label-free-quantitative-proteomics-workflow-for-discovery-driven-host

Label-Free Quantitative Proteomics Workflow for Discovery-Driven Host-Pathogen Interactions

The technological achievements of mass spectrometry (MS)-based quantitative proteomics opens many undiscovered avenues for analyzing an organism&#8217;s ...

University of Guelph

In addition to the psychoactive constituents that are typically associated with Cannabis sativa L., there exist numerous other specialized metabolites in this plant that are believed to contribute to its medicinal versatility. This study focused on two such compounds, known as cannflavin A and cannflavin B. These prenylated flavonoids specifically accumulate in C. sativa and are known to exhibit potent anti-inflammatory activity in various animal cell models. However, almost nothing is known about their biosynthesis. Using a combination of phylogenomic and biochemical approaches, an aromatic prenyltransferase from C. sativa (CsPT3) was identified that catalyzes the regiospecific addition of either geranyl diphosphate (GPP) or dimethylallyl diphosphate (DMAPP) to the methylated flavone, chrysoeriol, to produce cannflavins A and B, respectively. Further evidence is presented for an O-methyltransferase (CsOMT21) encoded within the C. sativa genome that specifically converts the widespread plant flavone known as luteolin to chrysoeriol, both of which accumulate in C. sativa. These results therefore imply the following reaction sequence for cannflavins A and B biosynthesis: luteolin ► chrysoeriol ► cannflavin A and cannflavin B. Taken together, the identification of these two unique enzymes represent a branch point from the general flavonoid pathway in C. sativa and offer a tractable route towards metabolic engineering strategies that are designed to produce these two medicinally relevant Cannabis compounds.

Biosynthesis of cannflavins A and B from Cannabis sativa L.

The innate immune system is a collective network of cell types involved in cell recruitment and activation using a robust and refined communication system. Engagement of receptor-mediated intracellular signaling initiates communication cascades by conveying information about the host cell status to surrounding cells for surveillance and protection. Comprehensive profiling of innate immune cells is challenging due to low cell numbers, high dynamic range of the cellular proteome, low abundance of secreted proteins, and the release of degradative enzymes (e.g., proteases). However, recent advances in mass spectrometry-based proteomics provides the capability to overcome these limitations through profiling the dynamics of cellular processes, signaling cascades, post-translational modifications, and interaction networks. Moreover, integration of technologies and molecular datasets provide a holistic view of a complex and intricate network of communications underscoring host defense and tissue homeostasis mechanisms. In this Review, we explore the diverse applications of mass spectrometry-based proteomics in innate immunity to define communication patterns of the innate immune cells during health and disease. We also provide a technical overview of mass spectrometry-based proteomic workflows, with a focus on bottom-up approaches, and we present the emerging role of proteomics in immune-based drug discovery while providing a perspective on new applications in the future.

Decoding communication patterns of the innate immune system by quantitative proteomics.

Nutrient adaptation is key in limiting environments for the promotion of microbial growth and survival. In microbial systems, iron is an essential component for many cellular processes, and bioavailability varies greatly among different conditions. In the bacterium, , the impact of iron limitation is known to alter transcriptional expression of iron-acquisition pathways and influence secretion of iron-binding siderophores, however, a comprehensive view of iron limitation at the protein level remains to be defined. Here, we apply a mass-spectrometry-based quantitative proteomics strategy to profile the global impact of iron limitation on the cellular proteome and extracellular environment (secretome) of . Our data define the impact of iron on proteins involved in transcriptional regulation and emphasize the modulation of a vast array of proteins associated with iron acquisition, transport, and binding. We also identify proteins in the extracellular environment associated with conventional and non-conventional modes of secretion, as well as vesicle release. In particular, we demonstrate a new role for Lon protease in promoting iron homeostasis outside of the cell. Characterization of a Lon protease mutant in  validates roles in bacterial growth, cell division, and virulence, and uncovers novel degradation candidates of Lon protease associated with improved iron utilization strategies in the absence of the enzyme. Overall, we provide evidence of unique connections between Lon and iron in a bacterial system and suggest a new role for Lon protease in the extracellular environment during nutrient limitation.

Iron Limitation in  Defines New Roles for Lon Protease in Homeostasis and Degradation by Quantitative Proteomics.

Mass spectrometry-based proteomics is a powerful and robust platform for studying the interactions between biological systems during health and disease. Bacterial infections represent a significant threat to global health and drive the pursuit of novel therapeutic strategies to combat emerging and resistant pathogens. During infection, the interplay between a host and pathogen determines the ability of the microbe to survive in a hostile environment and promotes an immune response by the host as a protective measure. It is the protein-level changes from either biological system that define the outcome of infection, and mass spectrometry-based proteomics provides a rapid and effective platform to identify such changes. In particular, proteomics detects alterations in protein abundance, quantifies protein secretion and/or release, measures an array of post-translational modifications that influence signaling cascades, and profiles protein-protein interactions through protein complex and/or network formation. Such information provides new insight into the role of known and novel bacterial effectors, as well as the outcome of host cell activation. In this Review, we highlight the diverse applications of mass spectrometry-based proteomics in profiling the relationship between bacterial pathogens and the host. Our work identifies a plethora of strategies for exploring mechanisms of infection from dual perspectives (i.e., host and pathogen) and we suggest opportunities to extrapolate the current knowledgebase to other biological systems for applications in therapeutic discovery.

Article	Year
Biosynthesis of cannflavins A and B from Cannabis sativa L. Phytochemistry\| PubMed ID: 31151063	2019
Decoding communication patterns of the innate immune system by quantitative proteomics. Journal of leukocyte biology\| PubMed ID: 31556465	2019
Iron Limitation in Defines New Roles for Lon Protease in Homeostasis and Degradation by Quantitative Proteomics. Frontiers in microbiology\| PubMed ID: 32390954	2020
Proteomics of host-bacterial interactions: new insights from dual perspectives. Canadian journal of microbiology\| PubMed ID: 33027598	2020

Arjun Sukumaran

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