16 research outputs found
Optimization of Formaldehyde Cross-Linking for Protein Interaction Analysis of Non-Tagged Integrin β1
Formaldehyde cross-linking of protein complexes combined with immunoprecipitation and mass spectrometry analysis is a promising technique for analysing protein-protein interactions, including those of transient nature. Here we used integrin β1 as a model to describe the application of formaldehyde cross-linking in detail, particularly focusing on the optimal parameters for cross-linking, the detection of formaldehyde cross-linked complexes, the utility of antibodies, and the identification of binding partners. Integrin β1 was found in a high molecular weight complex after formaldehyde cross-linking. Eight different anti-integrin β1 antibodies were used for pull-down experiments and no loss in precipitation efficiency after cross-linking was observed. However, two of the antibodies could not precipitate the complex, probably due to hidden epitopes. Formaldehyde cross-linked complexes, precipitated from Jurkat cells or human platelets and analyzed by mass spectrometry, were found to be composed of integrin β1, α4 and α6 or β1, α6, α2, and α5, respectively
CIP2A Inhibits PP2A in Human Malignancies
SummaryInhibition of protein phosphatase 2A (PP2A) activity has been identified as a prerequisite for the transformation of human cells. However, the molecular mechanisms by which PP2A activity is inhibited in human cancers are currently unclear. In this study, we describe a cellular inhibitor of PP2A with oncogenic activity. The protein, designated Cancerous Inhibitor of PP2A (CIP2A), interacts directly with the oncogenic transcription factor c-Myc, inhibits PP2A activity toward c-Myc serine 62 (S62), and thereby prevents c-Myc proteolytic degradation. In addition to its function in c-Myc stabilization, CIP2A promotes anchorage-independent cell growth and in vivo tumor formation. The oncogenic activity of CIP2A is demonstrated by transformation of human cells by overexpression of CIP2A. Importantly, CIP2A is overexpressed in two common human malignancies, head and neck squamous cell carcinoma (HNSCC) and colon cancer. Thus, our data show that CIP2A is a human oncoprotein that inhibits PP2A and stabilizes c-Myc in human malignancies
Albiglutide and cardiovascular outcomes in patients with type 2 diabetes and cardiovascular disease (Harmony Outcomes): a double-blind, randomised placebo-controlled trial
Background:
Glucagon-like peptide 1 receptor agonists differ in chemical structure, duration of action, and in their effects on clinical outcomes. The cardiovascular effects of once-weekly albiglutide in type 2 diabetes are unknown. We aimed to determine the safety and efficacy of albiglutide in preventing cardiovascular death, myocardial infarction, or stroke.
Methods:
We did a double-blind, randomised, placebo-controlled trial in 610 sites across 28 countries. We randomly assigned patients aged 40 years and older with type 2 diabetes and cardiovascular disease (at a 1:1 ratio) to groups that either received a subcutaneous injection of albiglutide (30–50 mg, based on glycaemic response and tolerability) or of a matched volume of placebo once a week, in addition to their standard care. Investigators used an interactive voice or web response system to obtain treatment assignment, and patients and all study investigators were masked to their treatment allocation. We hypothesised that albiglutide would be non-inferior to placebo for the primary outcome of the first occurrence of cardiovascular death, myocardial infarction, or stroke, which was assessed in the intention-to-treat population. If non-inferiority was confirmed by an upper limit of the 95% CI for a hazard ratio of less than 1·30, closed testing for superiority was prespecified. This study is registered with ClinicalTrials.gov, number NCT02465515.
Findings:
Patients were screened between July 1, 2015, and Nov 24, 2016. 10 793 patients were screened and 9463 participants were enrolled and randomly assigned to groups: 4731 patients were assigned to receive albiglutide and 4732 patients to receive placebo. On Nov 8, 2017, it was determined that 611 primary endpoints and a median follow-up of at least 1·5 years had accrued, and participants returned for a final visit and discontinuation from study treatment; the last patient visit was on March 12, 2018. These 9463 patients, the intention-to-treat population, were evaluated for a median duration of 1·6 years and were assessed for the primary outcome. The primary composite outcome occurred in 338 (7%) of 4731 patients at an incidence rate of 4·6 events per 100 person-years in the albiglutide group and in 428 (9%) of 4732 patients at an incidence rate of 5·9 events per 100 person-years in the placebo group (hazard ratio 0·78, 95% CI 0·68–0·90), which indicated that albiglutide was superior to placebo (p<0·0001 for non-inferiority; p=0·0006 for superiority). The incidence of acute pancreatitis (ten patients in the albiglutide group and seven patients in the placebo group), pancreatic cancer (six patients in the albiglutide group and five patients in the placebo group), medullary thyroid carcinoma (zero patients in both groups), and other serious adverse events did not differ between the two groups. There were three (<1%) deaths in the placebo group that were assessed by investigators, who were masked to study drug assignment, to be treatment-related and two (<1%) deaths in the albiglutide group.
Interpretation:
In patients with type 2 diabetes and cardiovascular disease, albiglutide was superior to placebo with respect to major adverse cardiovascular events. Evidence-based glucagon-like peptide 1 receptor agonists should therefore be considered as part of a comprehensive strategy to reduce the risk of cardiovascular events in patients with type 2 diabetes.
Funding:
GlaxoSmithKline
Quantitative Glycoproteomic Analysis Identifies Platelet-Induced Increase of Monocyte Adhesion via the Up-Regulation of Very Late Antigen 5
Physiological
stimuli, such as thrombin, or pathological stimuli,
such as lysophosphatidic acid (LPA), activate platelets circulating
in blood. Once activated, platelets bind to monocytes via P-selectin–PSGL-1
interactions but also release the stored contents of their granules.
These platelet releasates, in addition to direct platelet binding,
activate monocytes and facilitate their recruitment to atherosclerotic
sites. Consequently, understanding the changes platelet releasates
induce in monocyte membrane proteins is critical. We studied the glyco-proteome
changes of THP-1 monocytic cells affected by LPA- or thrombin-induced
platelet releasates. We employed lectin affinity chromatography combined
with filter aided sample preparation to achieve high glyco- and membrane
protein and protein sequence coverage. Using stable isotope labeling
by amino acids in cell culture, we quantified 1715 proteins, including
852 membrane and 500 glycoproteins, identifying the up-regulation
of multiple proteins involved in monocyte extracellular matrix binding
and transendothelial migration. Flow cytometry indicated expression
changes of integrin α5, integrin β1, PECAM-1, and PSGL-1.
The observed increase in monocyte adhesion to fibronectin was determined
to be mediated by the up-regulation of very late antigen 5 via a P-selectin–PSGL-1
independent mechanism. This novel aspect could be validated on CD14+
human primary monocytes, highlighting the benefits of the improved
enrichment method regarding high membrane protein coverage and reliable
quantification
Identification of Total Reversible Cysteine Oxidation in an Atherosclerosis Model Using a Modified Biotin Switch Assay
Oxidative
stress due to the imbalance of reactive oxygen species
(ROS) and the resulting reversible cysteine oxidation (Cys<sub>OX</sub>) are involved in the early proatherogenic aspect of atherosclerosis.
Given that the corresponding redox signaling pathways are still unclear,
a modified biotin switch assay was developed to quantify the reversible
Cys<sub>OX</sub> in an atherosclerosis model established by using
a monocytic cell line treated with platelet releasate. The accumulation
of ROS was observed in the model system and validated in human primary
monocytes. Through the application of the modified biotin switch assay,
we obtained the first reversible Cys<sub>OX</sub> proteome for this
model. A total of 75 peptides, corresponding to 53 proteins, were
quantified with oxidative modification. The bioinformatics analysis
of these Cys<sub>OX</sub>-containing proteins highlighted biological
processes including glycolysis, cytoskeleton arrangement, and redox
regulation. Moreover, the reversible oxidation of three glycolysis
enzymes was observed using this method, and the regulation influence
was verified by an enzyme activity assay. NADPH oxidase (NOX) inhibition
treatment, in conjunction with the modified biotin switch method,
was used to evaluate the global Cys<sub>OX</sub> status. In conclusion,
this versatile modified biotin switch assay provides an approach for
the quantification of all reversible Cys<sub>OX</sub> and for the
study of redox signaling in atherosclerosis as well as in diseases
in other biological systems
A Compartmentalized Phosphorylation/Dephosphorylation System That Regulates U snRNA Export from the Nucleus▿ †
PHAX (phosphorylated adaptor for RNA export) is the key regulator of U snRNA nuclear export in metazoa. Our previous work revealed that PHAX is phosphorylated in the nucleus and is exported as a component of the U snRNA export complex to the cytoplasm, where it is dephosphorylated (M. Ohno, A. Segref, A. Bachi, M. Wilm, and I. W. Mattaj, Cell 101:187-198, 2000). PHAX phosphorylation is essential for export complex assembly, whereas its dephosphorylation causes export complex disassembly. Thus, PHAX is subject to a compartmentalized phosphorylation/dephosphorylation cycle that contributes to transport directionality. However, neither essential PHAX phosphorylation sites nor the modifying enzymes that contribute to the compartmentalized system have been identified. Here, we identify PHAX phosphorylation sites that are necessary and sufficient for U snRNA export. Mutation of the phosphorylation sites inhibited U snRNA export in a dominant-negative way. We also show, by both biochemical and RNA interference knockdown experiments, that the nuclear kinase and the cytoplasmic phosphatase for PHAX are CK2 kinase and protein phosphatase 2A, respectively. Our results reveal the composition of the compartmentalized phosphorylation/dephosphorylation system that regulates U snRNA export. This finding was surprising in that such a specific system for U snRNA export regulation is composed of two such universal regulators, suggesting that this compartmentalized system is used more broadly for gene expression regulation
Quantitative Protein Sulfenic Acid Analysis Identifies Platelet Releasate-Induced Activation of Integrin β<sub>2</sub> on Monocytes via NADPH Oxidase
Physiological
stimuli such as thrombin, or pathological stimuli
such as lysophosphatidic acid (LPA), activate platelets. The activated
platelets bind to monocytes through P-selectin–PSGL-1 interactions
but also release the contents of their granules, commonly called “platelet
releasate”. It is known that monocytes in contact with platelet
releasate produce reactive oxygen species (ROS). Reversible cysteine
oxidation by ROS is considered to be a potential regulator of protein
function. In a previous study, we used THP-1 monocytic cells exposed
to LPA- or thrombin-induced platelet releasate and a modified biotin
switch assay to unravel the biological processes that are influenced
by reversible cysteine oxidation. To gain a better understanding of
the redox regulation of monocytes in atherosclerosis, we have now
altered the modified biotin switch to selectively quantify protein
sulfenic acid, a subpopulation of reversible cysteine oxidation. Using
arsenite as reducing agent in the modified biotin switch assay, we
were able to quantify 1161 proteins, in which more than 100 sulfenic
acid sites were identified. Bioinformatics analysis of the quantified
sulfenic acid sites highlighted the relevant, previously missed biological
process of monocyte transendothelial migration, which included integrin
β<sub>2</sub>. Flow cytometry validated the activation of LFA-1
(α<sub>L</sub>β<sub>2</sub>) and Mac-1 (α<sub>M</sub>β<sub>2</sub>), two subfamilies of integrin β<sub>2</sub> complexes, on human primary monocytes following platelet releasate
treatment. The activation of LFA-1 was mediated by ROS from NADPH
oxidase (NOX) activation. Production of ROS and activation of LFA-1
in human primary monocytes were independent of P-selectin–PSGL-1
interaction. Our results proved the modified biotin switch assay to
be a powerful tool with the ability to reveal new regulatory mechanisms
and identify new therapeutic targets
Quantitative Glycoproteomic Analysis Identifies Platelet-Induced Increase of Monocyte Adhesion via the Up-Regulation of Very Late Antigen 5
Development and Application of a Quantitative Multiplexed Small GTPase Activity Assay Using Targeted Proteomics
Small GTPases are a family of key
signaling molecules that are
ubiquitously expressed in various types of cells. Their activity is
often analyzed by western blot, which is limited by its multiplexing
capability, the quality of isoform-specific antibodies, and the accuracy
of quantification. To overcome these issues, a quantitative multiplexed
small GTPase activity assay has been developed. Using four different
binding domains, this assay allows the binding of up to 12 active
small GTPase isoforms simultaneously in a single experiment. To accurately
quantify the closely related small GTPase isoforms, a targeted proteomic
approach, i.e., selected/multiple reaction monitoring, was developed,
and its functionality and reproducibility were validated. This assay
was successfully applied to human platelets and revealed time-resolved
coactivation of multiple small GTPase isoforms in response to agonists
and differential activation of these isoforms in response to inhibitor
treatment. This widely applicable approach can be used for signaling
pathway studies and inhibitor screening in many cellular systems