here we used an expression cloning approach to identify genes in human cancer cells that are able to complement the loss of GPX4. We found that the flavoprotein apoptosis-inducing factor mitochondria-associated 2 (AIFM2) is a previously unrecognized anti-ferroptotic gene. AIFM2, which we renamed ferroptosis suppressor protein 1 (FSP1) and which was initially described as a pro-apoptotic gene11, confers protection against ferroptosis elicited by GPX4 deletion. We further demonstrate that the suppression of ferroptosis by FSP1 is mediated by ubiquinone (also known as coenzyme Q10 (CoQ10)): the reduced form, ubiquinol, traps lipid peroxyl radicals that mediate lipid peroxidation, whereas FSP1 catalyses the regeneration of CoQ10 using NAD(P)H. Pharmacological targeting of FSP1 strongly synergizes with GPX4 inhibitors to trigger ferroptosis in a number of cancer entities. In conclusion, the FSP1–CoQ10–NAD(P)H pathway exists as a stand-alone parallel system, which co-operates with GPX4 and glutathione to suppress phospholipid peroxidation and ferroptosis

A KLK6 activity-based probe reveals a role for KLK6 activity in pancreatic cancer cell invasion.

Pancreatic cancer has the lowest survival rate of all common cancers due to late diagnosis and limited treatment options. Serine hydrolases are known to mediate cancer progression and metastasis through initiation of signaling cascades and cleavage of extracellular matrix proteins, and the kallikrein-related peptidase (KLK) family of secreted serine proteases have emerging roles in pancreatic ductal adenocarcinoma (PDAC). However, the lack of reliable activity-based probes (ABPs) to profile KLK activity has hindered progress in validation of these enzymes as potential targets or biomarkers. Here, we developed potent and selective ABPs for KLK6 by using a positional scanning combinatorial substrate library and characterized their binding mode and interactions by X-ray crystallography. The optimized KLK6 probe IMP-2352 (kobs/I = 11,000 M-1 s-1) enabled selective detection of KLK6 activity in a variety of PDAC cell lines, and we observed that KLK6 inhibition reduced the invasiveness of PDAC cells that secrete active KLK6. KLK6 inhibitors were combined with N-terminomics to identify potential secreted protein substrates of KLK6 in PDAC cells, providing insights into KLK6-mediated invasion pathways. These novel KLK6 ABPs offer a toolset to validate KLK6 and associated signaling partners as targets or biomarkers across a range of diseases

Whole proteome profiling of N-Myristoyltransferase activity and inhibition using sortase A

N-myristoylation is the covalent addition of a 14-carbon saturated fatty acid (myristate) to the N-terminal glycine of specific protein substrates by N-myristoyltransferase (NMT) and plays an important role in protein regulation by controlling localization, stability, and interactions. We developed a novel method for whole-proteome profiling of free N-terminal glycines through labeling with S. Aureus sortase A (SrtA) and used it for assessment of target engagement by an NMT inhibitor. Analysis of the SrtA-labeling pattern with an engineered biotinylated depsipeptide SrtA substrate (Biotin-ALPET-Haa, Haa = 2-hydroxyacetamide) enabled whole proteome identification and quantification of de novo generated N-terminal Gly proteins in response to NMT inhibition by nanoLC-MS/MS proteomics, and was confirmed for specific substrates across multiple cell lines by gel-based analyses and ELISA. To achieve optimal signal over background noise we introduce a novel and generally applicable improvement to the biotin/avidin affinity enrichment step by chemically dimethylating commercial NeutrAvidin resin and combining this with two-step LysC on-bead/trypsin off-bead digestion, effectively eliminating avidin-derived tryptic peptides and enhancing identification of enriched peptides. We also report SrtA substrate specificity in whole-cell lysates for the first time, confirming SrtA promiscuity beyond its recognized preference for N-terminal glycine, and its usefulness as a tool for unbiased labeling of N-terminal glycine-containing proteins. Our new methodology is complementary to metabolic tagging strategies, providing the first approach for whole proteome gain-of signal readout for NMT inhibition in complex samples which are not amenable to metabolic tagging