Steven Blais

The Matrix Peptide Exporter HAF-1 Signals a Mitochondrial UPR by Activating the Transcription Factor ZC376.7 in C. elegans

Genetic analyses previously implicated the matrix-localized protease ClpP in signaling the stress of protein misfolding in the mitochondrial matrix to activate nuclear-encoded mitochondrial chaperone genes in C. elegans (UPR(mt)). Here, we report that haf-1, a gene encoding a mitochondria-localized ATP-binding cassette protein, is required for signaling within the UPR(mt) and for coping with misfolded protein stress. Peptide efflux from isolated mitochondria was ATP dependent and required HAF-1 and the protease ClpP. Defective UPR(mt) signaling in the haf-1-deleted worms was associated with failure of the bZIP protein, ZC376.7, to localize to nuclei in worms with perturbed mitochondrial protein folding, whereas zc376.7(RNAi) strongly inhibited the UPR(mt). These observations suggest a simple model whereby perturbation of the protein-folding environment in the mitochondrial matrix promotes ClpP-mediated generation of peptides whose haf-1-dependent export from the matrix contributes to UPR(mt) signaling across the mitochondrial inner membrane.

Ionotropic Glutamate Receptors IR64a and IR8a Form a Functional Odorant Receptor Complex In Vivo in Drosophila

Drosophila olfactory sensory neurons express either odorant receptors or ionotropic glutamate receptors (IRs). The sensory neurons that express IR64a, a member of the IR family, send axonal projections to either the DC4 or DP1m glomeruli in the antennal lobe. DC4 neurons respond specifically to acids/protons, whereas DP1m neurons respond to a broad spectrum of odorants. The molecular composition of IR64a-containing receptor complexes in either DC4 or DP1m neurons is not known, however. Here, we immunoprecipitated the IR64a protein from lysates of fly antennal tissue and identified IR8a as a receptor subunit physically associated with IR64a by mass spectrometry. IR8a mutants and flies in which IR8a was knocked down by RNAi in IR64a+ neurons exhibited defects in acid-evoked physiological and behavioral responses. Furthermore, we found that the loss of IR8a caused a significant reduction in IR64a protein levels. When expressed in Xenopus oocytes, IR64a and IR8a formed a functional ion channel that allowed ligand-evoked cation currents. These findings provide direct evidence that IR8a is a subunit that forms a functional olfactory receptor with IR64a in vivo to mediate odor detection.

Characterization of Tafazzin Splice Variants from Humans and Fruit Flies

The tafazzin gene encodes a phospholipid-lysophospholipid transacylase involved in cardiolipin metabolism, but it is not known why it forms multiple transcripts as a result of alternative splicing. Here we studied the intracellular localization, enzymatic activity, and metabolic function of four isoforms of human tafazzin and three isoforms of Drosophila tafazzin upon expression in different mammalian and insect systems. When expressed in HeLa cells, all isoforms were localized in mitochondria except for the B-form of Drosophila tafazzin, which was associated with multiple intracellular membranes. Among the human isoforms, only full-length tafazzin (FL) and tafazzin lacking exon 5 (Δ5) had transacylase activity, and only these two isoforms were able to restore a normal cardiolipin pattern, normal respiratory activity of mitochondria, and male fertility in tafazzin-deficient flies. Both FL and Δ5 were associated with large protein complexes in 293T cell mitochondria, but treatment with alkali and proteinase K suggested that the Δ5 isoform was more integrated into the hydrophobic core of the membrane than the FL isoform. Although all Drosophila isoforms showed transacylase activity in vitro, only the A-form supported cardiolipin remodeling in flies. The data suggest that humans express two mitochondrial isoenzymes of tafazzin that have similar transacylase activities but different membrane topologies. Furthermore, the data show that the expression of human tafazzin in flies creates cardiolipin with a Drosophila pattern, suggesting that the characteristic fatty acid profile of cardiolipin is not determined by the substrate specificity of tafazzin.

Homodimerization Controls the Fibroblast Growth Factor 9 Subfamily's Receptor Binding and Heparan Sulfate-Dependent Diffusion in the Extracellular Matrix

ABSTRACT Uncontrolled fibroblast growth factor (FGF) signaling can lead to human diseases, necessitating multiple layers of self-regulatory control mechanisms to keep its activity in check. Herein, we demonstrate that FGF9 and FGF20 ligands undergo a reversible homodimerization, occluding their key receptor binding sites. To test the role of dimerization in ligand autoinhibition, we introduced structure-based mutations into the dimer interfaces of FGF9 and FGF20. The mutations weakened the ability of the ligands to dimerize, effectively increasing the concentrations of monomeric ligands capable of binding and activating their cognate FGF receptor in vitro and in living cells. Interestingly, the monomeric ligands exhibit reduced heparin binding, resulting in their increased radii of heparan sulfate-dependent diffusion and biologic action, as evidenced by the wider dilation area of ex vivo lung cultures in response to implanted mutant FGF9-loaded beads. Hence, our data demonstrate that homodimerization autoregulates FGF9 and FGF20s receptor binding and concentration gradients in the extracellular matrix. Our study is the first to implicate ligand dimerization as an autoregulatory mechanism for growth factor bioactivity and sets the stage for engineering modified FGF9 subfamily ligands, with desired activity for use in both basic and translational research.

Blue Native PAGE and Mass Spectrometry Analysis of Ephrin Stimulation-Dependent Protein-Protein Interactions in NG108-EphB2 Cells

Receptor tyrosine kinases (RTK) are proteins that undergo dimerization and/or multimerization and autophosphorylation in response to ligand stimulation. Members of the RTK family are receptors for a series of growth factors that, upon stimulation, are able to start signaling events that promote cell growth and differentiation. A class of RTKs, the Eph receptors (EphRs), are found in a variety of cell types and play important roles in patterning the central and peripheral nervous systems, as well as in synapse and neural crest formation. Interaction of Eph receptors with their ephrin ligands activates signal transduction pathways that lead to cytoskeletal remodeling through formation of many stable or transient protein-protein interactions. However, these intracellular signal transduction pathways that lead to cytoskeletal remodeling are not well understood. Here, we combined Blue Native PAGE (BN-PAGE) and mass spectrometry (MS) to analyze protein-protein interactions as a result of ephrin stimulation. We analyzed both lysates and phosphotyrosine immunoprecipitate (pY99-IP) of unstimulated and ephrin-stimulated cells. Our experiments allowed us to characterize many constitutive homo- and hetero-protein complexes from the cell lysate. Furthermore, BN-PAGE and MS of the pY99-IPs from both unstimulated and stimulated cells allowed us to analyze protein-protein interactions that resulted upon ephrin stimulation. Combination of BN-PAGE and MS also has the potential for the analysis of stable and transient protein-protein interactions in other ligand-stimulated RTK-dependent signal transduction pathways.

Sequential Amyloid-β Degradation by the Matrix Metalloproteases MMP-2 and MMP-9

Background: Abnormal accumulation of Aβ in the brain is associated with neurodegeneration in Alzheimer disease. Results: Proteolytic degradation of Aβ by MMP-2/MMP-9 sequentially generates highly soluble, nontoxic, and nonfibrillogenic C-terminally truncated fragments normally present in CSF. Conclusion: The MMP-generated Aβ species likely reflects clearance mechanisms rather than active participation in the amyloidogenesis process. Significance: Matrix metalloproteases are important contributors to Aβ homeostasis. Matrix metalloproteases (MMPs) MMP-2 and MMP-9 have been implicated in the physiological catabolism of Alzheimers amyloid-β (Aβ). Conversely, their association with vascular amyloid deposits, blood-brain barrier disruption, and hemorrhagic transformations after ischemic stroke also highlights their involvement in pathological processes. To better understand this dichotomy, recombinant human (rh) MMP-2 and MMP-9 were incubated with Aβ40 and Aβ42, and the resulting proteolytic fragments were assessed via immunoprecipitation and quantitative mass spectrometry. Both MMPs generated Aβ fragments truncated only at the C terminus, ending at positions 34, 30, and 16. Using deuterated homologues as internal standards, we observed limited and relatively slow degradation of Aβ42 by rhMMP-2, although the enzyme cleaved >80% of Aβ40 during the 1st h of incubation. rhMMP-9 was significantly less effective, particularly in degrading Aβ(1–42), although the targeted peptide bonds were identical. Using Aβ(1–34) and Aβ(1–30), we demonstrated that these peptides are also substrates for both MMPs, cleaving Aβ(1–34) to produce Aβ(1–30) first and Aβ(1–16) subsequently. Consistent with the kinetics observed with full-length Aβ, rhMMP-9 degraded only a minute fraction of Aβ(1–34) and was even less effective in producing Aβ(1–16). Further degradation of Aβ(1–16) by either MMP-2 or MMP-9 was not observed even after prolonged incubation times. Notably, all MMP-generated C-terminally truncated Aβ fragments were highly soluble and did not exhibit fibrillogenic properties or induce cytotoxicity in human cerebral microvascular endothelial or neuronal cells supporting the notion that these truncated Aβ species are associated with clearance mechanisms rather than being key elements in the fibrillogenesis process.

Matrix Metalloproteinase 2 (MMP-2) Degrades Soluble Vasculotropic Amyloid-β E22Q and L34V Mutants, Delaying Their Toxicity for Human Brain Microvascular Endothelial Cells

Patients carrying mutations within the amyloid-β (Aβ) sequence develop severe early-onset cerebral amyloid angiopathy with some of the related variants manifesting primarily with hemorrhagic phenotypes. Matrix metalloproteases (MMPs) are typically associated with blood brain barrier disruption and hemorrhagic transformations after ischemic stroke. However, their contribution to cerebral amyloid angiopathy-related hemorrhage remains unclear. Human brain endothelial cells challenged with Aβ synthetic homologues containing mutations known to be associated in vivo with hemorrhagic manifestations (AβE22Q and AβL34V) showed enhanced production and activation of MMP-2, evaluated via Multiplex MMP antibody arrays, gel zymography, and Western blot, which in turn proteolytically cleaved in situ the Aβ peptides. Immunoprecipitation followed by mass spectrometry analysis highlighted the generation of specific C-terminal proteolytic fragments, in particular the accumulation of Aβ-(1–16), a result validated in vitro with recombinant MMP-2 and quantitatively evaluated using deuterium-labeled internal standards. Silencing MMP-2 gene expression resulted in reduced Aβ degradation and enhanced apoptosis. Secretion and activation of MMP-2 as well as susceptibility of the Aβ peptides to MMP-2 degradation were dependent on the peptide conformation, with fibrillar elements of AβE22Q exhibiting negligible effects. Our results indicate that MMP-2 release and activation differentially degrades Aβ species, delaying their toxicity for endothelial cells. However, taking into consideration MMP ability to degrade basement membrane components, these protective effects might also undesirably compromise blood brain barrier integrity and precipitate a hemorrhagic phenotype.

Structural Mimicry of A-Loop Tyrosine Phosphorylation by a Pathogenic FGF Receptor 3 Mutation

The K650E gain-of-function mutation in the tyrosine kinase domain of FGF receptor 3 (FGFR3) causes Thanatophoric Dysplasia type II, a neonatal lethal congenital dwarfism syndrome, and when acquired somatically, it contributes to carcinogenesis. In this report, we determine the crystal structure of the FGFR3 kinase domain harboring this pathogenic mutation and show that the mutation introduces a network of intramolecular hydrogen bonds to stabilize the active-state conformation. In the crystal, the mutant FGFR3 kinases are caught in the act of trans-phosphorylation on a kinase insert autophosphorylation site, emphasizing the fact that the K650E mutation circumvents the requirement for A-loop tyrosine phosphorylation in kinase activation. Analysis of this trans-phosphorylation complex sheds light onto the determinants of tyrosine trans-phosphorylation specificity. We propose that the targeted inhibition of this pathogenic FGFR3 kinase may be achievable by small molecule kinase inhibitors that selectively bind the active-state conformation of FGFR3 kinase.

DFG-out Mode of Inhibition by an Irreversible Type-1 Inhibitor Capable of Overcoming Gate-Keeper Mutations in FGF Receptors.

Drug-resistance acquisition through kinase gate-keeper mutations is a major hurdle in the clinic. Here, we determined the first crystal structures of the human FGFR4 kinase domain (FGFR4K) alone and complexed with ponatinib, a promiscuous type-2 (DFG-out) kinase inhibitor, and an oncogenic FGFR4K harboring the V550L gate-keeper mutation bound to FIIN-2, a new type-1 irreversible inhibitor. Remarkably, like ponatinib, FIIN-2 also binds in the DFG-out mode despite lacking a functional group necessary to occupy the pocket vacated upon the DFG-out flip. Structural analysis reveals that the covalent bond between FIIN-2 and a cysteine, uniquely present in the glycine-rich loop of FGFR kinases, facilitates the DFG-out conformation, which together with the internal flexibility of FIIN-2 enables FIIN-2 to avoid the steric clash with the gate-keeper mutation that causes the ponatinib resistance. The structural data provide a blueprint for the development of next generation anticancer inhibitors through combining the salient inhibitory mechanisms of ponatinib and FIIN-2.

Comparison of cardiolipins from Drosophila strains with mutations in putative remodeling enzymes

Cardiolipin is a dimeric phospholipid with a characteristic acyl composition that is generated by fatty acid remodeling after de novo synthesis. Several enzymes have been proposed to participate in acyl remodeling of cardiolipin. In order to compare the effect of these enzymes, we determined the pattern of cardiolipin molecular species in Drosophila strains with specific enzyme deletions, using MALDI-TOF mass spectrometry with internal standards. We established the linear range of the method for cardiolipin quantification, determined the relative signal intensities of several cardiolipin standards, and demonstrated satisfying signal-to-noise ratios in cardiolipin spectra from a single fly. Our data demonstrate changes in the cardiolipin composition during the Drosophila life cycle. Comparison of cardiolipin spectra, using vector algebra, showed that inactivation of tafazzin had a large effect on the molecular composition of cardiolipin, inactivation of calcium-independent phospholipase A(2) had a small effect, whereas inactivation of acyl-CoA:lysocardiolipin-acyltransferase and of the trifunctional enzyme did not affect the cardiolipin composition.