16 research outputs found
Phosphorylation stoichiometries of human eukaryotic initiation factors.
Eukaryotic translation initiation factors are the principal molecular effectors regulating the process converting nucleic acid to functional protein. Commonly referred to as eIFs (eukaryotic initiation factors), this suite of proteins is comprised of at least 25 individual subunits that function in a coordinated, regulated, manner during mRNA translation. Multiple facets of eIF regulation have yet to be elucidated; however, many of the necessary protein factors are phosphorylated. Herein, we have isolated, identified and quantified phosphosites from eIF2, eIF3, and eIF4G generated from log phase grown HeLa cell lysates. Our investigation is the first study to globally quantify eIF phosphosites and illustrates differences in abundance of phosphorylation between the residues of each factor. Thus, identification of those phosphosites that exhibit either high or low levels of phosphorylation under log phase growing conditions may aid researchers to concentrate their investigative efforts to specific phosphosites that potentially harbor important regulatory mechanisms germane to mRNA translation
Heparan sulfate differences in rheumatoid arthritis versus healthy sera
Heparan sulfate (HS) is a complex and highly variable polysaccharide, expressed ubiquitously on the cell surface as HS proteoglycans (HSPGs), and found in the extracellular matrix as free HS fragments. Its heterogeneity due to various acetylation and sulfation patterns endows a multitude of functions. In animal tissues, HS interacts with a wide range of proteins to mediate numerous biological activities; given its multiple roles in inflammation processes, characterization of HS in human serum has significant potential for elucidating disease mechanisms. Historically, investigation of HS was limited by its low concentration in human serum, together with the complexity of the serum matrix. In this study, we used a modified mass spectrometry method to examine HS disaccharide profiles in the serum of 50 women with rheumatoid arthritis (RA), and compared our results to 51 sera from healthy women. Using various purification methods and online LC–MS/MS, we discovered statistically significant differences in the sulfation and acetylation patterns between populations. Since early diagnosis of RA is considered important in decelerating the disease's progression, identification of specific biomolecule characterizations may provide crucial information towards developing new therapies for suppressing the disease in its early stages. This is the first report of potential glycosaminoglycan biomarkers for RA found in human sera, while acknowledging the obvious fact that a larger population set, and more stringent collection parameters, will need to be investigated in the future.The authors gratefully acknowledge
the financial support provided by the National Institutes of
Health (Grant GM 47356)
Dynactin-dependent cortical dynein and spherical spindle shape correlate temporally with meiotic spindle rotation in Caenorhabditis elegans.
Oocyte meiotic spindles orient with one pole juxtaposed to the cortex to facilitate extrusion of chromosomes into polar bodies. In Caenorhabditis elegans, these acentriolar spindles initially orient parallel to the cortex and then rotate to the perpendicular orientation. To understand the mechanism of spindle rotation, we characterized events that correlated temporally with rotation, including shortening of the spindle in the pole-to pole axis, which resulted in a nearly spherical spindle at rotation. By analyzing large spindles of polyploid C. elegans and a related nematode species, we found that spindle rotation initiated at a defined spherical shape rather than at a defined spindle length. In addition, dynein accumulated on the cortex just before rotation, and microtubules grew from the spindle with plus ends outward during rotation. Dynactin depletion prevented accumulation of dynein on the cortex and prevented spindle rotation independently of effects on spindle shape. These results support a cortical pulling model in which spindle shape might facilitate rotation because a sphere can rotate without deforming the adjacent elastic cytoplasm. We also present evidence that activation of spindle rotation is promoted by dephosphorylation of the basic domain of p150 dynactin
Novel Mass Spectrometric Method for Phosphorylation Quantification Using Cerium Oxide Nanoparticles and Tandem Mass Tags
The stoichiometry of protein phosphorylation significantly
impacts
protein function. The development of quantitative techniques in mass
spectrometry has generated the ability to systematically monitor the
regulation levels of various proteins. This study reports an integrated
methodology using cerium oxide nanoparticles and isobaric tandem mass
tag (TMT) labeling to assess absolute stoichiometries of protein phosphorylation.
This protocol was designed to directly measure the dephosphorylation
levels for a known phosphorylation site, therefore allowing for quantification
of phosphosites. Both the accuracy and precision of the method were
verified using standard peptides and protein tryptic digests. This
novel method was then applied to quantify phosphorylations on eukaryotic
initiation factor 3H (eIF3H), a protein integral to overall eukaryotic
protein translation initiation. To date, this is the first report
of assessment of protein phosphorylation quantification on eIF3
Differentiation of CC vs CXC Chemokine Dimers with GAG Octasaccharide Binding Partners: An Ion Mobility Mass Spectrometry Approach
Chemokines,
8 kDa proteins implicated in leukocyte migration via
oligomerization, bind to glycosaminoglycans (GAGs) during the inflammation
response as a means to regulate chemokine migration. Structural characterization
of chemokines non-covalently bound to GAGs provides physiologically
meaningful data in regard to routine inmmunosurveillance and disease
response. In order to analyze the structures resulting from the GAG:chemokine
interaction, we employed ion mobility mass spectrometry (IMMS) to
analyze monocyte chemoattractant protein‑1 (MCP‑1),
a CC chemokine, and interleukin‑8 (IL‑8), a CXC chemokine,
along with their individual interactions with GAG heparin octasaccharides.
We show that MCP‑1 and IL‑8 are physiologically present
as a dimer, with MCP‑1 having two variants of its dimeric form
and IL‑8 having only one. We also show that the MCP‑1
dimer adopts two conformations, one extended and one compact, when
bound to a dodecasulfated heparin octasaccharide. Binding of MCP‑1
to heparin octasaccharide isomers of varying sulfation patterns results
in similar arrival time distribution values, which suggests minimal
distinguishing features among the resultant complexes. Additionally,
tandem mass spectrometry (MS/MS) showed that the binding of MCP‑1
to a heparin octasaccharide has different dissociation patterns when
compared with the corresponding IL‑8 bound dimer. Overall,
IMMS and MS/MS were used to better define the structural tendencies
and differences associated with CC and CXC dimers when associated
with GAG octasaccharides
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Probing the orientation of inhibitor and epoxy-eicosatrienoic acid binding in the active site of soluble epoxide hydrolase
Soluble epoxide hydrolase (sEH) is an important therapeutic target of many diseases, such as chronic obstructive pulmonary disease (COPD) and diabetic neuropathic pain. It acts by hydrolyzing and thus regulating specific bioactive long chain polyunsaturated fatty acid epoxides (lcPUFA), like epoxyeicosatrienoic acids (EETs). To better predict which epoxides could be hydrolyzed by sEH, one needs to dissect the important factors and structural requirements that govern the binding of the substrates to sEH. This knowledge allows further exploration of the physiological role played by sEH. Unfortunately, a crystal structure of sEH with a substrate bound has not yet been reported. In this report, new photoaffinity mimics of a sEH inhibitor and EET were prepared and used in combination with peptide sequencing and computational modeling, to identify the binding orientation of different regioisomers and enantiomers of EETs into the catalytic cavity of sEH. Results indicate that the stereochemistry of the epoxide plays a crucial role in dictating the binding orientation of the substrate
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Dynactin-dependent cortical dynein and spherical spindle shape correlate temporally with meiotic spindle rotation in Caenorhabditis elegans.
Oocyte meiotic spindles orient with one pole juxtaposed to the cortex to facilitate extrusion of chromosomes into polar bodies. In Caenorhabditis elegans, these acentriolar spindles initially orient parallel to the cortex and then rotate to the perpendicular orientation. To understand the mechanism of spindle rotation, we characterized events that correlated temporally with rotation, including shortening of the spindle in the pole-to pole axis, which resulted in a nearly spherical spindle at rotation. By analyzing large spindles of polyploid C. elegans and a related nematode species, we found that spindle rotation initiated at a defined spherical shape rather than at a defined spindle length. In addition, dynein accumulated on the cortex just before rotation, and microtubules grew from the spindle with plus ends outward during rotation. Dynactin depletion prevented accumulation of dynein on the cortex and prevented spindle rotation independently of effects on spindle shape. These results support a cortical pulling model in which spindle shape might facilitate rotation because a sphere can rotate without deforming the adjacent elastic cytoplasm. We also present evidence that activation of spindle rotation is promoted by dephosphorylation of the basic domain of p150 dynactin
Methodology for measuring conformation of solvent-disrupted protein subunits using T-WAVE ion mobility MS: an investigation into eukaryotic initiation factors
The methodology developed in the research presented herein makes use of chaotropic solvents to gently dissociate subunits from an intact macromolecular complex and subsequently allows for the measurement of collision cross section (CCS) for both the recombinant (R-eIF3k) and solvent dissociated form of the subunit (S-eIF3k). In this particular case, the k Subunit from the eukaryotic initiation factor 3 (eIF3) was investigated in detail. Experimental and theoretical CCS values show both the recombinant and solvent disrupted forms of the protein to be essentially the same. The ultimate goal of the project is to structurally characterize all the binding partners of eIF3, determine which subunits interact directly, and investigate how subunits may change conformation when they form complexes with other proteins. Research presented herein is the first report showing retention of solution conformation of a protein as evidenced by CCS measurements of both recombinant and solvent disrupted versions of the same protein. (J Am Soc Mass Spectrom 2009, 20, 1699-1706) (C) 2009 Published by Elsevier Inc. on behalf of American Society for Mass Spectrometr