Faculty Bibliography

Genetically encoded calcium indicators (GECIs) permit imaging intracellular calcium transients. Among GECIs, the GFP-based GCaMPs are the most widely used because of their high sensitivity and rapid response to changes in intracellular calcium concentrations. Here we report that the fluorescence of GCaMPs-including GCaMP3, GCaMP5 and GCaMP6-can be converted from green to red following exposure to blue-green light (450-500 nm). This photoconversion occurs in both insect and mammalian cells and is enhanced in a low oxygen environment. The red fluorescent GCaMPs retained calcium responsiveness, albeit with reduced sensitivity. We identified several amino acid residues in GCaMP important for photoconversion and generated a GCaMP variant with increased photoconversion efficiency in cell culture. This light-induced spectral shift allows the ready labeling of specific, targeted sets of GCaMP-expressing cells for functional imaging in the red channel. Together, these findings indicate the potential for greater utility of existing GCaMP reagents, including transgenic animals.

Determination of a protein's N-terminal sequence can be important for the characterization of protein processing. To increase the confidence of protein N-terminal identification, chemical derivatization of the N-terminal amine group by (N-Succinimidyloxycarbonylmethyl)tris(2,4,6-trimethoxyphenyl)phosphonium bromide (TMPP) or dimethyl labeling followed by mass spectrometric analysis is commonly performed. Using this approach, proteins can be separated by SDS-PAGE, and the protein N-terminus of interest is labeled by TMPP or dimethyl in-gel before tryptic digestion and LC-MS analysis. The N-terminus of a protein can thus be easily identified because the N-terminal tryptic peptides are preferentially labeled. Peptides with N-terminal derivatization produce a better fragmentation pattern during tandem mass spectrometric analysis, which significantly facilitates sequencing of these peptides.

Identification of proteins by mass spectrometry is crucial for better understanding of many biological, biochemical, and biomedical processes. Here we describe two methods for the identification of electroblotted proteins by on-membrane digestion prior to analysis by mass spectrometry. These on-membrane methods take approximately half the time of in-gel digestion and provide better digestion efficiency, due to the better accessibility of the protease to the proteins adsorbed onto the nitrocellulose, and better protein sequence coverage, especially for membrane proteins where large and hydrophobic peptides are commonly present.

Measuring the synthesis of new proteins in the context of a much greater number of pre-existing proteins can be difficult. To overcome this obstacle, bioorthogonal noncanonical amino acid tagging (BONCAT) can be combined with stable isotope labeling by amino acid in cell culture (SILAC) for comparative proteomic analysis of de novo protein synthesis (BONLAC). In the present study, we show that alkyne resin-based isolation of L-azidohomoalanine (AHA) labeled proteins using azide/alkyne cycloaddition minimizes contamination from pre-existing proteins. Using this approach, we isolated and identified 7414 BONCAT-labeled proteins. The nascent proteome isolated by BONCAT was very similar to the steady-state proteome, though transcription factors were highly enriched by BONCAT. About 30% of the methionine residues were replaced by AHA in our BONCAT samples, which allowed for identification of methionine-containing peptides. There was no bias against low methionine proteins by BONCAT at the proteome level. When we applied the BONLAC approach to screen for brain-derived neurotrophic factor (BDNF)-induced protein synthesis, 53 proteins were found to be significantly up-regulated two hours after BDNF stimulation. Our study demonstrated that the newly synthesized proteome, even after a short period of stimulation, can be efficiently isolated by BONCAT and analyzed to a depth that is similar to that of the steady-state proteome.

Activity-dependent CREB phosphorylation and gene expression are critical for long-term neuronal plasticity. Local signaling at CaV1 channels triggers these events, but how information is relayed onward to the nucleus remains unclear. Here, we report a mechanism that mediates long-distance communication within cells: a shuttle that transports Ca(2+)/calmodulin from the surface membrane to the nucleus. We show that the shuttle protein is gammaCaMKII, its phosphorylation at Thr287 by betaCaMKII protects the Ca(2+)/CaM signal, and CaN triggers its nuclear translocation. Both betaCaMKII and CaN act in close proximity to CaV1 channels, supporting their dominance, whereas gammaCaMKII operates as a carrier, not as a kinase. Upon arrival within the nucleus, Ca(2+)/CaM activates CaMKK and its substrate CaMKIV, the CREB kinase. This mechanism resolves long-standing puzzles about CaM/CaMK-dependent signaling to the nucleus. The significance of the mechanism is emphasized by dysregulation of CaV1, gammaCaMKII, betaCaMKII, and CaN in multiple neuropsychiatric disorders.

Mass spectrometry is an indispensable tool for peptide and protein analysis owing to its speed, sensitivity, and versatility. It can be used to determine amino acid sequences of peptides, and to characterize a wide variety of post-translational modifications such as phosphorylation and glycosylation. Mass spectrometry can also be used to determine absolute and relative protein quantities, and can identify and quantify thousands of proteins from complex samples, which makes it an extremely powerful tool for systems biology studies. The main goals of this unit are to familiarize peptide and protein chemists and biologists with the types of mass spectrometers that are appropriate for the majority of their analytical needs, to describe the kinds of experiments that can be performed with these instruments on a routine basis, and to discuss the kinds of information that these experiments provide. Curr. Protoc. Mol. Biol. 108:10.21.1-10.21.30. (c) 2014 by John Wiley & Sons, Inc.

Oncogenic RAS (H-RAS(V12)) induces premature senescence in primary cells by triggering production of reactive oxygen species (ROS), but the molecular role of ROS in senescence remains elusive. We investigated whether inhibition of protein tyrosine phosphatases by ROS contributed to H-RAS(V12)-induced senescence. We identified protein tyrosine phosphatase 1B (PTP1B) as a major target of H-RAS(V12)-induced ROS. Inactivation of PTP1B was necessary and sufficient to induce premature senescence in H-RAS(V12)-expressing IMR90 fibroblasts. We identified phospho-Tyr 393 of argonaute 2 (AGO2) as a direct substrate of PTP1B. Phosphorylation of AGO2 at Tyr 393 inhibited loading with microRNAs (miRNAs) and thus miRNA-mediated gene silencing, which counteracted the function of H-RAS(V12)-induced oncogenic miRNAs. Overall, our data illustrate that premature senescence in H-RAS(V12)-transformed primary cells is a consequence of oxidative inactivation of PTP1B and inhibition of miRNA-mediated gene silencing.

BACKGROUND: Parkinson's disease (PD) is the second most common neurodegenerative movement disorder, caused by preferential dopaminergic neuronal cell death in the substantia nigra, a process also influenced by oxidative stress. L-3,4-dihydroxyphenylalanine (L-DOPA) represents the main treatment route for motor symptoms associated with PD however, its exact mode of action remains unclear. A spectrum of conflicting data suggests that L-DOPA may damage dopaminergic neurons due to oxidative stress whilst other data suggest that L-DOPA itself may induce low levels of oxidative stress, which in turn stimulates endogenous antioxidant mechanisms and neuroprotection. RESULTS: In this study we performed a two-dimensional gel electrophoresis (2DE)-based proteomic study to gain further insight into the mechanism by which L-DOPA can influence the toxic effects of H2O2 in neuronal cells. We observed that oxidative stress affects metabolic pathways as well as cytoskeletal integrity and that neuronal cells respond to oxidative conditions by enhancing numerous survival pathways. Our study underlines the complex nature of L-DOPA in PD and sheds light on the interplay between oxidative stress and L-DOPA. CONCLUSIONS: Oxidative stress changes neuronal metabolic routes and affects cytoskeletal integrity. Further, L-DOPA appears to reverse some H2O2-mediated effects evident at both the proteome and cellular level.

Although antipsychotic drugs can reduce psychotic behavior within a few hours, full efficacy is not achieved for several weeks, implying that there may be rapid, short-term changes in neuronal function, which are consolidated into long-lasting changes. We showed that the antipsychotic drug haloperidol, a dopamine receptor type 2 (D2R) antagonist, stimulated the kinase Akt to activate the mRNA translation pathway mediated by the mammalian target of rapamycin complex 1 (mTORC1). In primary striatal D2R-positive neurons, haloperidol-mediated activation of mTORC1 resulted in increased phosphorylation of ribosomal protein S6 (S6) and eukaryotic translation initiation factor 4E-binding protein (4E-BP). Proteomic mass spectrometry revealed marked changes in the pattern of protein synthesis after acute exposure of cultured striatal neurons to haloperidol, including increased abundance of cytoskeletal proteins and proteins associated with translation machinery. These proteomic changes coincided with increased morphological complexity of neurons that was diminished by inhibition of downstream effectors of mTORC1, suggesting that mTORC1-dependent translation enhances neuronal complexity in response to haloperidol. In vivo, we observed rapid morphological changes with a concomitant increase in the abundance of cytoskeletal proteins in cortical neurons of haloperidol-injected mice. These results suggest a mechanism for both the acute and long-term actions of antipsychotics.