Lindsey N. Anderson

Mycobacterium tuberculosis Ser/Thr Protein Kinase B Mediates an Oxygen-Dependent Replication Switch

Mtb growth and replication are sensitive to altered levels of the Ser/Thr kinase PknB, and this sensitivity increased under hypoxic conditions. Thus, PknB is a critical regulator of the oxygen-dependent replication switch of Mycobacterium tuberculosis.

Suite of activity-based probes for cellulose-degrading enzymes.

Microbial glycoside hydrolases play a dominant role in the biochemical conversion of cellulosic biomass to high-value biofuels. Anaerobic cellulolytic bacteria are capable of producing multicomplex catalytic subunits containing cell-adherent cellulases, hemicellulases, xylanases, and other glycoside hydrolases to facilitate the degradation of highly recalcitrant cellulose and other related plant cell wall polysaccharides. Clostridium thermocellum is a cellulosome-producing bacterium that couples rapid reproduction rates to highly efficient degradation of crystalline cellulose. Herein, we have developed and applied a suite of difluoromethylphenyl aglycone, N-halogenated glycosylamine, and 2-deoxy-2-fluoroglycoside activity-based protein profiling (ABPP) probes to the direct labeling of the C. thermocellum cellulosomal secretome. These activity-based probes (ABPs) were synthesized with alkynes to harness the utility and multimodal possibilities of click chemistry and to increase enzyme active site inclusion for liquid chromatography-mass spectrometry (LC-MS) analysis. We directly analyzed ABP-labeled and unlabeled global MS data, revealing ABP selectivity for glycoside hydrolase (GH) enzymes, in addition to a large collection of integral cellulosome-containing proteins. By identifying reactivity and selectivity profiles for each ABP, we demonstrate our ability to widely profile the functional cellulose-degrading machinery of the bacterium. Derivatization of the ABPs, including reactive groups, acetylation of the glycoside binding groups, and mono- and disaccharide binding groups, resulted in considerable variability in protein labeling. Our probe suite is applicable to aerobic and anaerobic microbial cellulose-degrading systems and facilitates a greater understanding of the organismal role associated with biofuel development.

Organelle-Specific Activity-Based Protein Profiling in Living Cells

A multimodal activity-based probe for targeting acidic organelles was developed to measure subcellular native enzymatic activity in cells by fluorescence microscopy and mass spectrometry. A cathepsin-reactive warhead conjugated to a weakly basic amine and a clickable alkyne, for subsequent appendage of a fluorophore or biotin reporter tag, accumulated in lysosomes as observed by structured illumination microscopy (SIM) in J774 mouse macrophage cells. Analysis of in vivo labeled J774 cells by mass spectrometry showed that the probe was very selective for cathepsins B and Z, two lysosomal cysteine proteases. Analysis of starvation-induced autophagy, a catabolic pathway involving lysosomes, showed a large increase in the number of tagged proteins and an increase in cathepsin activity. The organelle-targeting of activity-based probes holds great promise for the characterization of enzyme activities in the myriad diseases linked to specific subcellular locations, particularly the lysosome.

Systematic Survey of Serine Hydrolase Activity in Mycobacterium tuberculosis Defines Changes Associated with Persistence

The transition from replication to non-replication underlies much of Mycobacterium tuberculosis (Mtb) pathogenesis, as non- or slowly replicating Mtb are responsible for persistence and poor treatment outcomes. Therapeutic targeting of non-replicating populations is a priority for tuberculosis treatment, but few drug targets in non-replicating Mtb are currently known. Here, we directly measured the activity of the highly diverse and druggable serine hydrolases (SHs) during active replication and non-replication using activity-based proteomics. We predict SH activity for 78 proteins, including 27 proteins with unknown function, and identify 37 SHs that remain active in the absence of replication, providing a set of candidate persistence targets. Non-replication was associated with major shifts in SH activity. These activity changes were largely independent of SH abundance, indicating extensive post-translational regulation of SHs. By probing a large cross-section of druggable Mtb enzyme space during replication and non-replication, we identify new SHs and suggest new persistence targets.

Multiplexed Activity-based Protein Profiling of the Human Pathogen Aspergillus fumigatus Reveals Large Functional Changes upon Exposure to Human Serum

Background: A. fumigatus is an opportunistic pathogen responsible for pulmonary invasive aspergillosis. Results: Multiplexed ABPP revealed significant changes in A. fumigatus metabolism and stress response during culture with human serum over time. Conclusion: Changes in functional pathways indicate robust adaptation to environmental change. Significance: A. fumigatus grows under stress by altering metabolism, energy production, and protein biosynthesis, which is relevant for lung colonization. Environmental adaptability is critical for survival of the fungal human pathogen Aspergillus fumigatus in the immunocompromised host lung. We hypothesized that exposure of the fungal pathogen to human serum would lead to significant alterations to the organisms physiology, including metabolic activity and stress response. Shifts in functional pathway and corresponding enzyme reactivity of A. fumigatus upon exposure to the human host may represent much needed prognostic indicators of fungal infection. To address this, we employed a multiplexed activity-based protein profiling (ABPP) approach coupled to quantitative mass spectrometry-based proteomics to measure broad enzyme reactivity of the fungus cultured with and without human serum. ABPP showed a shift from aerobic respiration to ethanol fermentation and utilization over time in the presence of human serum, which was not observed in serum-free culture. Our approach provides direct insight into this pathogens ability to survive, adapt, and proliferate. Additionally, our multiplexed ABPP approach captured a broad swath of enzyme reactivity and functional pathways and provides a method for rapid assessment of the A. fumigatus response to external stimuli.

Hepatic Cytochrome P450 Activity, Abundance, and Expression Throughout Human Development

Cytochrome P450s are oxidative metabolic enzymes that play critical roles in the biotransformation of endogenous compounds and xenobiotics. The expression and activity of P450 enzymes varies considerably throughout human development; the deficit in our understanding of these dynamics limits our ability to predict environmental and pharmaceutical exposure effects. In an effort to develop a more comprehensive understanding of the ontogeny of P450 enzymes, we employed a multi-omic characterization of P450 transcript expression, protein abundance, and functional activity. Modified mechanism-based inhibitors of P450s were used as chemical probes for isolating active P450 proteoforms in human hepatic microsomes with developmental stages ranging from early gestation to late adult. High-resolution liquid chromatography–mass spectrometry was used to identify and quantify probe-labeled P450s, allowing for a functional profile of P450 ontogeny. Total protein abundance profiles and P450 rRNA was also measured, and our results reveal life-stage–dependent variability in P450 expression, abundance, and activity throughout human development and frequent discordant relationships between expression and activity. We have significantly expanded the knowledge of P450 ontogeny, particularly at the level of individual P450 activity. We anticipate that these results will be useful for enabling predictive therapeutic dosing, and for avoiding potentially adverse and harmful reactions during maturation from both therapeutic drugs and environmental xenobiotics.

Live Cell Discovery of Microbial Vitamin Transport and Enzyme-Cofactor Interactions.

The rapid completion of microbial genomes is inducing a conundrum in functional gene discovery. Novel methods are needed to shorten the gap between characterizing a microbial genome and experimentally validating bioinformatically predicted functions. Of particular importance are transport mechanisms, which shuttle nutrients such as B vitamins and metabolites across cell membranes and are required for the survival of microbes ranging from members of environmental microbial communities to pathogens. Methods to accurately assign function and specificity for a wide range of experimentally unidentified and/or predicted membrane-embedded transport proteins, along with characterization of intracellular enzyme-cofactor associations, are needed to enable a significantly improved understanding of microbial biochemistry and physiology, microbial interactions, and microbial responses to perturbations. Chemical probes derived from B vitamins B1, B2, and B7 have allowed us to experimentally address the aforementioned needs by identifying B vitamin transporters and intracellular enzyme-cofactor associations through live cell labeling of the filamentous anoxygenic photoheterotroph, Chloroflexus aurantiacus J-10-fl, known to employ mechanisms for both B vitamin biosynthesis and environmental salvage. Our probes provide a unique opportunity to directly link cellular activity and protein function back to ecosystem and/or host dynamics by identifying B vitamin transport and cofactor-dependent interactions required for survival.

Elucidation of roles for vitamin B12 in regulation of folate, ubiquinone, and methionine metabolism

Significance Using a chemical probe mimic of vitamin B12, we reveal a light- and B12-dependent DNA regulator, and make the unexpected discovery of B12 having regulatory involvement in microbial folate, ubiquinone, and methionine processes. These findings suggest a pivotal role for B12 in the control of cell growth, which may lead to coordination of cell behavior in complex multicellular systems. As key research questions emerge from host-associated and environmental microbiomes, we anticipate that B12 regulatory control of metabolism will be found to be generalizable, will be critical for coordination of individual microbe and community metabolism, and that organismal interdependencies for B12 may be pertinent to microbiome organization, stability, and overall function. Only a small fraction of vitamin B12-requiring organisms are able to synthesize B12 de novo, making it a common commodity in microbial communities. Initially recognized as an enzyme cofactor of a few enzymes, recent studies have revealed additional B12-binding enzymes and regulatory roles for B12. Here we report the development and use of a B12-based chemical probe to identify B12-binding proteins in a nonphototrophic B12-producing bacterium. Two unexpected discoveries resulted from this study. First, we identified a light-sensing B12-binding transcriptional regulator and demonstrated that it controls folate and ubiquinone biosynthesis. Second, our probe captured proteins involved in folate, methionine, and ubiquinone metabolism, suggesting that it may play a role as an allosteric effector of these processes. These metabolic processes produce precursors for synthesis of DNA, RNA, and protein. Thereby, B12 likely modulates growth, and by limiting its availability to auxotrophs, B12-producing organisms may facilitate coordination of community metabolism.

Activity-based protein profiling of secreted cellulolytic enzyme activity dynamics in Trichoderma reesei QM6a, NG14, and RUT-C30

Lignocellulosic biomass has great promise as a highly abundant and renewable source for the production of biofuels. However, the recalcitrant nature of lignocellulose toward hydrolysis into soluble sugars remains a significant challenge to harnessing the potential of this source of bioenergy. A primary method for deconstructing lignocellulose is via chemical treatments, high temperatures, and hydrolytic enzyme cocktails, many of which are derived from the fungus Trichoderma reesei. Herein, we use an activity-based probe for glycoside hydrolases to rapidly identify optimal conditions for maximum enzymatic lignocellulose deconstruction. We also demonstrate that subtle changes to enzyme composition and activity in various strains of T. reesei can be readily characterized by our probe approach. The approach also permits multimodal measurements, including fluorescent gel-based analysis of activity in response to varied conditions and treatments, and mass spectrometry-based quantitative identification of labelled proteins. We demonstrate the promise this probe approach holds to facilitate rapid production of enzyme cocktails for high-efficiency lignocellulose deconstruction to accommodate high-yield biofuel production.

Deficient expression of aldehyde dehydrogenase 1A1 is consistent with increased sensitivity of Gorlin syndrome patients to radiation carcinogenesis

Human phenotypes that are highly susceptible to radiation carcinogenesis have been identified. Sensitive phenotypes often display robust regulation of molecular features that modify biological response, which can facilitate identification of the pathways/networks that contribute to pathophysiological outcomes. Here we interrogate primary dermal fibroblasts isolated from Gorlin syndrome patients (GDFs), who display a pronounced inducible tumorigenic response to radiation, in comparison to normal human dermal fibroblasts (NHDFs). Our approach exploits newly developed thiol reactive probes to define changes in protein thiol profiles in live cell studies, which minimizes artifacts associated with cell lysis. Redox probes revealed deficient expression of an apparent 55 kDa protein thiol in GDFs from independent Gorlin syndrome patients, compared with NHDFs. Proteomics tentatively identified this protein as aldehyde dehydrogenase 1A1 (ALDH1A1), a key enzyme regulating retinoic acid synthesis, and ALDH1A1 protein deficiency in GDFs was confirmed by Western blot. A number of additional protein thiol differences in GDFs were identified, including radiation responsive annexin family members and lamin A/C. Collectively, candidates identified in our study have plausible implications for radiation health effects and cancer susceptibility.