Faculty Bibliography

Conventional stable isotope labeling with amino acids in cell culture (SILAC) requires extensive metabolic labeling of proteins and therefore is difficult to apply to cells that do not divide or are unstable in SILAC culture. Using two different sets of heavy amino acids for labeling allows for straightforward SILAC quantitation using partially labeled cells because the two cell populations are always equally labeled. Here we report the application of this labeling strategy to primary cultured neurons. We demonstrated that protein quantitation was not compromised by incomplete labeling of the neuronal proteins. We used this method to study neurotrophin-3 (NT-3) signaling in primary cultured neurons. Surprisingly our results indicate TrkB signaling is a major component of the signaling network induced by NT-3 in cortical neurons. In addition, involvement of proteins such as VAMP2, Scamp1, and Scamp3 suggests that NT-3 may lead to enhanced exocytosis of synaptic vesicles

Alzheimer's disease (AD) is the leading cause of dementia and the most common form of amyloidosis in humans. Extensive extracellular deposition of amyloid-beta (Abeta), a 40-42 amino acid degradation product of APP, is considered a hallmark feature of AD. Our attention is focused on the highly heterogeneous biochemical nature of the brain Abeta species, delving beyond Abeta40 and Abeta42, likely reflecting a complex balance between amyloidogenic and clearance pathways. We have fractionated water-soluble, detergent-soluble and formic acid soluble Abeta species from brains of transgenic mouse models of amyloid depostion and AD cases. Subsequently, we applied a combination of biochemical techniques including immunoprecipitation followed by identification of Abeta species with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Our biochemical data on the Abeta species present in sporadic AD cases and in transgenic mouse models highlight the presence of similar N-and C-terminally truncated fragments-likely reflecting the ability of multiple proteases to degrade Abeta in situ-and several post-translational modifications with still unclear roles in the amyloidogenesis mechanism. Notably, not all the brain Abeta peptides have identical solubility properties; whereas many of them are highly soluble in water-based physiologic solutions others require mild detergents or strong acids for extraction, suggesting their differential involvement in catabolic and fibrillogenic processes

Endoplasmic reticulum (ER) oxidation 1 (ERO1) transfers disulfides to protein disulfide isomerase (PDI) and is essential for oxidative protein folding in simple eukaryotes such as yeast and worms. Surprisingly, ERO1-deficient mammalian cells exhibit only a modest delay in disulfide bond formation. To identify ERO1-independent pathways to disulfide bond formation, we purified PDI oxidants with a trapping mutant of PDI. Peroxiredoxin IV (PRDX4) stood out in this list, as the related cytosolic peroxiredoxins are known to form disulfides in the presence of hydroperoxides. Mouse embryo fibroblasts lacking ERO1 were intolerant of PRDX4 knockdown. Introduction of wild-type mammalian PRDX4 into the ER rescued the temperature-sensitive phenotype of an ero1 yeast mutation. In the presence of an H(2)O(2)-generating system, purified PRDX4 oxidized PDI and reconstituted oxidative folding of RNase A. These observations implicate ER-localized PRDX4 in a previously unanticipated, parallel, ERO1-independent pathway that couples hydroperoxide production to oxidative protein folding in mammalian cells

Ezh2 functions as a histone H3 Lys 27 (H3K27) methyltransferase when comprising the Polycomb-Repressive Complex 2 (PRC2). Trimethylation of H3K27 (H3K27me3) correlates with transcriptionally repressed chromatin. The means by which PRC2 targets specific chromatin regions is currently unclear, but noncoding RNAs (ncRNAs) have been shown to interact with PRC2 and may facilitate its recruitment to some target genes. Here we show that Ezh2 interacts with HOTAIR and Xist. Ezh2 is phosphorylated by cyclin-dependent kinase 1 (CDK1) at threonine residues 345 and 487 in a cell cycle-dependent manner. A phospho-mimic at residue 345 increased HOTAIR ncRNA binding to Ezh2, while the phospho-mimic at residue 487 was ineffectual. An Ezh2 domain comprising T345 was found to be important for binding to HOTAIR and the 5' end of Xist

Agrin, released by motor neurons, promotes neuromuscular synapse formation by stimulating MuSK, a receptor tyrosine kinase expressed in skeletal muscle. Phosphorylated MuSK recruits docking protein-7 (Dok-7), an adaptor protein that is expressed selectively in muscle. In the absence of Dok-7, neuromuscular synapses fail to form, and mutations that impair Dok-7 are a major cause of congenital myasthenia in humans. How Dok-7 stimulates synaptic differentiation is poorly understood. Once recruited to MuSK, Dok-7 directly stimulates MuSK kinase activity. This unusual activity of an adapter protein is mediated by the N-terminal region of Dok-7, whereas most mutations that cause congenital myasthenia truncate the C-terminal domain. Here, we demonstrate that Dok-7 also functions downstream from MuSK, and we identify the proteins that are recruited to the C-terminal domain of Dok-7. We show that Agrin stimulates phosphorylation of two tyrosine residues in the C-terminal domain of Dok-7, which leads to recruitment of two adapter proteins: Crk and Crk-L. Furthermore, we show that selective inactivation of Crk and Crk-L in skeletal muscle leads to severe defects in neuromuscular synapses in vivo, revealing a critical role for Crk and Crk-L downstream from Dok-7 in presynaptic and postsynaptic differentiation

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

The canonical microRNA (miRNA) biogenesis pathway requires two RNaseIII enzymes: Drosha and Dicer. To understand their functions in mammals in vivo, we engineered mice with germline or tissue-specific inactivation of the genes encoding these two proteins. Changes in proteomic and transcriptional profiles that were shared in Dicer- and Drosha-deficient mice confirmed the requirement for both enzymes in canonical miRNA biogenesis. However, deficiency in Drosha or Dicer did not always result in identical phenotypes, suggesting additional functions. We found that, in early-stage thymocytes, Drosha recognizes and directly cleaves many protein-coding messenger RNAs (mRNAs) with secondary stem-loop structures. In addition, we identified a subset of miRNAs generated by a Dicer-dependent but Drosha-independent mechanism. These were distinct from previously described mirtrons. Thus, in mammalian cells, Dicer is required for the biogenesis of multiple classes of miRNAs. Together, these findings extend the range of function of RNaseIII enzymes beyond canonical miRNA biogenesis, and help explain the nonoverlapping phenotypes caused by Drosha and Dicer deficiency

Patients carrying mutations within the amyloid-beta (Abeta) 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 Abeta synthetic homologues containing mutations known to be associated in vivo with hemorrhagic manifestations (AbetaE22Q and AbetaL34V) 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 Abeta peptides. Immunoprecipitation followed by mass spectrometry analysis highlighted the generation of specific C-terminal proteolytic fragments, in particular the accumulation of Abeta-(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 Abeta degradation and enhanced apoptosis. Secretion and activation of MMP-2 as well as susceptibility of the Abeta peptides to MMP-2 degradation were dependent on the peptide conformation, with fibrillar elements of AbetaE22Q exhibiting negligible effects. Our results indicate that MMP-2 release and activation differentially degrades Abeta 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

Activation of the c-JUN N-terminal kinase (JNK) pathway is implicated in a number of important physiological processes, from embryonic morphogenesis to cell survival and apoptosis. JNK stimulatory phosphatase 1 (JSP1) is a member of the dual-specificity phosphatase subfamily of protein tyrosine phosphatases. In contrast to other dual-specificity phosphatases that catalyze the inactivation of mitogen-activated protein kinases, expression of JSP1 activates JNK-mediated signaling. JSP1 and its relative DUSP15 are unique among members of the protein tyrosine phosphatase family in that they contain a potential myristoylation site at the N-terminus (MGNGMXK). In this study, we investigated whether JSP1 was myristoylated and examined the functional consequences of myristoylation. Using mass spectrometry, we showed that wild-type JSP1, but not a JSP1 mutant in which Gly2 was mutated to Ala (JSP1-G2A), was myristoylated in cells. Although JSP1 maintained intrinsic phosphatase activity in the absence of myristoylation, the subcellular localization of the enzyme was altered. Compared with the wild type, the ability of nonmyristoylated JSP1 to induce JNK activation and phosphorylation of the transcription factor c-JUN was attenuated. Upon expression of wild-type JSP1, a subpopulation of cells, with the highest levels of the phosphatase, was induced to float off the dish and undergo apoptosis. In contrast, cells expressing similar levels of JSP1-G2A remained attached, further highlighting that the myristoylation mutant was functionally compromised.

Work from multiple laboratories has clarified how the structural domains of botulinum neurotoxin A (BoNT/A) disable neuronal exocytosis, but important questions remain unanswered. Because BoNT/A intoxication disables its own uptake, light chain (LC) does not accumulate in neurons at detectable levels. We have therefore designed, expressed and purified a series of BoNT/A atoxic derivatives (ad) that retain the wild type features required for native trafficking. BoNT/A1ad(ek) and BoNT/A1ad(tev) are full length derivatives rendered atoxic through double point mutations in the LC protease (E(224)>A; Y(366)>A). DeltaLC-peptide-BoNT/A(tev) and DeltaLC-GFP-BoNT/A(tev) are derivatives wherein the catalytic portion of the LC is replaced with a short peptide or with GFP plus the peptide. In all four derivatives, we have fused the S6 peptide sequence GDSLSWLLRLLN to the N-terminus of the proteins to enable site-specific attachment of cargo using Sfp phosphopantetheinyl transferase. Cargo can be attached in a manner that provides a homogeneous derivative population rather than a polydisperse mixture of singly and multiply-labeled molecular species. All four derivatives contain an introduced cleavage site for conversion into disulfide-bonded heterodimers. These constructs were expressed in a baculovirus system and the proteins were secreted into culture medium and purified to homogeneity in yields ranging from 1 to 30 mg per liter. These derivatives provide unique tools to study toxin trafficking in vivo, and to assess how the structure of cargo linked to the heavy chain (HC) influences delivery to the neuronal cytosol. Moreover, they create the potential to engineer BoNT-based molecular vehicles that can target therapeutic agents to the neuronal cytoplasm