10 research outputs found
Identifying Functional Cysteine Residues in the Mitochondria
The
mitochondria are dynamic organelles that regulate oxidative
metabolism and mediate cellular redox homeostasis. Proteins within
the mitochondria are exposed to large fluxes in the surrounding redox
environment. In particular, cysteine residues within mitochondrial
proteins sense and respond to these redox changes through oxidative
modifications of the cysteine thiol group. These oxidative modifications
result in a loss in cysteine reactivity, which can be monitored using
cysteine-reactive chemical probes and quantitative mass spectrometry
(MS). Analysis of cell lysates treated with cysteine-reactive probes
enable the identification of hundreds of cysteine residues, however,
the mitochondrial proteome is poorly represented (<10% of identified
peptides), due to the low abundance of mitochondrial proteins and
suppression of mitochondrial peptide MS signals by highly abundant
cytosolic peptides. Here, we apply a mitochondrial isolation and purification
protocol to substantially increase coverage of the mitochondrial cysteine
proteome. Over 1500 cysteine residues from ∼450 mitochondrial
proteins were identified, thereby enabling interrogation of an unprecedented
number of mitochondrial cysteines. Specifically, these mitochondrial
cysteines were ranked by reactivity to identify hyper-reactive cysteines
with potential catalytic and regulatory functional roles. Furthermore,
analyses of mitochondria exposed to nitrosative stress revealed previously
uncharacterized sites of protein <i>S</i>-nitrosation on
mitochondrial proteins. Together, the mitochondrial cysteine enrichment
strategy presented herein enables detailed characterization of protein
modifications that occur within the mitochondria during (patho)Âphysiological
fluxes in the redox environment
Investigating the Proteome Reactivity and Selectivity of Aryl Halides
Protein-reactive electrophiles are
critical to chemical proteomic
applications including activity-based protein profiling, site-selective
protein modification, and covalent inhibitor development. Here, we
explore the protein reactivity of a panel of aryl halides that function
through a nucleophilic aromatic substitution (S<sub>N</sub>Ar) mechanism.
We show that the reactivity of these electrophiles can be finely tuned
by varying the substituents on the aryl ring. We identify <i>p</i>-chloro- and fluoronitrobenzenes and dichlorotriazines
as covalent protein modifiers at low micromolar concentrations. Interestingly,
investigating the site of labeling of these electrophiles within complex
proteomes identified <i>p</i>-chloronitrobenzene as highly
cysteine selective, whereas the dichlorotriazine favored reactivity
with lysines. These studies illustrate the diverse reactivity and
amino-acid selectivity of aryl halides and enable the future application
of this class of electrophiles in chemical proteomics
Investigating the Proteome Reactivity and Selectivity of Aryl Halides
Protein-reactive electrophiles are
critical to chemical proteomic
applications including activity-based protein profiling, site-selective
protein modification, and covalent inhibitor development. Here, we
explore the protein reactivity of a panel of aryl halides that function
through a nucleophilic aromatic substitution (S<sub>N</sub>Ar) mechanism.
We show that the reactivity of these electrophiles can be finely tuned
by varying the substituents on the aryl ring. We identify <i>p</i>-chloro- and fluoronitrobenzenes and dichlorotriazines
as covalent protein modifiers at low micromolar concentrations. Interestingly,
investigating the site of labeling of these electrophiles within complex
proteomes identified <i>p</i>-chloronitrobenzene as highly
cysteine selective, whereas the dichlorotriazine favored reactivity
with lysines. These studies illustrate the diverse reactivity and
amino-acid selectivity of aryl halides and enable the future application
of this class of electrophiles in chemical proteomics
Bioinformatic and Biochemical Characterizations of C–S Bond Formation and Cleavage Enzymes in the Fungus <i>Neurospora crassa</i> Ergothioneine Biosynthetic Pathway
Ergothioneine
is a histidine thiol derivative. Its mycobacterial
biosynthetic pathway has five steps (EgtA-E catalysis) with two novel
reactions: a mononuclear nonheme iron enzyme (EgtB) catalyzed oxidative
C–S bond formation and a PLP-mediated C–S lyase (EgtE)
reaction. Our bioinformatic and biochemical analyses indicate that
the fungus <i>Neurospora crassa</i> has a more concise ergothioneine
biosynthetic pathway because its nonheme iron enzyme, Egt1, makes
use of cysteine instead of γ-Glu-Cys as the substrate. Such
a change of substrate preference eliminates the competition between
ergothioneine and glutathione biosyntheses. In addition, we have identified
the <i>N. crassa</i> C–S lyase (NCU11365) and reconstituted
its activity in vitro, which makes the future ergothioneine production
through metabolic engineering feasible
Selenium-Encoded Isotopic Signature Targeted Profiling
Selenium
(Se), as an essential trace element, plays crucial roles
in many organisms including humans. The biological functions of selenium
are mainly mediated by selenoproteins, a unique class of selenium-containing
proteins in which selenium is inserted in the form of selenocysteine.
Due to their low abundance and uneven tissue distribution, detection
of selenoproteins within proteomes is very challenging, and therefore
functional studies of these proteins are limited. In this study, we
developed a computational method, named as selenium-encoded isotopic
signature targeted profiling (SESTAR), which utilizes the distinct
natural isotopic distribution of selenium to assist detection of trace
selenium-containing signals from shotgun-proteomic data. SESTAR can
detect femtomole quantities of synthetic selenopeptides in a benchmark
test and dramatically improved detection of native selenoproteins
from tissue proteomes in a targeted profiling mode. By applying SESTAR
to screen publicly available datasets from Human Proteome Map, we
provide a comprehensive picture of selenoprotein distributions in
human primary hematopoietic cells and tissues. We further demonstrated
that SESTAR can aid chemical-proteomic strategies to identify additional
selenoprotein targets of RSL3, a canonical inducer of cell ferroptosis.
We believe SESTAR not only serves as a powerful tool for global profiling
of native selenoproteomes, but can also work seamlessly with chemical-proteomic
profiling strategies to enhance identification of target proteins,
post-translational modifications, or protein–protein interactions
Selenium-Encoded Isotopic Signature Targeted Profiling
Selenium
(Se), as an essential trace element, plays crucial roles
in many organisms including humans. The biological functions of selenium
are mainly mediated by selenoproteins, a unique class of selenium-containing
proteins in which selenium is inserted in the form of selenocysteine.
Due to their low abundance and uneven tissue distribution, detection
of selenoproteins within proteomes is very challenging, and therefore
functional studies of these proteins are limited. In this study, we
developed a computational method, named as selenium-encoded isotopic
signature targeted profiling (SESTAR), which utilizes the distinct
natural isotopic distribution of selenium to assist detection of trace
selenium-containing signals from shotgun-proteomic data. SESTAR can
detect femtomole quantities of synthetic selenopeptides in a benchmark
test and dramatically improved detection of native selenoproteins
from tissue proteomes in a targeted profiling mode. By applying SESTAR
to screen publicly available datasets from Human Proteome Map, we
provide a comprehensive picture of selenoprotein distributions in
human primary hematopoietic cells and tissues. We further demonstrated
that SESTAR can aid chemical-proteomic strategies to identify additional
selenoprotein targets of RSL3, a canonical inducer of cell ferroptosis.
We believe SESTAR not only serves as a powerful tool for global profiling
of native selenoproteomes, but can also work seamlessly with chemical-proteomic
profiling strategies to enhance identification of target proteins,
post-translational modifications, or protein–protein interactions
Selenium-Encoded Isotopic Signature Targeted Profiling
Selenium
(Se), as an essential trace element, plays crucial roles
in many organisms including humans. The biological functions of selenium
are mainly mediated by selenoproteins, a unique class of selenium-containing
proteins in which selenium is inserted in the form of selenocysteine.
Due to their low abundance and uneven tissue distribution, detection
of selenoproteins within proteomes is very challenging, and therefore
functional studies of these proteins are limited. In this study, we
developed a computational method, named as selenium-encoded isotopic
signature targeted profiling (SESTAR), which utilizes the distinct
natural isotopic distribution of selenium to assist detection of trace
selenium-containing signals from shotgun-proteomic data. SESTAR can
detect femtomole quantities of synthetic selenopeptides in a benchmark
test and dramatically improved detection of native selenoproteins
from tissue proteomes in a targeted profiling mode. By applying SESTAR
to screen publicly available datasets from Human Proteome Map, we
provide a comprehensive picture of selenoprotein distributions in
human primary hematopoietic cells and tissues. We further demonstrated
that SESTAR can aid chemical-proteomic strategies to identify additional
selenoprotein targets of RSL3, a canonical inducer of cell ferroptosis.
We believe SESTAR not only serves as a powerful tool for global profiling
of native selenoproteomes, but can also work seamlessly with chemical-proteomic
profiling strategies to enhance identification of target proteins,
post-translational modifications, or protein–protein interactions
Selenium-Encoded Isotopic Signature Targeted Profiling
Selenium
(Se), as an essential trace element, plays crucial roles
in many organisms including humans. The biological functions of selenium
are mainly mediated by selenoproteins, a unique class of selenium-containing
proteins in which selenium is inserted in the form of selenocysteine.
Due to their low abundance and uneven tissue distribution, detection
of selenoproteins within proteomes is very challenging, and therefore
functional studies of these proteins are limited. In this study, we
developed a computational method, named as selenium-encoded isotopic
signature targeted profiling (SESTAR), which utilizes the distinct
natural isotopic distribution of selenium to assist detection of trace
selenium-containing signals from shotgun-proteomic data. SESTAR can
detect femtomole quantities of synthetic selenopeptides in a benchmark
test and dramatically improved detection of native selenoproteins
from tissue proteomes in a targeted profiling mode. By applying SESTAR
to screen publicly available datasets from Human Proteome Map, we
provide a comprehensive picture of selenoprotein distributions in
human primary hematopoietic cells and tissues. We further demonstrated
that SESTAR can aid chemical-proteomic strategies to identify additional
selenoprotein targets of RSL3, a canonical inducer of cell ferroptosis.
We believe SESTAR not only serves as a powerful tool for global profiling
of native selenoproteomes, but can also work seamlessly with chemical-proteomic
profiling strategies to enhance identification of target proteins,
post-translational modifications, or protein–protein interactions
Selenium-Encoded Isotopic Signature Targeted Profiling
Selenium
(Se), as an essential trace element, plays crucial roles
in many organisms including humans. The biological functions of selenium
are mainly mediated by selenoproteins, a unique class of selenium-containing
proteins in which selenium is inserted in the form of selenocysteine.
Due to their low abundance and uneven tissue distribution, detection
of selenoproteins within proteomes is very challenging, and therefore
functional studies of these proteins are limited. In this study, we
developed a computational method, named as selenium-encoded isotopic
signature targeted profiling (SESTAR), which utilizes the distinct
natural isotopic distribution of selenium to assist detection of trace
selenium-containing signals from shotgun-proteomic data. SESTAR can
detect femtomole quantities of synthetic selenopeptides in a benchmark
test and dramatically improved detection of native selenoproteins
from tissue proteomes in a targeted profiling mode. By applying SESTAR
to screen publicly available datasets from Human Proteome Map, we
provide a comprehensive picture of selenoprotein distributions in
human primary hematopoietic cells and tissues. We further demonstrated
that SESTAR can aid chemical-proteomic strategies to identify additional
selenoprotein targets of RSL3, a canonical inducer of cell ferroptosis.
We believe SESTAR not only serves as a powerful tool for global profiling
of native selenoproteomes, but can also work seamlessly with chemical-proteomic
profiling strategies to enhance identification of target proteins,
post-translational modifications, or protein–protein interactions
Synthesis, characterization, and computational study of three-coordinate SNS-copper(I) complexes based on bis-thione precursors
<div><p>A series of tridentate pincer ligands, each possessing two sulfur and one nitrogen donor (SNS), based on bis-imidazolyl or bis-triazolyl salts were metallated with CuCl<sub>2</sub> to give new tridentate SNS pincer copper(I) complexes [(SNS)Cu]<sup>+</sup>. These orange complexes exhibit a three-coordinate pseudo-trigonal-planar geometry in copper. During the formation of these copper(I) complexes, disproportionation is observed as the copper(II) salt precursor is converted into the Cu(I) [(SNS)Cu]<sup>+</sup> cation and the [CuCl<sub>4</sub>]<sup>2–</sup> counteranion. The [(SNS)Cu]<sup>+</sup> complexes were characterized with single crystal X-ray diffraction, electrospray mass spectrometry, EPR spectroscopy, attenuated total reflectance infrared spectroscopy, UV–Vis spectroscopy, cyclic voltammetry, and elemental analysis. The EPR spectra are consistent with anisotropic Cu(II) signals with four hyperfine splittings in the lower-field region (<i>g</i><sub>||</sub>) and <i>g</i> values consistent with the presence of the tetrachlorocuprate. Various electronic transitions are apparent in the UV–Vis spectra of the complexes and originate in the copper-containing cations and anions. Density functional calculations support the nature of the SNS binding, allowing assignment of a number of features present in the UV–Vis and IR spectra and cyclic voltammograms of these complexes.</p></div