Biology subjects

Lakemeyer, M.

Publications and source records attributed to Lakemeyer, M..

7 recordsLinked to original sources

bioRxiv2026

Beyond the Catalytic Serine: Selective Protease Engagement with Covalent Macrocyclic Activity-based Probes

Activity-based probes (ABPs) are widely used to profile serine protease activity - enzymes central to diverse physiological and pathological processes - but most rely on covalent modification of the conserved catalytic serine residue, often resulting in poor selectivity across related proteases. Here, we introduce covalent macrocyclic activity-based probes (cmABPs) that selectively target non-catalytic residues within serine protease active sites. By combining phage display with systematic electrophile scanning, we identify macrocyclic scaffolds that position sulfur(VI) fluoride (SuFEx) electrophiles to covalently engage alternative nucleophiles such as lysine and tyrosine. Applied to plasma kallikrein, this approach yielded a macrocyclic scaffold that was converted into covalent probes via fluorosulfate scanning. Remarkably, small changes in electrophile structure produced large, tuneable differences in covalent kinetics, with benzenesulfonyl fluoride derivative 23 achieving rapid and complete protein modification. Biochemical and mass spectrometry analyses confirmed selective modification of an active-site lysine by 23, along with robust performance in complex biological samples. Extension to urokinase plasminogen activator further demonstrates the generality of this strategy. More broadly, this work establishes electrophile scanning within macrocyclic scaffolds as a general approach for tuning covalent reactivity and provides a blueprint for designing selective probes that move beyond catalytic-residue targeting.

biochemistry↗

bioRxiv2025

Small shifts, big ripples: multi-omics studies reveal how ambient temperatures govern Chlamydomonas cellular responses

Photosynthetic protists, known as microalgae, face increasing temperatures due to climate change. The green biflagellate alga Chlamydomonas reinhardtii serves as a model for thermoregulation. While responses to thermal stress are well characterized, much less is known about the impact of ambient temperature shifts. Understanding microalgal responses to environmental temperature changes is critical, as these primary producers drive ecosystem productivity and food web dynamics. Here, C. reinhardtii grew mixotrophically at ambient temperatures from 18 {degrees}C to 33 {degrees}C. Transcriptomic profiling revealed extensive reorganization, with over 5,000 transcripts significantly affected, including those involved in algal-bacterial interactions, photoreception, lipid metabolism, photosynthesis, cilia formation, and the secretome. CO2 transfer rates and acetate levels measured at 18 {degrees}C and 28 {degrees}C suggest decreased photoautotrophic algal growth at 28 {degrees}C at first. Antagonistic bacterial activity was sustained longer at lower temperatures. Proteomic analyses of isolated cilia and secreted proteins corroborate major abundance changes within these sub-proteomes, particularly in ciliary intraflagellar transport complexes and mating-related proteins in the secretome. Together, these molecular alterations resulted in pronounced changes in growth, the lengths of cells and cilia swimming behavior, mating ability and bacterial antagonism. These data reveal major cellular responses caused by ambient, even short-term temperature shifts.

cell biology↗

bioRxiv2025

Inhibitor fluorination pattern tunes chemically induced protein dimerization

Chemically induced dimerization of proteins is a powerful approach to regulate biomolecular functions through small molecule ligands acting as "molecular glues". Here, we demonstrate that simple, thienopyrimidinone scaffold-based inhibitors efficiently promote homodimerization of an essential oxidoreductase from the human pathogenic parasite Trypanosoma brucei through selective covalent attachment and self-assembly. A fluorine walk strategy, commonly used to optimize small molecule properties, resulted in tuning induced dimer affinity across two orders of magnitude. NMR spectroscopy, MD simulations, chromatography, multi-angle light scattering, mass spectrometry, calorimetry, and functional assays reveal how the inhibitor fluorination pattern alters the dynamics and interactions of the enzyme-bound inhibitor and surface-exposed aromatic protein side chains, affecting both enzyme inhibition kinetics and induced dimerization. This work highlights how site-specific fluorination can modulate protein interactions and offers a framework for the design of novel molecular glues with broad applications in chemical biology and drug development.

biophysics↗

bioRxiv2025

Identification of a secreted protease from Bacteroides fragilis that induces intestinal pain and inflammation by cleavage of PAR2

Protease-activated receptor 2 (PAR2) is a central regulator of intestinal barrier function, inflammation and pain. Upregulated intestinal proteolysis and PAR2-signaling are implicated in inflammatory bowel diseases (IBDs) and irritable bowel syndrome (IBS). To identify potential bacterial regulators of PAR2 activity, we developed a functional assay for PAR2 processing and used it to screen conditioned media from a library of diverse gut commensal microbes. We found that multiple bacteria secrete proteases that cleave host PAR2. Using chemoproteomic profiling with a covalent irreversible inhibitor, we identified a previously uncharacterized Bacteroides fragilis serine protease Bfp1, and showed that it cleaves and activates PAR2 in multicellular and murine models. PAR2 cleavage by Bfp1 disrupts the intestinal barrier, sensitizes nociceptors, and triggers colonic inflammation and abdominal pain. Collectively, our findings uncover Bfp1-mediated PAR2-processing as a new axis of host-commensal-interaction in the gut that has the potential to be targeted for therapeutic intervention in IBD or IBS.

microbiology↗

bioRxiv2022

Chemoproteomic identification of a dipeptidyl peptidase 4 (DPP4) homolog in Bacteroides thetaiotaomicron important for envelope integrity and fitness

Serine hydrolases play important roles in signaling and human metabolism, yet little is known about the functions of these enzymes in gut commensal bacteria. Using bioinformatics and chemoproteomics, we identify serine hydrolases in the gut commensal Bacteroides thetaiotaomicron that are specific to the Bacteroidetes phylum. Two are predicted homologs of the human protease dipeptidyl peptidase 4 (hDPP4), a key enzyme that regulates insulin signaling. Functional studies reveal that BT4193 is a true homolog of hDPP4 while the other is misannotated and is a proline-specific triaminopeptidase. We demonstrate that BT4193 is important for envelope integrity and is inhibited by FDA-approved type 2 diabetes drugs that target hDPP4. Loss of BT4193 reduces B. thetaiotaomicron fitness during in vitro growth within a diverse community. Taken together, our findings suggest that serine hydrolases contribute to gut microbiota dynamics and may be off-targets for existing drugs that could cause unintended impact on the microbiota.

microbiology↗

bioRxiv2022

Antibiotic acyldepsipeptides stimulate the Streptomyces Clp-ATPase/ClpP complex for accelerated proteolysis

Clp proteases consist of a proteolytic, tetradecameric core ClpP and AAA+ Clp-ATPases. Streptomycetes, producers of a plethora of secondary metabolites, encode up to five different ClpP homologs and the composition of their unusually complex Clp protease machinery has remained unsolved. Here, we report on the composition of the house-keeping Clp protease in Streptomyces, consisting of a hetero-tetradecameric core built of ClpP1, ClpP2 and the cognate Clp-ATPases ClpX, ClpC1 or ClpC2, all interacting with ClpP2 only. ADEP antibiotics dysregulate the Clp protease for unregulated proteolysis. We observed that ADEP binds Streptomyces ClpP1, but not ClpP2, thereby not only triggering the degradation of non-native protein substrates but also accelerating Clp-ATPase-dependent proteolysis. The explanation is the concomitant binding of ADEP and Clp-ATPases to opposite sides of the ClpP1P2 barrel, hence revealing a third, so far unknown mechanism of ADEP action, i.e., the accelerated proteolysis of native protein substrates by the Clp protease. SignificanceClp proteases are antibiotic and anti-cancer drug targets. Composed of the proteolytic core ClpP and a regulatory Clp-ATPase, the protease machinery is important for protein homeostasis and regulatory proteolysis. The acyldepsipeptide antibiotic ADEP targets ClpP and has shown promise for treating multi-resistant and persistent bacterial infections. The molecular mechanism of ADEP is multi-layered. Here, we present a new way how ADEP can deregulate the Clp protease system. Clp-ATPases and ADEP bind to opposite sides of Streptomyces ClpP, accelerating the degradation of natural Clp protease substrates. We also demonstrate the composition of the major Streptomyces Clp protease complex, a heteromeric ClpP1P2 core with the Clp-ATPases ClpX, ClpC1 or ClpC2 exclusively bound to ClpP2, and the killing mechanism of ADEP in Streptomyces.

microbiology↗

bioRxiv2020

Toxoplasma gondii serine hydrolases regulate parasite lipid mobilization during growth and replication within the host

The intracellular protozoan parasite Toxoplasma gondii must scavenge cholesterol and other lipids from the host to facilitate intracellular growth and replication. Enzymes responsible for neutral lipid synthesis have been identified but there is no evidence for enzymes that catalyze lipolysis of cholesterol esters and esterified lipids. Here we characterize several T. gondii serine hydrolases with esterase and thioesterase activities that were previously thought to be depalmitoylating enzymes. We find they do not cleave palmitoyl thiol esters but rather hydrolyze short chain lipid esters. Deletion of one of the hydrolases results in alterations in levels of multiple lipids species. We also identify small molecule inhibitors of these hydrolases and show that treatment of parasites results in phenotypic defects reminiscent of parasites exposed to excess cholesterol or oleic acid. Together, these data characterize enzymes necessary for processing lipids critical for infection and highlight the potential for targeting parasite hydrolases for therapeutic applications. HighlightsO_LIBioinformatic and biochemical characterization of T. gondii serine hydrolases reveals substrate preference between enzymes with similar catalytic fold C_LIO_LIT. gondii serine hydrolases previously thought to be depalmitoylases are lipid metabolizing enzymes C_LIO_LIT. gondii lipid metabolism pathways utilize enzymes that are viable therapeutic targets C_LI

microbiology↗