Veronica G. Anania

Transcription Factor ATF4 Induces NLRP1 Inflammasome Expression during Endoplasmic Reticulum Stress.

Perturbation of endoplasmic reticulum (ER) homeostasis triggers the ER stress response (also known as Unfolded Protein Response), a hallmark of many pathological disorders. However the connection between ER stress and inflammation remains largely unexplored. Recent data suggest that ER stress controls the activity of inflammasomes, key signaling platforms that mediate innate immune responses. Here we report that expression of NLRP1, a core inflammasome component, is specifically up-regulated during severe ER stress conditions in human cell lines. Both IRE1α and PERK, but not the ATF6 pathway, modulate NLRP1 gene expression. Furthermore, using mutagenesis, chromatin immunoprecipitation and CRISPR-Cas9-mediated genome editing technology, we demonstrate that ATF4 transcription factor directly binds to NLRP1 promoter during ER stress. Although involved in different types of inflammatory responses, XBP-1 splicing was not required for NLRP1 induction. This study provides further evidence that links ER stress with innate

Complementary Proteomic Tools for the Dissection of Apoptotic Proteolysis Events

Proteolysis is a key regulatory event that controls intracellular and extracellular signaling through irreversible changes in a proteins structure that greatly alters its function. Here we describe a platform for profiling caspase substrates which encompasses two highly complementary proteomic techniques--the first is a differential gel based approach termed Global Analyzer of SILAC-derived Substrates of Proteolysis (GASSP) and the second involves affinity enrichment of peptides containing a C-terminal aspartic acid residue. In combination, these techniques have enabled the profiling of a large cellular pool of apoptotic-mediated proteolytic events across a wide dynamic range. By applying this integrated proteomic work flow to analyze proteolytic events resulting from the induction of intrinsic apoptosis in Jurkat cells via etoposide treatment, 3346 proteins were quantified, of which 360 proteins were identified as etoposide-induced proteolytic substrates, including 160 previously assigned caspase substrates. In addition to global profiling, a targeted approach using BAX HCT116 isogenic cell lines was utilized to dissect pre- and post-mitochondrial extrinsic apoptotic cleavage events. By employing apoptotic activation with a pro-apoptotic receptor agonist (PARA), a limited set of apoptotic substrates including known caspase substrates such as BH3 interacting-domain death agonist (BID) and Poly (ADP-ribose) polymerase (PARP)-1, and novel substrates such as Basic Transcription Factor 3, TRK-fused gene protein (TFG), and p62/Sequestosome were also identified.

Peptide Level Immunoaffinity Enrichment Enhances Ubiquitination Site Identification on Individual Proteins

Ubiquitination is a process that involves the covalent attachment of the 76-residue ubiquitin protein through its C-terminal di-glycine (GG) to lysine (K) residues on substrate proteins. This post-translational modification elicits a wide range of functional consequences including targeting proteins for proteasomal degradation, altering subcellular trafficking events, and facilitating protein-protein interactions. A number of methods exist for identifying the sites of ubiquitination on proteins of interest, including site-directed mutagenesis and affinity-purification mass spectrometry (AP-MS). Recent publications have also highlighted the use of peptide-level immunoaffinity enrichment of K-GG modified peptides from whole cell lysates for global characterization of ubiquitination sites. Here we investigated the utility of this technique for focused mapping of ubiquitination sites on individual proteins. For a series of membrane-associated and cytoplasmic substrates including erbB-2 (HER2), Dishevelled-2 (DVL2), and T cell receptor α (TCRα), we observed that K-GG peptide immunoaffinity enrichment consistently yielded additional ubiquitination sites beyond those identified in protein level AP-MS experiments. To assess this quantitatively, SILAC-labeled lysates were prepared and used to compare the abundances of individual K-GG peptides from samples prepared in parallel. Consistently, K-GG peptide immunoaffinity enrichment yielded greater than fourfold higher levels of modified peptides than AP-MS approaches. Using this approach, we went on to characterize inducible ubiquitination on multiple members of the T-cell receptor complex that are functionally affected by endoplasmic reticulum (ER) stress. Together, these data demonstrate the utility of immunoaffinity peptide enrichment for single protein ubiquitination site analysis and provide insights into the ubiquitination of HER2, DVL2, and proteins in the T-cell receptor complex.

Combinatorial Approach for Large-scale Identification of Linked Peptides from Tandem Mass Spectrometry Spectra

The combination of chemical cross-linking and mass spectrometry has recently been shown to constitute a powerful tool for studying protein–protein interactions and elucidating the structure of large protein complexes. However, computational methods for interpreting the complex MS/MS spectra from linked peptides are still in their infancy, making the high-throughput application of this approach largely impractical. Because of the lack of large annotated datasets, most current approaches do not capture the specific fragmentation patterns of linked peptides and therefore are not optimal for the identification of cross-linked peptides. Here we propose a generic approach to address this problem and demonstrate it using disulfide-bridged peptide libraries to (i) efficiently generate large mass spectral reference data for linked peptides at a low cost and (ii) automatically train an algorithm that can efficiently and accurately identify linked peptides from MS/MS spectra. We show that using this approach we were able to identify thousands of MS/MS spectra from disulfide-bridged peptides through comparison with proteome-scale sequence databases and significantly improve the sensitivity of cross-linked peptide identification. This allowed us to identify 60% more direct pairwise interactions between the protein subunits in the 20S proteasome complex than existing tools on cross-linking studies of the proteasome complexes. The basic framework of this approach and the MS/MS reference dataset generated should be valuable resources for the future development of new tools for the identification of linked peptides.

A Novel Peptide-Based SILAC Method to Identify the Posttranslational Modifications Provides Evidence for Unconventional Ubiquitination in the ER-Associated Degradation Pathway.

The endoplasmic reticulum-associated degradation (ERAD) pathway is responsible for disposing misfolded proteins from the endoplasmic reticulum by inducing their ubiquitination and degradation. Ubiquitination is conventionally observed on lysine residues and has been demonstrated on cysteine residues and protein N-termini. Ubiquitination is fundamental to the ERAD process; however, a mutant T-cell receptor α (TCRα) lacking lysine residues is targeted for the degradation by the ERAD pathway. We have shown that ubiquitination of lysine-less TCRα occurs on internal, non-lysine residues and that the same E3 ligase conjugates ubiquitin to TCRα in the presence or absence of lysine residues. Mass-spectrometry indicates that WT-TCRα is ubiquitinated on multiple lysine residues. Recent publications have provided indirect evidence that serine and threonine residues may be modified by ubiquitin. Using a novel peptide-based stable isotope labeling in cell culture (SILAC) approach, we show that specific lysine-less TCRα peptides become modified. In this study, we demonstrate that it is possible to detect both ester and thioester based ubiquitination events, although the exact linkage on lysine-less TCRα remains elusive. These findings demonstrate that SILAC can be used as a tool to identify modified peptides, even those with novel modifications that may not be detected using conventional proteomic work flows or informatics algorithms.

Palmitoylation of MIR2 Is Required for Its Function

ABSTRACT Kaposis sarcoma-associated herpesvirus (KSHV) encodes two RING finger E3 ubiquitin ligases (MIR1 and MIR2) that mediate ubiquitination and degradation of cellular proteins important for the establishment of an efficient antiviral immune response. MIR1 and MIR2 share 30% sequence identity; however, their substrate preferences are varied. MIR1 has been shown to primarily downregulate major histocompatibility complex class I (MHC-I), whereas MIR2 can downregulate a wide range of cell surface proteins. Many of the MIR substrates are thought to be present in lipid raft microdomains, a subregion of the plasma membrane known to be important for a wide range of signal transduction events. Palmitoylation is a posttranslational modification that increases recruitment of transmembrane proteins to lipid rafts. In this study, we investigated the importance of palmitoylation for MIR function. We present evidence that MIR2-mediated downregulation of MHC-I and platelet endothelial cell adhesion molecule 1 (PECAM-1) but not other substrates is inhibited in the presence of the drug 2-bromohexadecanoic acid (2-Br), a chemical inhibitor of palmitoylation. Biochemical analysis indicates that MIR2 is directly palmitoylated on cysteine 146. Mutation of this cysteine to a phenylalanine prevents MIR2 palmitoylation and blocks the ability of MIR2 to downregulate MHC-I and PECAM-I but not B7.2 and intercellular adhesion molecule 1 (ICAM-I), consistent with the phenotype observed after 2-Br treatment. Unpalmitoylated MIR2 does not interact with MHC-I and is thus unable to ubiquitinate and downregulate MHC-I from the cell surface. Furthermore, we observed that MIR2 is palmitoylated in vivo during lytic infection. Palmitoylation may act to regulate MIR2 function and localization during viral infection by allowing MIR2 to properly interact with and downregulate multiple substrates known to play an important role in the host immune response.

SUMO deconjugation is required for arsenic-triggered ubiquitylation of PML

A chemotherapeutic triggers the degradation of an oncoprotein by inducing a switch from SUMO2 to SUMO1 at a specific lysine residue. SUMO switching for degradation Promyelocytic leukemia protein (PML) organizes various proteins into structures called PML nuclear bodies. Acute promyelocytic leukemia is caused by a fusion protein consisting of PML and the transcription factor RARα. Degradation of this oncoprotein can be induced by arsenic trioxide chemotherapy through modification of PML by SUMO1, SUMO2, and ubiquitin. Fasci et al. found that in untreated cells, SUMO2 was constantly added to Lys65 in PML and constantly removed from this residue by the deconjugating enzyme SENP1. In cells exposed to arsenic trioxide, SUMO1, rather than SUMO2, was conjugated to Lys65, which led to the formation of SUMO2 chains on a different residue, Lys160, that in turn triggered the ubiquitylation of PML, as well as reorganization of PML nuclear bodies. These results further define how arsenic trioxide induces the degradation of the RARα-PML oncoprotein, by promoting an exchange of SUMO paralogs on a “switch” residue that stimulates SUMO modification on a “chain” residue. Acute promyelocytic leukemia is characterized by a chromosomal translocation that produces an oncogenic fusion protein of the retinoic acid receptor α (RARα) and promyelocytic leukemia protein (PML). Arsenic trioxide chemotherapy of this cancer induces the PML moiety to organize nuclear bodies, where the oncoprotein is degraded. This process requires the participation of two SUMO paralogs (SUMO1 and SUMO2) to promote PML ubiquitylation mediated by the ubiquitin E3 ligase RNF4 and reorganization of PML nuclear bodies. We demonstrated that the ubiquitylation of PML required the SUMO deconjugation machinery, primarily the deconjugating enzyme SENP1, and was suppressed by expression of non-deconjugatable SUMO2. We hypothesized that constitutive SUMO2 conjugation and deconjugation occurred basally and that arsenic trioxide treatment caused the exchange of SUMO2 for SUMO1 on a fraction of Lys65 in PML. On the basis of data obtained with mutational analysis and quantitative proteomics, we propose that the SUMO switch at Lys65 of PML enhanced nuclear body formation, subsequent SUMO2 conjugation to Lys160, and consequent RNF4-dependent ubiquitylation of PML. Our work provides insights into how the SUMO system achieves selective SUMO paralog modification and highlights the crucial role of SENPs in defining the specificity of SUMO signaling.

Complementary methods for the identification of substrates of proteolysis.

Proteolysis describes the cleavage of proteins into smaller components, which in vivo occurs typically to either activate or impair the functionality of cellular proteins. Proteolysis can occur during cellular homeostasis or can be induced due to external stress stimuli such as heat, biological or chemical insult, and is mediated by the activity of cellular enzymes, namely, proteases. Proteolytic cleavage of proteins can influence protein activation by exposing an active site or disrupting inhibitor binding. Conversely, proteolytic cleavage of many proteins has also been shown to lead to protein degradation resulting in inactivation of the substrate. Thousands of proteolytic events are known to take place in regulated cellular processes such as apoptosis and pyroptosis, however, their individual contribution to these processes remains poorly understood. Additionally, many cellular homeostatic processes are regulated by proteolytic events, however, in some cases, few proteolytic substrates have been identified. To gain further insight into the mechanism of action of these cellular processes, and to characterize biomarkers of cell death and other pathological indications, it is imperative to utilize a complete arsenal of tools for studying proteolysis events in vivo and in vitro. In this chapter, we focus on alternative methodologies to N-terminomics for profiling substrates of proteolysis and describe an additional suite of tools including orthogonal biophysical separation techniques such as COFRADIC or GASSP, and affinity capture tools that can enrich for newly formed C-termini (C-terminomics) generated as a result of caspase-mediated proteolysis.

Uncovering a dual regulatory role for caspases during endoplasmic reticulum stress-induced cell death

Many diseases are associated with endoplasmic reticulum (ER) stress, which results from an accumulation of misfolded proteins. This triggers an adaptive response called the “unfolded protein response” (UPR), and prolonged exposure to ER stress leads to cell death. Caspases are reported to play a critical role in ER stress-induced cell death but the underlying mechanisms by which they exert their effect continue to remain elusive. To understand the role caspases play during ER stress, a systems level approach integrating analysis of the transcriptome, proteome, and proteolytic substrate profile was employed. This quantitative analysis revealed transcriptional profiles for most human genes, provided information on protein abundance for 4476 proteins, and identified 445 caspase substrates. Based on these data sets many caspase substrates were shown to be downregulated at the protein level during ER stress suggesting caspase activity inhibits their cellular function. Additionally, RNA sequencing revealed a role for caspases in regulation of ER stress-induced transcriptional pathways and gene set enrichment analysis showed expression of multiple gene targets of essential transcription factors to be upregulated during ER stress upon inhibition of caspases. Furthermore, these transcription factors were degraded in a caspase-dependent manner during ER stress. These results indicate that caspases play a dual role in regulating the cellular response to ER stress through both post-translational and transcriptional regulatory mechanisms. Moreover, this study provides unique insight into progression of the unfolded protein response into cell death, which may help identify therapeutic strategies to treat ER stress-related diseases.

Proteomic tools for the characterization of cell death mechanisms in drug discovery

This review focuses on the use of proteomic tools for the characterization of cell death mechanisms that have contributed to drug discovery efforts. Resistance to cell death plays a major role in the development of many diseases, including numerous types of malignancies. Using a multitude of proteomic approaches, including protein–protein interaction studies, phosphorylation site mapping, ubiquitination site identification, and differential quantitative approaches, various cellular death pathways such as apoptosis and necroptosis have been investigated. These studies have aided in the development of therapeutic strategies or allowed dissection of clinical results to evaluate the success of clinical trials in addition to contributing to our understanding of these biological pathways. Here, we address the new wave of discoveries enabled by advancements in mass spectrometric technology and bioinformatic infrastructure that will hopefully lead to clinically efficacious strategies to overcome resistance to apoptosis and therefore offer improved treatment options for patients.