21 research outputs found
GNAi2/gip2-Regulated Transcriptome and Its Therapeutic Significance in Ovarian Cancer
Increased expression of GNAi2, which encodes the α-subunit of G-protein i2, has been correlated with the late-stage progression of ovarian cancer. GNAi2, also referred to as the proto-oncogene gip2, transduces signals from lysophosphatidic acid (LPA)-activated LPA-receptors to oncogenic cellular responses in ovarian cancer cells. To identify the oncogenic program activated by gip2, we carried out micro-array-based transcriptomic and bioinformatic analyses using the ovarian cancer cell-line SKOV3, in which the expression of GNAi2/gip2 was silenced by specific shRNA. A cut-off value of 5-fold change in gene expression (p < 0.05) indicated that a total of 264 genes were dependent upon gip2-expression with 136 genes coding for functional proteins. Functional annotation of the transcriptome indicated the hitherto unknown role of gip2 in stimulating the expression of oncogenic/growth-promoting genes such as KDR/VEGFR2, CCL20, and VIP. The array results were further validated in a panel of High-Grade Serous Ovarian Carcinoma (HGSOC) cell lines that included Kuramochi, OVCAR3, and OVCAR8 cells. Gene set enrichment analyses using DAVID, STRING, and Cytoscape applications indicated the potential role of the gip2-stimulated transcriptomic network involved in the upregulation of cell proliferation, adhesion, migration, cellular metabolism, and therapy resistance. The results unravel a multi-modular network in which the hub and bottleneck nodes are defined by ACKR3/CXCR7, IL6, VEGFA, CYCS, COX5B, UQCRC1, UQCRFS1, and FYN. The identification of these genes as the critical nodes in GNAi2/gip2 orchestrated onco-transcriptome establishes their role in ovarian cancer pathophysiology. In addition, these results also point to these nodes as potential targets for novel therapeutic strategies
Fluorescence Analysis of the Lipid Binding-Induced Conformational Change of Apolipoprotein E4
Apolipoprotein (apo) E is thought to undergo conformational
changes
in the N-terminal helix bundle domain upon lipid binding, modulating
its receptor binding activity. In this study, site-specific fluorescence
labeling of the N-terminal (S94) and C-terminal (W264 or S290) helices
in apoE4 by pyrene maleimide or acrylodan was employed to probe the
conformational organization and lipid binding behavior of the N- and
C-terminal domains. Guanidine denaturation experiments monitored by
acrylodan fluorescence demonstrated the less organized, more solvent-exposed
structure of the C-terminal helices compared to the N-terminal helix
bundle. Pyrene excimer fluorescence together with gel filtration chromatography
indicated that there are extensive intermolecular helix–helix
contacts through the C-terminal helices of apoE4. Comparison of increases
in pyrene fluorescence upon binding of pyrene-labeled apoE4 to egg
phosphatidylcholine small unilamellar vesicles suggests a two-step
lipid-binding process; apoE4 initially binds to a lipid surface through
the C-terminal helices followed by the slower conformational reorganization
of the N-terminal helix bundle domain. Consistent with this, fluorescence
resonance energy transfer measurements from Trp residues to acrylodan
attached at position 94 demonstrated that upon binding to the lipid
surface, opening of the N-terminal helix bundle occurs at the same
rate as the increase in pyrene fluorescence of the N-terminal domain.
Such a two-step mechanism of lipid binding of apoE4 is likely to apply
to mostly phospholipid-covered lipoproteins such as VLDL. However,
monitoring pyrene fluorescence upon binding to HDL<sub>3</sub> suggests
that not only apoE–lipid interactions but also protein–protein
interactions are important for apoE4 binding to HDL<sub>3</sub>
Effects of the Iowa and Milano Mutations on Apolipoprotein A‑I Structure and Dynamics Determined by Hydrogen Exchange and Mass Spectrometry
The Iowa point mutation in apolipoprotein A-I (G26R)
leads to a
systemic amyloidosis condition, and the Milano mutation (R173C) is
associated with hypoalphalipoproteinemia, a reduced plasma level of
high-density lipoprotein. To probe the structural effects that lead
to these outcomes, we used amide hydrogen–deuterium exchange
coupled with a fragment separation/mass spectrometry analysis (HX
MS). The Iowa mutation inserts an arginine residue into the nonpolar
face of an α-helix that spans residues 7–44 and causes
changes in structure and structural dynamics. This helix unfolds,
and other helices in the N-terminal helix bundle domain are destabilized.
The segment encompassing residues 116–158, largely unstructured
in wild-type apolipoprotein A-I, becomes helical. The helix spanning
residues 81–115 is destabilized by 2 kcal/mol, increasing the
small fraction of time it is transiently unfolded to ≥1%, which
allows proteolysis at residue 83 in vivo over time, releasing an amyloid-forming
peptide. The Milano mutation situated on the polar face of the helix
spanning residues 147–178 destabilizes the helix bundle domain
only moderately, but enough to allow cysteine-mediated dimerization
that leads to the altered functionality of this variant. These results
show how the HX MS approach can provide a powerful means of monitoring,
in a nonperturbing way and at close to amino acid resolution, the
structural, dynamic, and energetic consequences of biologically interesting
point mutations