Faculty Bibliography

The molecular basis by which receptor tyrosine kinases (RTKs) recruit and phosphorylate Src Homology 2 (SH2) domain-containing substrates has remained elusive. We used X-ray crystallography, NMR spectroscopy, and cell-based assays to demonstrate that recruitment and phosphorylation of Phospholipase Cgamma (PLCgamma), a prototypical SH2 containing substrate, by FGF receptors (FGFR) entails formation of an allosteric 2:1 FGFR-PLCgamma complex. We show that the engagement of pTyr-binding pocket of the cSH2 domain of PLCgamma by the phosphorylated tail of an FGFR kinase induces a conformational change at the region past the cSH2 core domain encompassing Tyr-771 and Tyr-783 to facilitate the binding/phosphorylation of these tyrosines by another FGFR kinase in trans. Our data overturn the current paradigm that recruitment and phosphorylation of substrates are carried out by the same RTK monomer in cis and disclose an obligatory role for receptor dimerization in substrate phosphorylation in addition to its canonical role in kinase activation.

Stimulus-triggered protein synthesis is critical for brain health and function. However, due to technical hurdles, de novo neuronal translation is predominantly studied in cultured cells, whereas electrophysiological and circuit analyses often are performed in brain slices. The different properties of these two experimental systems create an information gap about stimulus-induced alterations in the expression of new proteins in mature circuits. To address this, we adapted two existing techniques, BONCAT and SILAC, to a combined proteomic technique, BONLAC, for use in acute adult hippocampal slices. Using BDNF-induced protein synthesis as a proof of concept, we found alterations in expression of proteins involved in neurotransmission, trafficking, and cation binding that differed from those found in a similar screen in cultured neurons. Our results indicate important differences between cultured neurons and slices, and suggest that BONLAC could be used to dissect proteomic changes underlying synaptic events in adult circuits.

Amyloid beta (Abeta) is the major constituent of the brain deposits found in parenchymal plaques and cerebral blood vessels of patients with Alzheimer's disease (AD). Several lines of investigation support the notion that synaptic pathology, one of the strongest correlates to cognitive impairment, is related to the progressive accumulation of neurotoxic Abeta oligomers. Since the process of oligomerization/fibrillization is concentration-dependent, it is highly reliant on the homeostatic mechanisms that regulate the steady state levels of Abeta influencing the delicate balance between rate of synthesis, dynamics of aggregation, and clearance kinetics. Emerging new data suggest that reduced Abeta clearance, particularly in the aging brain, plays a critical role in the process of amyloid formation and AD pathogenesis. Using well-defined monomeric and low molecular mass oligomeric Abeta1-40 species stereotaxically injected into the brain of C57BL/6 wild-type mice in combination with biochemical and mass spectrometric analyses in CSF, our data clearly demonstrate that Abeta physiologic removal is extremely fast and involves local proteolytic degradation leading to the generation of heterogeneous C-terminally cleaved proteolytic products, while providing clear indication of the detrimental role of oligomerization for brain Abeta efflux. Immunofluorescence confocal microscopy studies provide insight into the cellular pathways involved in the brain removal and cellular uptake of Abeta. The findings indicate that clearance from brain interstitial fluid follows local and systemic paths and that in addition to the blood-brain barrier, local enzymatic degradation and the bulk flow transport through the choroid plexus into the CSF play significant roles. Our studies highlight the diverse factors influencing brain clearance and the participation of various routes of elimination opening up new research opportunities for the understanding of altered mechanisms triggering AD pathology and for the potential design of combined therapeutic strategies.

Neuronal cell death, in neurodegenerative disorders, is mediated through a spectrum of biological processes. Excessive amounts of free radicals, such as reactive oxygen species (ROS), has detrimental effects on neurons leading to cell damage via peroxidation of unsaturated fatty acids in the cell membrane. Edaravone (3-methyl-1-phenyl-2-pyrazolin-5-one) has been used for neurological recovery in several countries, including Japan and China, and it has been suggested that Edaravone may have cytoprotective effects in neurodegeneration. Edaravone protects nerve cells in the brain by reducing ROS and inhibiting apoptosis. To gain further insight into the cytoprotective effects of Edaravone against oxidative stress condition we have performed comparative two-dimensional gel electrophoresis (2DE)-based proteomic analyses on SH-SY5Y neuroblastoma cells exposed to oxidative stress and in combination with Edaravone. We showed that Edaravone can reverse the cytotoxic effects of H2O2 through its specific mechanism. We observed that oxidative stress changes metabolic pathways and cytoskeletal integrity. Edaravone seems to reverse the H2O2-mediated effects at both the cellular and protein level via induction of Peroxiredoxin-2.

Mutations in genes encoding Isocitrate Dehydrogenase (IDH) isoforms are found in80%of low-grade gliomas (LGGs). Sequencing of LGGs has revealed branching cancer genetics; mutant IDH1 astrocytomas contain p53 and ATRX loss of function mutations, while IDH1-mutated oligodendrogliomas have a different set of mutations that includes chr 1p/19q co-deletion. The IDH mutation is a gain-of-function change at its catalytic core that results in the production of (R)-2-hydroxyglutarate, an oncometabolite, which causes characteristic DNA and histone hypermethylation changes that may contribute to tumorigenesis. Mouse models have thus far failed to demonstrate the role of IDH1 mutations in LGG formation. To test the hypothesis that mutant IDH1 is a driver of gliomagenesis, we use human embryonic stem cell (hESC)-derived neural stem cells (NSCs) to overexpress mutant IDH1 protein in combination with p53 and ATRX knockdown. We have generated twelve NSC lines that harbor combinations of mutant IDH1, wt IDH1 or an empty vector, in combination with ATRX and/or p53 knockdown. Our preliminary data indicate that mutantIDH1 does not alter the proliferative capacity of NSCs, as shown by cell cycle analysis and Ki67 staining, but paradoxically increases their apoptotic rate (15.8% vs 5.9% n = 4), a phenotype that is exacerbated by ATRX knockdown, as detected by annexin V and TUNEL staining (17.7% vs 2.6% n = 3). shRNA-mediated knockdown of p53 salvages the pro-apoptotic phenotype of mutant IDH1 and ATRX NSCs. Furthermore, initial observations suggest that mutant IDH1 biases NSCs toward glial fates, as evidenced by upregulation of the CD44 cell surface marker. We are currently testing the effects of IDH1 mutation on i) NSC differentiation to astrocytic and neuronal lineages, ii) NSC metabolism via metabolomics profiling and iii) in vivo tumorigenesis. We propose that mutant IDH1 alters the differentiation program of human NSCs toward glial rather than neuronal fates

Metabolomics is an emerging field that involves qualitative and quantitative measurements of small molecule metabolites in a biological system. These measurements can be useful for developing biomarkers for diagnosis, prognosis, or predicting response to therapy. Currently, a wide variety of metabolomics approaches, including nontargeted and targeted profiling, are used across laboratories on a routine basis. A diverse set of analytical platforms, such as NMR, gas chromatography-mass spectrometry, Orbitrap mass spectrometry, and time-of-flight-mass spectrometry, which use various chromatographic and ionization techniques, are used for resolution, detection, identification, and quantitation of metabolites from various biological matrices. However, few attempts have been made to standardize experimental methodologies or comparative analyses across different laboratories. The Metabolomics Research Group of the Association of Biomolecular Resource Facilities organized a "round-robin" experiment type of interlaboratory study, wherein human plasma samples were spiked with different amounts of metabolite standards in 2 groups of biologic samples (A and B). The goal was a study that resembles a typical metabolomics analysis. Here, we report our efforts and discuss challenges that create bottlenecks for the field. Finally, we discuss benchmarks that could be used by laboratories to compare their methodologies.

There is an increasing need in biology and clinical medicine to robustly and reliably measure tens-to-hundreds of peptides and proteins in clinical and biological samples with high sensitivity, specificity, reproducibility and repeatability. Previously, we demonstrated that LC-MRM-MS with isotope dilution has suitable performance for quantitative measurements of small numbers of relatively abundant proteins in human plasma, and that the resulting assays can be transferred across laboratories while maintaining high reproducibility and quantitative precision. Here we significantly extend that earlier work, demonstrating that 11 laboratories using 14 LC-MS systems can develop, determine analytical figures of merit, and apply highly multiplexed MRM-MS assays targeting 125 peptides derived from 27 cancer-relevant proteins and 7 control proteins to precisely and reproducibly measure the analytes in human plasma. To ensure consistent generation of high quality data we incorporated a system suitability protocol (SSP) into our experimental design. The SSP enabled real-time monitoring of LC-MRM-MS performance during assay development and implementation, facilitating early detection and correction of chromatographic and instrumental problems. Low to sub-nanogram/mL sensitivity for proteins in plasma was achieved by one-step immunoaffinity depletion of 14 abundant plasma proteins prior to analysis. Median intra- and inter-laboratory reproducibility was <20%, sufficient for most biological studies and candidate protein biomarker verification. Digestion recovery of peptides was assessed and quantitative accuracy improved using heavy isotope labeled versions of the proteins as internal standards. Using the highly multiplexed assay, participating laboratories were able to precisely and reproducibly determine the levels of a series of analytes in blinded samples used to simulate an inter-laboratory clinical study of patient samples. Our study further establishes that LC-MRM-MS using stable isotope dilution, with appropriate attention to analytical validation and appropriate quality c`ontrol measures, enables sensitive, specific, reproducible and quantitative measurements of proteins and peptides in complex biological matrices such as plasma.

Matrix metalloproteases MMP-2 and MMP-9 have been implicated in the physiologic catabolism of Alzheimer amyloid-beta (Abeta). Conversely, their association with vascular amyloid deposits, blood-brain barrier disruption, and hemorrhagic transformations after ischemic stroke also highlights their involvement in pathologic processes. To better understand this dichotomy, recombinant human (rh) MMP-2 and MMP-9 were incubated with Abeta40 and Abeta42 and the resulting proteolytic fragments assessed via immunoprecipitation and quantitative mass spectrometry. Both MMPs generated Abeta 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 Abeta42 by rhMMP-2 while the enzyme cleaved >80% of Abeta40 during the first hour of incubation. rhMMP-9 was significantly less effective, particularly in degrading Abeta1-42, although the targeted peptide bonds were identical. Using Abeta1-34 and Abeta1-30, we demonstrated that these peptides are also substrates for both MMPs, cleaving Abeta1-34 to produce Abeta1-30 first and Abeta1-16 subsequently. Consistent with the kinetics observed with full-length Abeta, rhMMP-9 degraded only a minute fraction of Abeta1-34 and was even less effective in producing Abeta1-16. Further degradation of Abeta1-16 by either MMP-2 or MMP-9 was not observed even after prolonged incubation times. Notably, all MMP-generated C-terminally truncated Abeta fragments were highly soluble, did not exhibit fibrillogenic properties or induce cytotoxicity in human cerebral microvascular endothelial or neuronal cells supporting the notion that these truncated Abeta species are associated with clearance mechanisms rather than being key elements in the fibrillogenesis process.