Investigating asparagine-linked protein glycosylation in eukaryotic and prokaryotic systems

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Chemistry, 2006.Vita.Includes bibliographical references.N-linked protein glycosylation is characterized by the formation of a -glycosylamine linkage to an asparagine residue within the Asn-Xaa-Ser/Thr consensus sequence. This modification is found in organisms from eukaryotic, archaeal and bacterial domains and is implicated in numerous cellular processes. Recently, a system of N-linked glycosylation was characterized in a gram-negative bacterium, Campylobacter jejuni. Glycosylation in this organism involves the transfer of a heptasaccharide from an undecaprenyl-pyrophosphate (Und-PP) carrier onto the asparagine side-chain of a protein. The genes in the 'pgl gene cluster' encode all of the proteins necessary for the biosynthesis of the glycan donor and its ultimate transfer to protein. The heptasaccharide donor has been characterized as GalNAc-al,4- GalNAc-al,4-(Glcpl1,3)-GalNAc-al,4-GalNAc-al,4-GalNAc-al,3-Bac-al ,PP-Und, where Bac is bacillosamine (2,4-diacetamido-2,4,6-trideoxyglucose). A synthetic route was developed to access bacillosamine-phosphate, which was incorporated into UDP-bacillosamine (UDP-Bac) and undecaprenyl-pyrophosphate-bacillosamine (Und-PP-Bac), which are substrates for the Pgl enzymes. Using the synthetic UDP-Bac, the role of the PglC glycophosphoryltransferase was elucidated in vitro.(cont.) The activities of the Pgl glycosyltransferases, PglA, PglJ, PglH and PglI were validated using the synthetic Und-PP-Bac substrate and it was discovered that PglH is a polymerase that catalyzes the transfer of the three terminal GalNAc residues. PglB is the oligosaccharyl transferase of the bacterial system. Using the synthetic glycan donor, PglB was shown to act in vitro on a short peptide substrate. This in vitro system enables detailed mechanistic investigations into the action of this intriguing enzyme. N-linked glycosylation in eukaryotes is catalyzed by oligosaccharyl transferase (OT), a multimeric protein complex localized in the lumen of the endoplasmic reticulum. The Stt3p protein of the eukaryotic OT cluster is homologous to the bacterial PglB enzyme. With the goal of inhibiting OT in a cellular environment, a family of peptidomimetic inhibitors with nanomolar affinity was synthesized. These inhibitors were evaluated for cellular inhibition of OT using a novel, high-throughput assay that monitors the production of a reporter glycoprotein, secreted alkaline phosphatase. The results from the screening yielded a hydrophobic peptidomimetic compound as a potential candidate for further studies into the in vivo inhibition of OT.by Eranthie Weerapana.Ph.D

Citrullination Inactivates Nicotinamide-N-methyltransferase

Nicotinamide-N-methyl transferase (NNMT) catalyzes the irreversible methylation of nicotinamide (NAM) to form N-methyl nicotinamide (MeNAM) using SAM as a methyl donor. NNMT is implicated in several chronic disease conditions, including cancers, kidney disease, cardiovascular disease, and Parkinson\u27s disease. Although phosphorylation of NNMT in gastric tumors is reported, the functional effects of this post-translational modification has not been investigated. We previously reported that citrullination of NNMT by Protein Arginine Deiminases (PADs) abolished its methyltransferase activity. Herein, we investigate the mechanism of inactivation. Using tandem MS, we identified three sites of citrullination in NNMT. With this information in hand, we used a combination of site-directed mutagenesis, kinetics, and CD experiments to demonstrate that citrullination of R132 leads to a structural perturbation that ultimately promotes NNMT inactivation

The Development of Benzimidazole-Based Clickable Probes for the Efficient Labeling of Cellular Protein Arginine Deiminases (PADs)

Citrullination is the post-translational hydrolysis of peptidyl-arginines to form peptidyl-citrulline, a reaction that is catalyzed by the protein arginine deiminases (PADs), a family of calcium-regulated enzymes. Aberrantly increased protein citrullination is associated with a slew of autoimmune diseases (e.g., rheumatoid arthritis (RA), multiple sclerosis, lupus, and ulcerative colitis) and certain cancers. Given the clear link between increased PAD activity and human disease, the PADs are therapeutically relevant targets. Herein, we report the development of next generation cell permeable and clickable probes (BB-Cl-Yne and BB-F-Yne) for covalent labeling of the PADs both in vitro and in cell-based systems. Using advanced chemoproteomic technologies, we also report the off targets of both BB-Cl-Yne and BB-F-Yne. The probes are highly specific for the PADs, with relatively few off targets, especially BB-F-Yne, suggesting the preferential use of the fluoroacetamidine warhead in next generation irreversible PAD inhibitors. Notably, these compounds can be used in a variety of modalities, including the identification of off targets of the parent compounds and as activity-based protein profiling probes in target engagement assays to demonstrate the efficacy of PAD inhibitors

Halogen Bonding Increases the Potency and Isozyme-selectivity of Protein Arginine Deiminase 1 Inhibitors

Protein Arginine Deiminases (PADs) hydrolyze the side chain of arginine to form citrulline. Aberrant PAD activity is associated with rheumatoid arthritis, multiple sclerosis, lupus, and certain cancers. These pathologies established the PADs as therapeutic targets and multiple PAD inhibitors are known. Herein, we describe the first highly potent PAD1-selective inhibitors (1 and 19). Detailed structure-activity relationships indicate that their potency and selectivity is due to the formation of a halogen bond with PAD1. Importantly, these inhibitors inhibit histone H3 citrullination in HEK293TPAD1 cells and mouse zygotes with excellent potency. Based on this scaffold, we also developed a PAD1-selective activity-based probe that shows remarkable cellular efficacy and proteome selectivity. Based on their potency and selectivity we expect that 1 and 19 will be widely used chemical tools to understand PAD1 biology

Calcium Regulates the Nuclear Localization of Protein Arginine Deiminase 2

Protein arginine deiminases (PADs) are calcium-dependent enzymes that mediate the post-translational conversion of arginine into citrulline. Dysregulated PAD activity is associated with numerous autoimmune disorders and cancers. In breast cancer, PAD2 citrullinates histone H3R26 and activates the transcription of estrogen receptor target genes. However, PAD2 lacks a canonical nuclear localization sequence, and it is unclear how this enzyme is transported into the nucleus. Here, we show for the first time that PAD2 translocates into the nucleus in response to calcium signaling. Using BioID2, a proximity-dependent biotinylation method for identifying interacting proteins, we found that PAD2 preferentially associates with ANXA5 in the cytoplasm. Binding of calcium to PAD2 weakens this cytoplasmic interaction, which generates a pool of calcium-bound PAD2 that can interact with Ran. We hypothesize that this latter interaction promotes the translocation of PAD2 into the nucleus. These findings highlight a critical role for ANXA5 in regulating PAD2 and identify an unusual mechanism whereby proteins translocate between the cytosol and nucleus

CRISPR-based oligo recombineering prioritizes apicomplexan cysteines for drug discovery

Nucleophilic amino acids are important in covalent drug development yet underutilized as anti-microbial targets. Chemoproteomic technologies have been developed to mine chemically accessible residues via their intrinsic reactivity towards electrophilic probes but cannot discern which chemically reactive sites contribute to protein function and should therefore be prioritized for drug discovery. To address this, we have developed a CRISPR-based oligo recombineering (CORe) platform to support the rapid identification, functional prioritization and rational targeting of chemically reactive sites in haploid systems. Our approach couples protein sequence and function with biological fitness of live cells. Here we profile the electrophile sensitivity of proteinogenic cysteines in the eukaryotic pathogen Toxoplasma gondii and prioritize functional sites using CORe. Electrophile-sensitive cysteines decorating the ribosome were found to be critical for parasite growth, with target-based screening identifying a parasite-selective anti-malarial lead molecule and validating the apicomplexan translation machinery as a target for ongoing covalent ligand development

Small-molecule inhibition of a depalmitoylase enhances Toxoplasma host-cell invasion.

Although there have been numerous advances in our understanding of how apicomplexan parasites such as Toxoplasma gondii enter host cells, many of the signaling pathways and enzymes involved in the organization of invasion mediators remain poorly defined. We recently performed a forward chemical-genetic screen in T. gondii and identified compounds that markedly enhanced infectivity. Although molecular dissection of invasion has benefited from the use of small-molecule inhibitors, the mechanisms underlying induction of invasion by small-molecule enhancers have never been described. Here we identify the Toxoplasma ortholog of human APT1, palmitoyl protein thioesterase-1 (TgPPT1), as the target of one class of small-molecule enhancers. Inhibition of this uncharacterized thioesterase triggered secretion of invasion-associated organelles, increased motility and enhanced the invasive capacity of tachyzoites. We demonstrate that TgPPT1 is a bona fide depalmitoylase, thereby establishing an important role for dynamic and reversible palmitoylation in host-cell invasion by T. gondii

KEAP1-modifying small molecule reveals muted NRF2 signaling responses in neural stem cells from Huntington's disease patients

The activity of the transcription factor nuclear factor-erythroid 2 p45-derived factor 2 (NRF2) is orchestrated and amplified through enhanced transcription of antioxidant and antiinflammatory target genes. The present study has characterized a triazole-containing inducer of NRF2 and elucidated the mechanism by which this molecule activates NRF2 signaling. In a highly selective manner, the compound covalently modifies a critical stress-sensor cysteine (C151) of the E3 ligase substrate adaptor protein Kelch-like ECH-associated protein 1 (KEAP1), the primary negative regulator of NRF2. We further used this inducer to probe the functional consequences of selective activation of NRF2 signaling in Huntington's disease (HD) mouse and human model systems. Surprisingly, we discovered a muted NRF2 activation response in human HD neural stem cells, which was restored by genetic correction of the disease-causing mutation. In contrast, selective activation of NRF2 signaling potently repressed the release of the proinflammatory cytokine IL-6 in primary mouse HD and WT microglia and astrocytes. Moreover, in primary monocytes from HD patients and healthy subjects, NRF2 induction repressed expression of the proinflammatory cytokines IL-1, IL-6, IL-8, and TNFα. Together, our results demonstrate a multifaceted protective potential of NRF2 signaling in key cell types relevant to HD pathology

Applications of Copper-Catalyzed Click Chemistry in Activity-Based Protein Profiling

Activity-based protein profiling (ABPP) is a chemical proteomic technique that enables the interrogation of protein activity directly within complex proteomes. Given the dominant role of posttranslational modifications in regulating protein function in vivo, ABPP provides a direct readout of activity that is not attained through traditional proteomic methods. ABPP relies on the design of covalent binding probes that either target a specific enzyme or a class of enzymes with related function. These covalent warheads are coupled to either fluorophores or biotin groups for visualization and enrichment of these active proteins. The advent of bioorthogonal chemistries, in particular, the copper (I)-catalyzed azide-alkyne cycloaddition (CuAAC), has benefitted the field of ABPP by achieving the following: (1) replacing bulky reporter groups with smaller alkyne or azide groups to promote cell permeability; (2) adding modularity to the system such that a single probe can be diversified with a variety of reporter groups without the need to develop new synthetic routes; and (3) enabling the conjugation of complex linkers to facilitate quantitative proteomic analyses. Here, we summarize recent examples of CuAAC in ABPP that serve to illustrate the contribution of bioorthogonal chemistry to advancing discoveries in this field