17 research outputs found
Systematic Investigation of Dose-Dependent Protein Thermal Stability Changes to Uncover the Mechanisms of the Pleiotropic Effects of Metformin
Metformin is a widely
used drug to treat type II diabetes. Beyond
lowering blood sugar, it has been reported to have pleiotropic effects
such as suppressing cancer growth and attenuating cell oxidative stress
and inflammation. However, the underlying mechanisms of these effects
remain to be explored. Here, we systematically study the thermal stability
changes of proteins in liver cells (HepG2) induced by a wide dosage
range of metformin by using the proteome integral solubility alteration
(PISA) assay. The current results demonstrate that, besides the most
accepted target of metformin (complex I), low concentrations of metformin
(such as 0.2 ÎĽM) stabilize the complex IV subunits, suggesting
its important role in the sugar-lowering effect. Low-dose metformin
also results in stability alterations of ribosomal proteins, correlating
with its inhibitive effect on cell proliferation. We further find
that low-concentration metformin impacts mitochondrial cargo and vesicle
transport, while high-concentration metformin affects cell redox responses
and cell membrane protein sorting. This study provides mechanistic
insights into the molecular mechanisms of lowering blood sugar and
the pleiotropic effects of metformin
Systematic Investigation of Dose-Dependent Protein Thermal Stability Changes to Uncover the Mechanisms of the Pleiotropic Effects of Metformin
Metformin is a widely
used drug to treat type II diabetes. Beyond
lowering blood sugar, it has been reported to have pleiotropic effects
such as suppressing cancer growth and attenuating cell oxidative stress
and inflammation. However, the underlying mechanisms of these effects
remain to be explored. Here, we systematically study the thermal stability
changes of proteins in liver cells (HepG2) induced by a wide dosage
range of metformin by using the proteome integral solubility alteration
(PISA) assay. The current results demonstrate that, besides the most
accepted target of metformin (complex I), low concentrations of metformin
(such as 0.2 ÎĽM) stabilize the complex IV subunits, suggesting
its important role in the sugar-lowering effect. Low-dose metformin
also results in stability alterations of ribosomal proteins, correlating
with its inhibitive effect on cell proliferation. We further find
that low-concentration metformin impacts mitochondrial cargo and vesicle
transport, while high-concentration metformin affects cell redox responses
and cell membrane protein sorting. This study provides mechanistic
insights into the molecular mechanisms of lowering blood sugar and
the pleiotropic effects of metformin
Simultaneous Quantitation of Glycoprotein Degradation and Synthesis Rates by Integrating Isotope Labeling, Chemical Enrichment, and Multiplexed Proteomics
Protein glycosylation
is essential for cell survival and regulates
many cellular events. Reversible glycosylation is also dynamic in
biological systems. The functions of glycoproteins are regulated by
their dynamics to adapt the ever-changing inter- and intracellular
environments. Glycans on proteins not only mediate a variety of protein
activities, but also creates a steric hindrance for protecting the
glycoproteins from degradation by proteases. In this work, a novel
strategy integrating isotopic labeling, chemical enrichment and multiplexed
proteomics was developed to simultaneously quantify the degradation
and synthesis rates of many glycoproteins in human cells. We quantified
the synthesis rates of 847 N-glycoproteins and the degradation rates
of 704 glycoproteins in biological triplicate experiments, including
many important glycoproteins such as CD molecules. Through comparing
the synthesis and degradation rates, we found that most proteins have
higher synthesis rates since cells are still growing throughout the
time course, while a small group of proteins with lower synthesis
rates mainly participate in adhesion, locomotion, localization, and
signaling. This method can be widely applied in biochemical and biomedical
research and provide insights into elucidating glycoprotein functions
and the molecular mechanism of many biological events
Global and Site-Specific Analysis Revealing Unexpected and Extensive Protein S‑GlcNAcylation in Human Cells
Protein glycosylation
is highly diverse and essential for mammalian
cell survival. Heterogeneous glycans may be bound to different amino
acid residues, forming multiple types of protein glycosylation. In
this work, unexpected protein S-GlcNAcylation on cysteine residues
was observed to extensively exist in human cells through global and
site-specific analysis of protein GlcNAcylation by mass spectrometry.
Three independent experiments produced similar results of many cysteine
residues bound to <i>N</i>-acetylglucosamine (GlcNAc). Among
well-localized S-GlcNAcylation sites, several motifs with an acidic
amino acid around the sites were identified, which strongly suggests
that a particular type of enzyme is responsible for this modification.
Clustering results show that glycoproteins modified with S-GlcNAc
are mainly involved in cell–cell adhesion and gene expression.
For the first time, we found that proteins were extensively bound
to GlcNAc through the side chains of cysteine residues in human cells,
and the current discovery further advances our understanding of protein
glycosylation
Systematic Analysis of Fatty Acids in Human Cells with a Multiplexed Isobaric Tag (TMT)-Based Method
Fatty acids (FAs) are essential components
in cells and are involved
in many cellular activities. Abnormal FA metabolism has been reported
to be related to human diseases such as cancer and cardiovascular
diseases. Identification and quantification of FAs provide insights
into their functions in biological systems, but it is very challenging
to analyze them due to their structures and properties. In this work,
we developed a novel method by integrating FAs tagged with stable
isotope labeled aminoxy tandem mass tags (aminoxyTMTs) and mass spectrometric
analysis in the positive mode. On the basis of their structures, the
aminoxyTMT reagents reacted with the carboxylic acid group of the
FAs, resulting in an amine group with high proton affinity covalently
attached to the analytes. This enabled the analysis of FAs under the
positive electrospray ionization–mass spectrometry (ESI–MS)
mode, which is normally more popular and sensitive compared to the
negative mode. More importantly, the multiplexed TMT tags allowed
us to quantify FAs from several samples simultaneously, which increased
the experimental throughput and quantification accuracy. FAs extracted
from three types of breast cells, i.e., MCFÂ 10A (normal), MCF7
(minimally invasive) and MDA-MB-231 (highly invasive) cells, were
labeled with the six-plexed aminoxyTMTs and quantified by LC–MS/MS.
The results demonstrated that the abundances of some FAs, such as
C22:5 and C20:3, were markedly increased in MCF7 and MDA-MB-231 cancer
cells compared to normal MCFÂ 10A cells. For the first time, aminoxyTMT
reagents were exploited to label FAs for their identification and
quantification in complex biological samples in the positive MS mode.
The current method enabled us to confidently identify FAs and to accurately
quantify them from several samples simultaneously. Because this method
does not have sample restrictions, it can be extensively applied for
biological and biomedical research
Global Analysis of Secreted Proteins and Glycoproteins in <i>Saccharomyces cerevisiae</i>
Protein secretion
is essential for numerous cellular activities,
and secreted proteins in bodily fluids are a promising and noninvasive
source of biomarkers for disease detection. Systematic analysis of
secreted proteins and glycoproteins will provide insight into protein
function and cellular activities. Yeast (<i>Saccharomyces cerevisiae</i>) is an excellent model system for eukaryotic cells, but global analysis
of secreted proteins and glycoproteins in yeast is challenging due
to the low abundances of secreted proteins and contamination from
high-abundance intracellular proteins. Here, by using mild separation
of secreted proteins from cells, we comprehensively identified and
quantified secreted proteins and glycoproteins through inhibition
of glycosylation and mass spectrometry-based proteomics. In biological
triplicate experiments, 245 secreted proteins were identified, and
comparison with previous experimental and computational results demonstrated
that many identified proteins were located in the extracellular space.
Most quantified secreted proteins were down-regulated from cells treated
with an N-glycosylation inhibitor (tunicamycin). The quantitative
results strongly suggest that the secretion of these down-regulated
proteins was regulated by glycosylation, while the secretion of proteins
with minimal abundance changes was contrarily irrelevant to protein
glycosylation, likely being secreted through nonclassical pathways.
Glycoproteins in the yeast secretome were globally analyzed for the
first time. A total of 27 proteins were quantified in at least two
protein and glycosylation triplicate experiments, and all except one
were down-regulated under N-glycosylation inhibition, which is solid
experimental evidence to further demonstrate that the secretion of
these proteins is regulated by their glycosylation. These results
provide valuable insight into protein secretion, which will further
advance protein secretion and disease studies
Comprehensive Analysis of Protein N‑Glycosylation Sites by Combining Chemical Deglycosylation with LC–MS
Glycosylation
is one of the most important protein modifications
in biological systems. It plays a critical role in protein folding,
trafficking, and stability as well as cellular events such as immune
response and cell-to-cell communication. Aberrant protein glycosylation
is correlated with several diseases including diabetes, cancer, and
infectious diseases. The heterogeneity of glycans makes comprehensive
identification of protein glycosylation sites very difficult by MS
because it is challenging to match mass spectra to peptides that contain
different types of unknown glycans. We combined a chemical deglycosylation
method with LC–MS-based proteomics techniques to comprehensively
identify protein N-glycosylation sites in yeast. On the basis of the
differences in chemical properties between the amide bond of the N-linkage
and the glycosidic bond of the O-linkage of sugars, O-linked sugars
were removed and only the innermost N-linked GlcNAc remained, which
served as a mass tag for MS analysis. This chemical deglycosylation
method allowed for the identification of 555 protein N-glycosylation
sites in yeast by LC–MS, which is 46% more than those obtained
from the parallel experiments using the Endo H cleavage method. A
total of 250 glycoproteins were identified, including 184 membrane
proteins. This method can be extensively used for other biological
samples
Global Analysis of Secreted Proteins and Glycoproteins in <i>Saccharomyces cerevisiae</i>
Protein secretion
is essential for numerous cellular activities,
and secreted proteins in bodily fluids are a promising and noninvasive
source of biomarkers for disease detection. Systematic analysis of
secreted proteins and glycoproteins will provide insight into protein
function and cellular activities. Yeast (<i>Saccharomyces cerevisiae</i>) is an excellent model system for eukaryotic cells, but global analysis
of secreted proteins and glycoproteins in yeast is challenging due
to the low abundances of secreted proteins and contamination from
high-abundance intracellular proteins. Here, by using mild separation
of secreted proteins from cells, we comprehensively identified and
quantified secreted proteins and glycoproteins through inhibition
of glycosylation and mass spectrometry-based proteomics. In biological
triplicate experiments, 245 secreted proteins were identified, and
comparison with previous experimental and computational results demonstrated
that many identified proteins were located in the extracellular space.
Most quantified secreted proteins were down-regulated from cells treated
with an N-glycosylation inhibitor (tunicamycin). The quantitative
results strongly suggest that the secretion of these down-regulated
proteins was regulated by glycosylation, while the secretion of proteins
with minimal abundance changes was contrarily irrelevant to protein
glycosylation, likely being secreted through nonclassical pathways.
Glycoproteins in the yeast secretome were globally analyzed for the
first time. A total of 27 proteins were quantified in at least two
protein and glycosylation triplicate experiments, and all except one
were down-regulated under N-glycosylation inhibition, which is solid
experimental evidence to further demonstrate that the secretion of
these proteins is regulated by their glycosylation. These results
provide valuable insight into protein secretion, which will further
advance protein secretion and disease studies
Site-Specific Quantification of Surface N‑Glycoproteins in Statin-Treated Liver Cells
The
frequent modification of cell-surface proteins by N-linked
glycans is known to be correlated with many biological processes.
Aberrant glycosylation on surface proteins is associated with different
cellular statuses and disease progression. However, it is extraordinarily
challenging to comprehensively and site-specifically analyze glycoproteins
located only on the cell surface. Currently mass spectrometry (MS)-based
proteomics provides the possibility to analyze the N-glycoproteome,
but effective separation and enrichment methods are required for the
analysis of surface glycoproteins prior to MS measurement. The introduction
of bio-orthogonal groups into proteins accelerates research in the
robust visualization, identification, and quantification of proteins.
Here we have comprehensively evaluated different sugar analogs in
the analysis of cell-surface N-glycoproteins by combining copper-free
click chemistry and MS-based proteomics. Comparison of three sugar
analogs, N-azidoacetylgalactosamine (GalNAz), N-azidoacetylglucosamine
(GlcNAz), and N-azidoacetylmannosamine (ManNAz), showed that metabolic
labeling with GalNAz resulted in the greatest number of glycoproteins
and glycosylation sites in biological duplicate experiments. GalNAz
was then employed for the quantification experiment in statin-treated
HepG2 liver cells, and 280 unique N-glycosylated sites were quantified
from 168 surface proteins. The quantification results demonstrated
that many glycosylation sites on surface proteins were down-regulated
in statin-treated cells compared to untreated cells because statin
prevents the synthesis of dolichol, which is essential for the formation
of dolichol-linked precursor oligosaccharides. Several glycosylation
sites in proteins that participate in the Alzheimer’s disease
pathway were down-regulated. This method can be extensively applied
for the global analysis of the cell-surface N-glycoproteome
Quantitative Structural Proteomics Unveils the Conformational Changes of Proteins under the Endoplasmic Reticulum Stress
Protein structures are decisive for their activities
and interactions
with other molecules. Global analysis of protein structures and conformational
changes cannot be achieved by commonly used abundance-based proteomics.
Here, we integrated cysteine covalent labeling, selective enrichment,
and quantitative proteomics to study protein structures and structural
changes on a large scale. This method was applied to globally investigate
protein structures in HEK293T cells and protein structural changes
in the cells with the tunicamycin (Tm)-induced endoplasmic reticulum
(ER) stress. We quantified several thousand cysteine residues, which
contain unprecedented and valuable information of protein structures.
Combining this method with pulsed stable isotope labeling by amino
acids in cell culture, we further analyzed the folding state differences
between pre-existing and newly synthesized proteins in cells under
the Tm treatment. Besides newly synthesized proteins, unexpectedly,
many pre-existing proteins were found to become unfolded upon ER stress,
especially those related to gene transcription and protein translation.
Furthermore, the current results reveal that N-glycosylation plays
a more important role in the folding process of the tertiary and quaternary
structures than the secondary structures for newly synthesized proteins.
Considering the importance of cysteine in protein structures, this
method can be extensively applied in the biological and biomedical
research fields