Melissa M. Dix

Global Mapping of the Topography and Magnitude of Proteolytic Events in Apoptosis

Proteolysis is a key regulatory process that promotes the (in)activation, translocation, and/or degradation of proteins. As such, there is considerable interest in methods to comprehensively characterize proteolytic pathways in biological systems. Here, we describe a robust and versatile proteomic platform that enables direct visualization of the topography and magnitude of proteolytic events on a global scale. We use this method to generate a proteome-wide map of proteolytic events induced by the intrinsic apoptotic pathway. This profile contained 91 characterized caspase substrates as well as 170 additional proteins not previously known to be cleaved during apoptosis. Surprisingly, the vast majority of proteolyzed proteins, regardless of the extent of cleavage, yielded persistent fragments that correspond to discrete protein domains, suggesting that the generation of active effector proteins may be a principal function of apoptotic proteolytic cascades.

Activity-based protein profiling for biochemical pathway discovery in cancer

Large-scale profiling methods have uncovered numerous gene and protein expression changes that correlate with tumorigenesis. However, determining the relevance of these expression changes and which biochemical pathways they affect has been hindered by our incomplete understanding of the proteome and its myriad functions and modes of regulation. Activity-based profiling platforms enable both the discovery of cancer-relevant enzymes and selective pharmacological probes to perturb and characterize these proteins in tumour cells. When integrated with other large-scale profiling methods, activity-based proteomics can provide insight into the metabolic and signalling pathways that support cancer pathogenesis and illuminate new strategies for disease diagnosis and treatment.

A road map to evaluate the proteome-wide selectivity of covalent kinase inhibitors

Kinases are principal components of signal transduction pathways and the focus of intense basic and drug discovery research. Irreversible inhibitors that covalently modify non-catalytic cysteines in kinase active-sites have emerged as valuable probes and approved drugs. Many protein classes, however, possess functional cysteines and therefore understanding the proteome-wide selectivity of covalent kinase inhibitors is imperative. Here, we accomplish this objective using activity-based protein profiling coupled with quantitative mass spectrometry to globally map the targets, both specific and non-specific, of covalent kinase inhibitors in human cells. Many of the specific off-targets represent non-kinase proteins that, interestingly, possess conserved, active-site cysteines. We define windows of selectivity for covalent kinase inhibitors and show that, when these windows are exceeded, rampant proteome-wide reactivity and kinase target-independent cell death conjointly occur. Our findings, taken together, provide an experimental roadmap to illuminate opportunities and surmount challenges for the development of covalent kinase inhibitors.

Functional Interplay between Caspase Cleavage and Phosphorylation Sculpts the Apoptotic Proteome

Caspase proteases are principal mediators of apoptosis, where they cleave hundreds of proteins. Phosphorylation also plays an important role in apoptosis, although the extent to which proteolytic and phosphorylation pathways crosstalk during programmed cell death remains poorly understood. Using a quantitative proteomic platform that integrates phosphorylation sites into the topographical maps of proteins, we identify a cohort of over 500 apoptosis-specific phosphorylation events and show that they are enriched on cleaved proteins and clustered around sites of caspase proteolysis. We find that caspase cleavage can expose new sites for phosphorylation, and, conversely, that phosphorylation at the +3 position of cleavage sites can directly promote substrate proteolysis by caspase-8. This study provides a global portrait of the apoptotic phosphoproteome, revealing heretofore unrecognized forms of functional crosstalk between phosphorylation and caspase proteolytic pathways that lead to enhanced rates of protein cleavage and the unveiling of new sites for phosphorylation.

The hereditary spastic paraplegia-related enzyme DDHD2 is a principal brain triglyceride lipase

Significance Many rare human genetic disorders are caused by mutations in genes that code for proteins of poorly characterized function. Determining the functions of these proteins is critical for understanding and devising potential treatments for human diseases. In this article, we discover using a combination of mouse genetic models, selective inhibitors, and lipid profiling that the DDHD2 enzyme, mutations of which cause a neurological disease termed complex hereditary spastic paraplegia (HSP), acts as a major brain triglyceride hydrolase. Mice lacking DDHD2 have elevated brain triglycerides and lipid droplet accumulation in neurons. We have thus discovered that the brain possesses a specialized pathway for triglyceride metabolism, disruption of which leads to biochemical and cellular changes that may contribute to complex HSP. Complex hereditary spastic paraplegia (HSP) is a genetic disorder that causes lower limb spasticity and weakness and intellectual disability. Deleterious mutations in the poorly characterized serine hydrolase DDHD2 are a causative basis for recessive complex HSP. DDHD2 exhibits phospholipase activity in vitro, but its endogenous substrates and biochemical functions remain unknown. Here, we report the development of DDHD2−/− mice and a selective, in vivo-active DDHD2 inhibitor and their use in combination with mass spectrometry-based lipidomics to discover that DDHD2 regulates brain triglycerides (triacylglycerols, or TAGs). DDHD2−/− mice show age-dependent TAG elevations in the central nervous system, but not in several peripheral tissues. Large lipid droplets accumulated in DDHD2−/− brains and were localized primarily to the intracellular compartments of neurons. These metabolic changes were accompanied by impairments in motor and cognitive function. Recombinant DDHD2 displays TAG hydrolase activity, and TAGs accumulated in the brains of wild-type mice treated subchronically with a selective DDHD2 inhibitor. These findings, taken together, indicate that the central nervous system possesses a specialized pathway for metabolizing TAGs, disruption of which leads to massive lipid accumulation in neurons and complex HSP syndrome.

Immunomodulatory lysophosphatidylserines are regulated by ABHD16A and ABHD12 interplay

Lysophosphatidylserines (lyso-PSs) are a class of signaling lipids that regulate immunological and neurological processes. The metabolism of lyso-PSs remains poorly understood in vivo. Recently, we determined that ABHD12 is a major brain lyso-PS lipase, implicating lyso-PSs in the neurological disease polyneuropathy, hearing loss, ataxia, retinitis pigmentosa and cataract (PHARC), which is caused by null mutations in the ABHD12 gene. Here, we couple activity-based profiling with pharmacological and genetic methods to annotate the poorly characterized enzyme ABHD16A as a phosphatidylserine (PS) lipase that generates lyso-PS in mammalian systems. We describe a small-molecule inhibitor of ABHD16A that depletes lyso-PSs from cells, including lymphoblasts derived from subjects with PHARC. In mouse macrophages, disruption of ABHD12 and ABHD16A respectively increases and decreases both lyso-PSs and lipopolysaccharide-induced cytokine production. Finally, Abhd16a(-/-) mice have decreased brain lyso-PSs, which runs counter to the elevation in lyso-PS in Abhd12(-/-) mice. Our findings illuminate an ABHD16A-ABHD12 axis that dynamically regulates lyso-PS metabolism in vivo, designating these enzymes as potential targets for treating neuroimmunological disorders.

Comparative Assessment of Large-Scale Proteomic Studies of Apoptotic Proteolysis

Two proteomic methods were recently introduced to globally map proteolytic cleavage events in biological systems, one that characterized proteolyzed proteins by differential gel migration (PROTOMAP) and the other by enzymatic tagging and enrichment of the nascent N-terminal peptides generated by proteolysis (Subtiligase). Both technologies were applied to apoptosis, and each uncovered hundreds of novel proteolytic events. An initial survey, however, revealed only minimal overlap in the two data sets. In this article, we perform an in-depth comparative analysis of the PROTOMAP and Subtiligase results that assimilates the complementary information acquired by each method. This analysis uncovered substantial agreement between the PROTOMAP and Subtiligase data sets, which in integrated form yield a highly enriched portrait of the proteome-wide impact of proteolysis in apoptosis. We discuss the respective strengths of each proteomic method and the potential for these technologies to expand the scope and sensitivity of large-scale studies of proteolysis in biological systems.

Selective Inhibitor of Platelet-Activating Factor Acetylhydrolases 1b2 and 1b3 That Impairs Cancer Cell Survival

Platelet-activating factor acetylhydrolases (PAFAHs) 1b2 and 1b3 are poorly characterized serine hydrolases that form a complex with a noncatalytic protein (1b1) to regulate brain development, spermatogenesis, and cancer pathogenesis. Determining physiological substrates and biochemical functions for the PAFAH1b complex would benefit from selective chemical probes that can perturb its activity in living systems. Here, we report a class of tetrahydropyridine reversible inhibitors of PAFAH1b2/3 discovered using a fluorescence polarization-activity-based protein profiling (fluopol-ABPP) screen of the NIH 300,000+ compound library. The most potent of these agents, P11, exhibited IC50 values of ∼40 and 900 nM for PAFAH1b2 and 1b3, respectively. We confirm selective inhibition of PAFAH1b2/3 in cancer cells by P11 using an ABPP protocol adapted for in situ analysis of reversible inhibitors and show that this compound impairs tumor cell survival, supporting a role for PAFAH1b2/3 in cancer.

Chemical Proteomics Identifies Druggable Vulnerabilities in a Genetically Defined Cancer

The transcription factor NRF2 is a master regulator of the cellular antioxidant response, and it is often genetically activated in non-small-cell lung cancers (NSCLCs) by, for instance, mutations in the negative regulator KEAP1. While direct pharmacological inhibition of NRF2 has proven challenging, its aberrant activation rewires biochemical networks in cancer cells that may create special vulnerabilities. Here, we use chemical proteomics to map druggable proteins that are selectively expressed in KEAP1-mutant NSCLC cells. Principal among these is NR0B1, an atypical orphan nuclear receptor that we show engages in a multimeric protein complex to regulate the transcriptional output of KEAP1-mutant NSCLC cells. We further identify small molecules that covalently target a conserved cysteine within the NR0B1 protein interaction domain, and we demonstrate that these compounds disrupt NR0B1 complexes and impair the anchorage-independent growth of KEAP1-mutant cancer cells. Our findings designate NR0B1 as a druggable transcriptional regulator that supports NRF2-dependent lung cancers.

Global Identification of Caspase Substrates Using PROTOMAP (Protein Topography and Migration Analysis Platform)

Delineation of the natural substrate scope of proteases is important for understanding the functions of proteolytic pathways in physiology and disease. Herein we describe the protocol for PROTOMAP, a technique that combines SDS-PAGE with tandem mass spectrometry to globally identify shifts in protein migration indicative of proteolytic processing. When applied to cells undergoing apoptosis, this unbiased global method provides a snapshot of the topography and magnitude of proteolytic events associated with programmed cell death.