14 research outputs found
Discovery of a Highly Selective Caspase‑3 Substrate for Imaging Live Cells
Caspases are a family of cysteine
proteases that are well-known
for their roles in apoptosis and inflammation. Recent studies provide
evidence that caspases are also integral to many additional cellular
processes, such as differentiation and proliferation. Likewise, aberrant
caspase activity has been implicated in the progression of several
diseases, including neurodegenerative disorders, cancer, cardiovascular
disease, and sepsis. These observations establish the importance of
caspases to a diverse array of physiological functions and future
endeavors will undoubtedly continue to elucidate additional processes
that require caspase activity. Unfortunately, the existence of 11
functional human caspases, with overlapping substrate specificities,
confounds the ability to confidently assign one or more isoforms to
biological phenomena. Herein, we characterize a first-in-class FRET
substrate that is selectively recognized by active caspase-3 over
other initiator and executioner caspases. We further apply this substrate
to specifically image caspase-3 activity in live cells undergoing
apoptosis
Selective Detection and Inhibition of Active Caspase‑3 in Cells with Optimized Peptides
Caspases
are a family of cysteine-aspartyl proteases that are well
recognized for their essential roles in apoptosis and inflammation.
Recently, caspases have also been linked to the promotion of other
biologically important phenomena, such as cellular differentiation
and proliferation. Dysregulation of the multifaceted and indispensable
activities of caspases has been globally linked to several diseases,
including cancer and neurodegenerative disorders; however, the specific
caspase members responsible for these diseases have yet to be assigned.
Activity-based probes (ABPs) and peptide-based inhibitors are instrumental
in the detection and control of protease activity and serve as alternative
methods to genetic approaches. Such molecules aid in the interrogation
of specific proteases within cellular and animal models as well as
help elucidate aberrant proteolytic function correlated to disease
phenotypes. No ABPs or inhibitors have been discovered that specifically
target one of the eleven human caspases in a cellular context. Therefore,
ascribing distinct contributions to an individual caspase activity
within naturally occurring biological systems is not possible. Herein,
we describe a peptide series optimized for the selective detection
and inhibition of active caspase-3 in cells. These compounds exhibit
low nanomolar potency against caspase-3 with >120-fold selectivity
over caspase-7 which shares 77% active site identity. Our ability
to individually target wild-type active caspase-3 for detection and
cell permeable inhibition is a valuable proof-of-concept methodology
that can be readily employed to probe the significance of caspase-3
in apoptosis, neurological disorders, cardiovascular diseases, and
sepsis
Identification and Co-complex Structure of a New <i>S. pyogenes</i> SpeB Small Molecule Inhibitor
The secreted <i>Streptococcus
pyogenes</i> cysteine protease
SpeB is implicated in host immune system evasion and bacterial virulence.
We present a small molecule inhibitor of SpeB <b>2477</b> identified
from a high-throughput screen based on the hydrolysis of a fluorogenic
peptide substrate Ac-AIK-AMC. <b>2477</b> inhibits other SpeB-related
proteases but not human caspase-3, suggesting that the molecule targets
proteases with the papain-like structural fold. A 1.59 Å X-ray
crystal structure of <b>2477</b> bound to the SpeB active site
reveals the mechanism of inhibition and the essential constituents
of <b>2477</b> necessary for binding. An assessment against
a panel of <b>2477</b> derivatives confirms our structural findings
and shows that a carbamate and nitrile on <b>2477</b> are required
for SpeB inhibition, as these moieties provide an extensive network
of electrostatic and hydrogen-bonding interactions with SpeB active
site residues. Surprisingly, despite <b>2477</b> having a reduced
inhibitory potential against papain, the majority of <b>2477</b>-related compounds inhibit papain to a much greater and broader extent
than SpeB. These findings indicate that SpeB is more stringently selective
than papain for this panel of small molecule inhibitors. On the basis
of our structural and biochemical characterization, we propose modifications
to <b>2477</b> for subsequent rounds of inhibitor design that
will impart specificity to SpeB over other papain-like proteases,
including alterations of the compound to exploit the differences in
CA protease active site pocket sizes and electrostatics
Selective Detection of Caspase‑3 versus Caspase‑7 Using Activity-Based Probes with Key Unnatural Amino Acids
Caspases are required for essential
biological functions, most
notably apoptosis and pyroptosis, but also cytokine production, cell
proliferation, and differentiation. One of the most well studied members
of this cysteine protease family includes executioner caspase-3, which
plays a central role in cell apoptosis and differentiation. Unfortunately,
there exists a dearth of chemical tools to selectively monitor caspase-3
activity under complex cellular and <i>in vivo</i> conditions
due to its close homology with executioner caspase-7. Commercially
available activity-based probes and substrates rely on the canonical
DEVD tetrapeptide sequence, which both caspases-3 and -7 recognize
with similar affinity, and thus the individual contributions of caspase-3
and/or -7 toward important cellular processes are irresolvable. Here,
we analyzed a variety of permutations of the DEVD peptide sequence
in order to discover peptides with biased activity and recognition
of caspase-3 versus caspases-6, -7, -8, and -9. Through this study,
we identify fluorescent and biotinylated probes capable of selective
detection of caspase-3 using key unnatural amino acids. Likewise,
we determined the X-ray crystal structures of caspases-3, -7, and
-8 in complex with our lead peptide inhibitor to elucidate the binding
mechanism and active site interactions that promote the selective
recognition of caspase-3 over other highly homologous caspase family
members
Quantitative Metaproteomics and Activity-Based Probe Enrichment Reveals Significant Alterations in Protein Expression from a Mouse Model of Inflammatory Bowel Disease
Tandem
mass spectrometry based shotgun proteomics of distal gut
microbiomes is exceedingly difficult due to the inherent complexity
and taxonomic diversity of the samples. We introduce two new methodologies
to improve metaproteomic studies of microbiome samples. These methods
include the stable isotope labeling in mammals to permit protein quantitation
across two mouse cohorts as well as the application of activity-based
probes to enrich and analyze both host and microbial proteins with
specific functionalities. We used these technologies to study the
microbiota from the adoptive T cell transfer mouse model of inflammatory
bowel disease (IBD) and compare these samples to an isogenic control,
thereby limiting genetic and environmental variables that influence
microbiome composition. The data generated highlight quantitative
alterations in both host and microbial proteins due to intestinal
inflammation and corroborates the observed phylogenetic changes in
bacteria that accompany IBD in humans and mouse models. The combination
of isotope labeling with shotgun proteomics resulted in the total
identification of 4434 protein clusters expressed in the microbial
proteomic environment, 276 of which demonstrated differential abundance
between control and IBD mice. Notably, application of a novel cysteine-reactive
probe uncovered several microbial proteases and hydrolases overrepresented
in the IBD mice. Implementation of these methods demonstrated that
substantial insights into the identity and dysregulation of host and
microbial proteins altered in IBD can be accomplished and can be used
in the interrogation of other microbiome-related diseases
Quantitative Metaproteomics and Activity-Based Probe Enrichment Reveals Significant Alterations in Protein Expression from a Mouse Model of Inflammatory Bowel Disease
Tandem
mass spectrometry based shotgun proteomics of distal gut
microbiomes is exceedingly difficult due to the inherent complexity
and taxonomic diversity of the samples. We introduce two new methodologies
to improve metaproteomic studies of microbiome samples. These methods
include the stable isotope labeling in mammals to permit protein quantitation
across two mouse cohorts as well as the application of activity-based
probes to enrich and analyze both host and microbial proteins with
specific functionalities. We used these technologies to study the
microbiota from the adoptive T cell transfer mouse model of inflammatory
bowel disease (IBD) and compare these samples to an isogenic control,
thereby limiting genetic and environmental variables that influence
microbiome composition. The data generated highlight quantitative
alterations in both host and microbial proteins due to intestinal
inflammation and corroborates the observed phylogenetic changes in
bacteria that accompany IBD in humans and mouse models. The combination
of isotope labeling with shotgun proteomics resulted in the total
identification of 4434 protein clusters expressed in the microbial
proteomic environment, 276 of which demonstrated differential abundance
between control and IBD mice. Notably, application of a novel cysteine-reactive
probe uncovered several microbial proteases and hydrolases overrepresented
in the IBD mice. Implementation of these methods demonstrated that
substantial insights into the identity and dysregulation of host and
microbial proteins altered in IBD can be accomplished and can be used
in the interrogation of other microbiome-related diseases
Quantitative Metaproteomics and Activity-Based Probe Enrichment Reveals Significant Alterations in Protein Expression from a Mouse Model of Inflammatory Bowel Disease
Tandem
mass spectrometry based shotgun proteomics of distal gut
microbiomes is exceedingly difficult due to the inherent complexity
and taxonomic diversity of the samples. We introduce two new methodologies
to improve metaproteomic studies of microbiome samples. These methods
include the stable isotope labeling in mammals to permit protein quantitation
across two mouse cohorts as well as the application of activity-based
probes to enrich and analyze both host and microbial proteins with
specific functionalities. We used these technologies to study the
microbiota from the adoptive T cell transfer mouse model of inflammatory
bowel disease (IBD) and compare these samples to an isogenic control,
thereby limiting genetic and environmental variables that influence
microbiome composition. The data generated highlight quantitative
alterations in both host and microbial proteins due to intestinal
inflammation and corroborates the observed phylogenetic changes in
bacteria that accompany IBD in humans and mouse models. The combination
of isotope labeling with shotgun proteomics resulted in the total
identification of 4434 protein clusters expressed in the microbial
proteomic environment, 276 of which demonstrated differential abundance
between control and IBD mice. Notably, application of a novel cysteine-reactive
probe uncovered several microbial proteases and hydrolases overrepresented
in the IBD mice. Implementation of these methods demonstrated that
substantial insights into the identity and dysregulation of host and
microbial proteins altered in IBD can be accomplished and can be used
in the interrogation of other microbiome-related diseases
Triflic Acid Treatment Enables LC-MS/MS Analysis of Insoluble Bacterial Biomass
The
lysis and extraction of soluble bacterial proteins from cells is a
common practice for proteomics analyses, but insoluble bacterial biomasses
are often left behind. Here, we show that with triflic acid treatment,
the insoluble bacterial biomass of Gram<sup>–</sup> and Gram<sup>+</sup> bacteria can be rendered soluble. We use LC-MS/MS shotgun
proteomics to show that bacterial proteins in the soluble and insoluble
postlysis fractions differ significantly. Additionally, in the case
of Gram<sup>–</sup> Pseudomonas aeruginosa, triflic acid treatment enables the enrichment of cell-envelope-associated
proteins. Finally, we apply triflic acid to a human microbiome sample
to show that this treatment is robust and enables the identification
of a new, complementary subset of proteins from a complex microbial
mixture
Triflic Acid Treatment Enables LC-MS/MS Analysis of Insoluble Bacterial Biomass
The
lysis and extraction of soluble bacterial proteins from cells is a
common practice for proteomics analyses, but insoluble bacterial biomasses
are often left behind. Here, we show that with triflic acid treatment,
the insoluble bacterial biomass of Gram<sup>–</sup> and Gram<sup>+</sup> bacteria can be rendered soluble. We use LC-MS/MS shotgun
proteomics to show that bacterial proteins in the soluble and insoluble
postlysis fractions differ significantly. Additionally, in the case
of Gram<sup>–</sup> Pseudomonas aeruginosa, triflic acid treatment enables the enrichment of cell-envelope-associated
proteins. Finally, we apply triflic acid to a human microbiome sample
to show that this treatment is robust and enables the identification
of a new, complementary subset of proteins from a complex microbial
mixture
Selective Inhibition of Initiator versus Executioner Caspases Using Small Peptides Containing Unnatural Amino Acids
Caspases
are fundamental to many essential biological processes,
including apoptosis, differentiation, and inflammation. Unregulated
caspase activity is also implicated in the development and progression
of several diseases, such as cancer, neurodegenerative disorders,
and sepsis. Unfortunately, it is difficult to determine exactly which
caspase(s) of the 11 isoforms that humans express is responsible for
specific biological functions. This lack of resolution is primarily
due to highly homologous active sites and overlapping substrates.
Currently available peptide-based inhibitors and probes are based
on specificity garnered from peptide substrate libraries. For example,
the canonical tetrapeptide LETD was discovered as the canonical sequence
that is optimally recognized by caspase-8; however, LETD-based inhibitors
and substrates promiscuously bind to other isoforms with equal affinity,
including caspases-3, -6, and -9. In order to mitigate this problem,
we report the identification of a new series of compounds that are
>100-fold selective for inhibiting the initiator caspases-8 and
-9
over the executioner caspases-3, -6, and -7