3 research outputs found
Biochemical and Cellular Characterization of the Function of Fluorophosphonate-Binding Hydrolase H (FphH) in <i>Staphylococcus aureus</i> Support a Role in Bacterial Stress Response
The development of
new treatment options for bacterial
infections
requires access to new targets for antibiotics and antivirulence strategies.
Chemoproteomic approaches are powerful tools for profiling and identifying
novel druggable target candidates, but their functions often remain
uncharacterized. Previously, we used activity-based protein profiling
in the opportunistic pathogen Staphylococcus aureus to identify active serine hydrolases termed fluorophosphonate-binding
hydrolases (Fph). Here, we provide the first characterization of S. aureus FphH, a conserved, putative carboxylesterase (referred
to as yvaK in Bacillus subtilis)
at the molecular and cellular level. First, phenotypic characterization
of fphH-deficient transposon mutants revealed phenotypes
during growth under nutrient deprivation, biofilm formation, and intracellular
survival. Biochemical and structural investigations revealed that
FphH acts as an esterase and lipase based on a fold well suited to
act on a small to long hydrophobic unbranched lipid group within its
substrate and can be inhibited by active site-targeting oxadiazoles.
Prompted by a previous observation that fphH expression
was upregulated in response to fusidic acid, we found that FphH can
deacetylate this ribosome-targeting antibiotic, but the lack of FphH
function did not infer major changes in antibiotic susceptibility.
In conclusion, our results indicate a functional role of this hydrolase
in S. aureus stress responses, and hypothetical functions
connecting FphH with components of the ribosome rescue system that
are conserved in the same gene cluster across Bacillales are discussed. Our atomic characterization of FphH will facilitate
the development of specific FphH inhibitors and probes to elucidate
its physiological role and validity as a drug target
Design of Selective Substrates and Activity-Based Probes for Hydrolase Important for Pathogenesis 1 (HIP1) from <i>Mycobacterium tuberculosis</i>
Although
serine proteases are important mediators of <i>Mycobacterium
tuberculosis</i> (Mtb) virulence, there are currently no tools
to selectively block or visualize members of this family of enzymes.
Selective reporter substrates or activity-based probes (ABPs) could
provide a means to monitor infection and response to therapy using
imaging methods. Here, we use a combination of substrate selectivity
profiling and focused screening to identify optimized reporter substrates
and ABPs for the Mtb “Hydrolase important for pathogenesis
1” (Hip1) serine protease. Hip1 is a cell-envelope-associated
enzyme with minimal homology to host proteases, making it an ideal
target for probe development. We identified substituted 7-amino-4-chloro-3-(2-bromoethoxy)isocoumarins
as irreversible inhibitor scaffolds. Furthermore, we used specificity
data to generate selective reporter substrates and to further optimize
a selective chloroisocoumarin inhibitor. These new reagents are potentially
useful in delineating the roles of Hip1 during pathogenesis or as
diagnostic imaging tools for specifically monitoring Mtb infections
Inhibition of NGLY1 Inactivates the Transcription Factor Nrf1 and Potentiates Proteasome Inhibitor Cytotoxicity
Proteasome inhibitors
are used to treat blood cancers such as multiple
myeloma (MM) and mantle cell lymphoma. The efficacy of these drugs
is frequently undermined by acquired resistance. One mechanism of
proteasome inhibitor resistance may involve the transcription factor
Nuclear Factor, Erythroid 2 Like 1 (NFE2L1, also referred to as Nrf1),
which responds to proteasome insufficiency or pharmacological inhibition
by upregulating proteasome subunit gene expression. This “bounce-back”
response is achieved through a unique mechanism. Nrf1 is constitutively
translocated into the ER lumen, N-glycosylated, and then targeted
for proteasomal degradation via the ER-associated degradation (ERAD)
pathway. Proteasome inhibition leads to accumulation of cytosolic
Nrf1, which is then processed to form the active transcription factor.
Here we show that the cytosolic enzyme N-glycanase 1 (NGLY1, the human
PNGase) is essential for Nrf1 activation in response to proteasome
inhibition. Chemical or genetic disruption of NGLY1 activity results
in the accumulation of misprocessed Nrf1 that is largely excluded
from the nucleus. Under these conditions, Nrf1 is inactive in regulating
proteasome subunit gene expression in response to proteasome inhibition.
Through a small molecule screen, we identified a cell-active NGLY1
inhibitor that disrupts the processing and function of Nrf1. The compound
potentiates the cytotoxicity of carfilzomib, a clinically used proteasome
inhibitor, against MM and T cell-derived acute lymphoblastic leukemia
(T-ALL) cell lines. Thus, NGLY1 inhibition prevents Nrf1 activation
and represents a new therapeutic approach for cancers that depend
on proteasome homeostasis