4 research outputs found
Covalent Modulators of the Vacuolar ATPase
The
vacuolar H<sup>+</sup> ATPase (V-ATPase) is a complex multisubunit
machine that regulates important cellular processes through controlling
acidity of intracellular compartments in eukaryotes. Existing small-molecule
modulators of V-ATPase either are restricted to targeting one membranous
subunit of V-ATPase or have poorly understood mechanisms of action.
Small molecules with novel and defined mechanisms of inhibition are
thus needed to functionally characterize V-ATPase and to fully evaluate
the therapeutic relevance of V-ATPase in human diseases. We have discovered
electrophilic quinazolines that covalently modify a soluble catalytic
subunit of V-ATPase with high potency and exquisite proteomic selectivity
as revealed by fluorescence imaging and chemical proteomic activity-based
profiling. The site of covalent modification was mapped to a cysteine
residue located in a region of V-ATPase subunit A that is thought
to regulate the dissociation of V-ATPase. We further demonstrate that
a previously reported V-ATPase inhibitor, 3-bromopyruvate, also targets
the same cysteine residue and that our electrophilic quinazolines
modulate the function of V-ATPase in cells. With their well-defined
mechanism of action and high proteomic specificity, the described
quinazolines offer a powerful set of chemical probes to investigate
the physiological and pathological roles of V-ATPase
Chemical Proteomic Profiling of Human Methyltransferases
Methylation
is a fundamental mechanism used in Nature to modify
the structure and function of biomolecules, including proteins, DNA,
RNA, and metabolites. Methyl groups are predominantly installed into
biomolecules by a large and diverse class of <i>S</i>-adenosyl
methionine (SAM)-dependent methyltransferases (MTs), of which there
are ∼200 known or putative members in the human proteome. Deregulated
MT activity contributes to numerous diseases, including cancer, and
several MT inhibitors are in clinical development. Nonetheless, a
large fraction of the human MT family remains poorly characterized,
underscoring the need for new technologies to characterize MTs and
their inhibitors in native biological systems. Here, we describe a
suite of <i>S</i>-adenosyl homocysteine (SAH) photoreactive
probes and their application in chemical proteomic experiments to
profile and enrich a large number of MTs (>50) from human cancer
cell
lysates with remarkable specificity over other classes of proteins.
We further demonstrate that the SAH probes can enrich MT-associated
proteins and be used to screen for and assess the selectivity of MT
inhibitors, leading to the discovery of a covalent inhibitor of nicotinamide <i>N</i>-methyltransferase (NNMT), an enzyme implicated in cancer
and metabolic disorders. The chemical proteomics probes and methods
for their utilization reported herein should prove of value for the
functional characterization of MTs, MT complexes, and MT inhibitors
in mammalian biology and disease
A Screen for Protein–Protein Interactions in Live Mycobacteria Reveals a Functional Link between the Virulence-Associated Lipid Transporter LprG and the Mycolyltransferase Antigen 85A
Outer
membrane lipids in pathogenic mycobacteria are important for virulence
and survival. Although the biosynthesis of these lipids has been extensively
studied, mechanisms responsible for their assembly in the outer membrane
are not understood. In the study of Gram-negative outer membrane assembly,
protein–protein interactions define transport mechanisms, but
analogous interactions have not been explored in mycobacteria. Here
we identified interactions with the lipid transport protein LprG.
Using site-specific photo-cross-linking in live mycobacteria, we mapped
three major interaction interfaces within LprG. We went on to identify
proteins that cross-link at the entrance to the lipid binding pocket,
an area likely relevant to LprG transport function. We verified LprG
site-specific interactions with two hits, the conserved lipoproteins
LppK and LppI. We further showed that LprG interacts physically and
functionally with the mycolyltransferase Ag85A, as loss of either
protein leads to similar defects in cell growth and mycolylation.
Overall, our results support a model in which protein interactions
coordinate multiple pathways in outer membrane biogenesis and connect
lipid biosynthesis to transport
A Screen for Protein–Protein Interactions in Live Mycobacteria Reveals a Functional Link between the Virulence-Associated Lipid Transporter LprG and the Mycolyltransferase Antigen 85A
Outer
membrane lipids in pathogenic mycobacteria are important for virulence
and survival. Although the biosynthesis of these lipids has been extensively
studied, mechanisms responsible for their assembly in the outer membrane
are not understood. In the study of Gram-negative outer membrane assembly,
protein–protein interactions define transport mechanisms, but
analogous interactions have not been explored in mycobacteria. Here
we identified interactions with the lipid transport protein LprG.
Using site-specific photo-cross-linking in live mycobacteria, we mapped
three major interaction interfaces within LprG. We went on to identify
proteins that cross-link at the entrance to the lipid binding pocket,
an area likely relevant to LprG transport function. We verified LprG
site-specific interactions with two hits, the conserved lipoproteins
LppK and LppI. We further showed that LprG interacts physically and
functionally with the mycolyltransferase Ag85A, as loss of either
protein leads to similar defects in cell growth and mycolylation.
Overall, our results support a model in which protein interactions
coordinate multiple pathways in outer membrane biogenesis and connect
lipid biosynthesis to transport