Markus Grammel

Chemical reporters for biological discovery

Functional tools are needed to understand complex biological systems. Here we review how chemical reporters in conjunction with bioorthogonal labeling methods can be used to image and retrieve nucleic acids, proteins, glycans, lipids and other metabolites in vitro, in cells as well as in whole organisms. By tagging these biomolecules, researchers can now monitor their dynamics in living systems and discover specific substrates of cellular pathways. These advances in chemical biology are thus providing important tools to characterize biological pathways and are poised to facilitate our understanding of human diseases.

Comparative Analysis of Cleavable Azobenzene-Based Affinity Tags for Bioorthogonal Chemical Proteomics

The advances in bioorthogonal ligation methods have provided new opportunities for proteomic analysis of newly synthesized proteins, posttranslational modifications, and specific enzyme families using azide/alkyne-functionalized chemical reporters and activity-based probes. Efficient enrichment and elution of azide/alkyne-labeled proteins with selectively cleavable affinity tags are essential for protein identification and quantification applications. Here, we report the synthesis and comparative analysis of Na₂S₂O₄-cleavable azobenzene-based affinity tags for bioorthogonal chemical proteomics. We demonstrated that ortho-hydroxyl substituent is required for efficient azobenzene-bond cleavage and show that these cleavable affinity tags can be used to identify newly synthesized proteins in bacteria targeted by amino acid chemical reporters as well as their sites of modification on endogenously expressed proteins. The azobenzene-based affinity tags are compatible with in-gel, in-solution, and on-bead enrichment strategies and should afford useful tools for diverse bioorthogonal proteomic applications.

Orthogonal Alkynyl Amino Acid Reporter for Selective Labeling of Bacterial Proteomes during Infection

Bacterial pathogens have evolved sophisticated mechanisms to evade host defenses and cause disease. [1] The emergence of new and antibiotic-resistant bacterial pathogens demands a better understanding of virulence mechanisms for antibacterial drug discovery. While the discovery of bacterial toxins, quorum sensing and protein secretion pathways has revealed some key virulence mechanisms, the precise mechanisms by which intracellular bacterial pathogens subvert host immune responses are still unclear. [1] The analysis of individual virulence factors has demonstrated that bacterial pathogens alter their protein expression to infect and replicate in host tissues. [2] However, the system-wide identification and analysis of bacterial proteins that are uniquely expressed or secreted during infection is paramount for understanding mechanisms of bacterial pathogenesis. [2, 3] Comparative genomics and mutagenesis studies have revealed bacterial genes that are important for infection, but their precise biochemical mechanisms and temporal expression pattern can be elusive due to posttranscriptional regulation. [2, 3] Direct biochemical analysis of bacterial proteomes during infection is needed. [3] The large excess of host proteins in mixed pathogen-host lysates presents a significant challenge for proteomic analysis of bacterial proteins during infections [3] and even after physical isolation of intact bacteria significant amounts of host proteins still remain. [4, 5] This is particularly important since many bacterial virulence factors are often expressed at low levels. [2] New strategies are therefore required to selectively enrich bacterial proteins from host proteomes for their analysis during infection.

Copper-catalyzed azide-alkyne cycloaddition (click chemistry)-based Detection of Global Pathogen-host AMPylation on Self-assembled Human Protein Microarrays

AMPylation (adenylylation) is a recently discovered mechanism employed by infectious bacteria to regulate host cell signaling. However, despite significant effort, only a few host targets have been identified, limiting our understanding of how these pathogens exploit this mechanism to control host cells. Accordingly, we developed a novel nonradioactive AMPylation screening platform using high-density cell-free protein microarrays displaying human proteins produced by human translational machinery. We screened 10,000 unique human proteins with Vibrio parahaemolyticus VopS and Histophilus somni IbpAFic2, and identified many new AMPylation substrates. Two of these, Rac2, and Rac3, were confirmed in vivo as bona fide substrates during infection with Vibrio parahaemolyticus. We also mapped the site of AMPylation of a non-GTPase substrate, LyGDI, to threonine 51, in a region regulated by Src kinase, and demonstrated that AMPylation prevented its phosphorylation by Src. Our results greatly expanded the repertoire of potential host substrates for bacterial AMPylators, determined their recognition motif, and revealed the first pathogen-host interaction AMPylation network. This approach can be extended to identify novel substrates of AMPylators with different domains or in different species and readily adapted for other post-translational modifications.

Identification of lysine acetyltransferase substrates using bioorthogonal chemical proteomics.

Bioorthogonal chemical proteomics is a valuable method to identify enzyme-specific substrates, a challenging task by traditional biochemical standards. The addition of recombinant enzyme and alkynyl chemical reporter to complex protein mixtures, such as cell lysates, allows the detection and identification of modified substrates. Proteins that have been modified with the chemical reporter can be selectively labeled with fluorescent dyes for detection or affinity tags for biochemical enrichment and subsequent identification by mass spectrometry. Here, we describe the detection and identification of substrates of the lysine acetyltransferase p300 in nuclear extracts using the chemical reporter 4-pentynoyl-CoA.