Anti-citrullinated protein antibodies
(ACPAs) are a hallmark of
rheumatoid arthritis (RA) and are routinely used for disease diagnosis.
Protein citrullination is also increased in cancer and other autoimmune
disorders, suggesting that citrullinated proteins may serve as biomarkers
for diseases beyond RA. To identify these citrullinated proteins,
we developed biotin-conjugated phenylglyoxal (biotin-PG). Using this
probe and our platform technology, we identified >50 intracellular
citrullinated proteins. More than 20 of these are involved in RNA
splicing, suggesting, for the first time, that citrullination modulates
RNA biology. Overall, this chemical proteomic platform will play a
key role in furthering our understanding of protein citrullination
in rheumatoid arthritis and potentially a wider spectrum of inflammatory
diseases.
Activity-based protein profiling (ABPP) is a chemoproteomic tool for detecting active enzymes in complex biological systems. We used ABPP to identify secreted bacterial and host serine hydrolases that are active in animals infected with the cholera pathogen Vibrio cholerae. Four V. cholerae proteases were consistently active in infected rabbits, and one, VC0157 (renamed IvaP), was also active in human cholera stool. Inactivation of IvaP influenced the activity of other secreted V. cholerae and rabbit enzymes in vivo, while genetic disruption of all four proteases increased the abundance and binding of an intestinal lectin—intelectin—to V. cholerae in infected rabbits. Intelectin also bound to other enteric bacterial pathogens, suggesting it may constitute a previously unrecognized mechanism of bacterial surveillance in the intestine that is inhibited by pathogen-secreted proteases. Our work demonstrates the power of activity-based proteomics to reveal host-pathogen enzymatic dialogue in an animal model of infection.
Putting a number on it: Cleavable linkers are widely utilized in proteomics applications. In particular, the azobenzene-based linker cleaves under mild conditions that are mass-spectrometry-compatible. Here, we adapt this linker for quantitative proteomic applications by incorporating an isotopic label. These light- and heavy-tagged linkers enable the identification and quantitation of labeled peptides from multiple proteomes.
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.
Summary
In the nematode, Caenorhabditis elegans, inactivating mutations in the insulin/IGF-1 receptor, DAF-2, result in a 2-fold increase in lifespan mediated by DAF-16, a FOXO-family transcription factor. Downstream protein activities that directly regulate longevity during impaired Insulin/IGF-1 signaling (IIS) are poorly characterized. Here, we use global cysteine-reactivity profiling to identify protein-activity changes during impaired IIS. Upon confirming that cysteine reactivity is a good predictor of functionality in C. elegans, we profiled cysteine-reactivity changes between daf-2 and daf-16;daf-2 mutants, and identified 40 proteins that display a >2-fold change. Subsequent RNAi-mediated knockdown studies revealed that lbp-3 and K02D7.1 knockdown caused significant increases in lifespan and dauer formation. The proteins encoded by these two genes, LBP-3 and K02D7.1, are implicated in intracellular fatty-acid transport and purine metabolism, respectively. These studies demonstrate that cysteine-reactivity profiling can be complementary to abundance-based transcriptomic and proteomic studies, serving to identify uncharacterized mediators of C. elegans longevity.
The elucidation of protein activities dysregulated during the aging process is a vital step toward the discovery of signaling networks and metabolic pathways directly implicated in aging. Genetic studies in the model organism, Caenorhabditis elegans, generated a daf‐2 mutant that has a significantly extended lifespan. The daf‐2‐ gene mutant activates DAF‐16, a transcription factor, which initiates gene expression changes that mediate the life‐extension phenotype of this mutant. Identifying protein‐activity changes that are downstream effects of DAF‐16 activation will illuminate cellular pathways directly implicated in the aging process. We propose to directly monitor protein activity changes using the tools of activity‐based protein profiling. We are focusing our initial efforts on cysteine‐mediated protein activities that encompass a subset of diverse protein classes including proteases, kinases, ubiquitinating proteins, and metabolic enzymes. Combining small‐molecule probes that selectively bind to active members of these enzyme families, together with quantitative mass spectrometrybased proteomics, we are comparing protein activity in daf‐2 and daf‐2/daf‐16 mutant nematodes. Current work is focused on verifying the identity of these dysregulated proteins and understanding the role of these protein activities in slowing down the aging process in daf‐2 nematodes.
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