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In JoVE (1)
Other Publications (5)
Articles by Kalina Dimova in JoVE
JoVE Clinical and Translational Medicine
Skeletal Muscle Gender Dimorphism from Proteomics
1Center for Proteomics, Smith College, 2Department of Molecular Biophysics and Biochemistry, Yale University, 3Department of Chemistry, Smith College, 4Department of Biological Sciences and Center for Proteomics, Smith College
A straight-forward set of methods to isolate and determine the identity of the most abundant proteins expressed in skeletal muscle. About 800 spots are discerned on a two-dimensional gel from 10 mg muscle; this allows for the determination of gender-specific protein expression. These methods will give equivalent results in most tissues.
Other articles by Kalina Dimova on PubMed
Characterization of the Munc13-calmodulin Interaction by Photoaffinity Labeling
Sensing of and response to transient increases in the residual presynaptic Ca2+ levels are important adaptive mechanisms that define the short-term plasticity characteristics of neurons. Due to their essential function in synaptic vesicle priming and in the modulation of synaptic strength, Munc13 proteins have emerged as key regulators of these adaptive mechanisms. Indeed, Munc13-1 and ubMunc13-2 contain a conserved calmodulin (CaM) binding site and the Ca2+ -dependent interaction of these Munc13 isoforms with CaM constitutes a molecular mechanism that transduces residual Ca2+ signaling to the synaptic exocytotic machinery. Here, we used Munc13-derived model peptides in photoaffinity labeling (PAL) experiments to demonstrate the stoichiometric and Ca2+ -dependent CaM binding of the other members of the Munc13 family, bMunc13-2 and Munc13-3, via structurally distinct non-conserved binding sites. A PAL-based Ca2+ titration assay revealed that all Munc13 isoforms can form a complex with CaM already at low Ca2+ concentrations just above resting levels, underscoring the Ca2+ sensor/effector function of this interaction in short-term synaptic plasticity phenomena.
Proteolipid Protein is Required for Transport of Sirtuin 2 into CNS Myelin
Mice lacking the expression of proteolipid protein (PLP)/DM20 in oligodendrocytes provide a genuine model for spastic paraplegia (SPG-2). Their axons are well myelinated but exhibit impaired axonal transport and progressive degeneration, which is difficult to attribute to the absence of a single myelin protein. We hypothesized that secondary molecular changes in PLP(null) myelin contribute to the loss of PLP/DM20-dependent neuroprotection and provide more insight into glia-axonal interactions in this disease model. By gel-based proteome analysis, we identified >160 proteins in purified myelin membranes, which allowed us to systematically monitor the CNS myelin proteome of adult PLP(null) mice, before the onset of disease. We identified three proteins of the septin family to be reduced in abundance, but the nicotinamide adenine dinucleotide (NAD+)-dependent deacetylase sirtuin 2 (SIRT2) was virtually absent. SIRT2 is expressed throughout the oligodendrocyte lineage, and immunoelectron microscopy revealed its association with myelin. Loss of SIRT2 in PLP(null) was posttranscriptional, suggesting that PLP/DM20 is required for its transport into the myelin compartment. Because normal SIRT2 activity is controlled by the NAD+/NADH ratio, its function may be coupled to the axo-glial metabolism and the long-term support of axons by oligodendrocytes.
Structural Insights into the Calmodulin-Munc13 Interaction Obtained by Cross-linking and Mass Spectrometry
Munc13 proteins are essential regulators of synaptic vesicle priming and play a key role in adaptive synaptic plasticity phenomena. We recently identified and characterized the Ca(2+)-dependent interaction of Munc13 and calmodulin (CaM) as the molecular mechanism linking changes in residual Ca(2+) concentrations to presynaptic vesicle priming and short-term plasticity. Here, we used peptidic photoprobes covering the established CaM-binding motif of Munc13 for photoaffinity labeling (PAL) of CaM, followed by structural characterization of the covalent photoadducts. Our innovative analytical workflow based on isotopically labeled CaM and mass spectrometry revealed that, in the bound state, the hydrophobic anchor residue of the CaM-binding motif in Munc13s contacts two distinct methionine residues in the C-terminal domain of CaM. To address the orientation of the peptide during binding, we obtained additional distance constraints from the mass spectrometric analysis of chemically cross-linked CaM-Munc13 peptide adducts. The constraints from both complementary cross-linking approaches were integrated into low-resolution three-dimensional structure models of the CaM-Munc13 peptide complexes. Our experimental data are best compatible with the structure of the complex formed by CaM and a CaM-binding peptide derived from neuronal NO synthase and show that Munc13-1 and ubMunc13-2 bind to CaM in an antiparallel orientation through a 1-5-8 motif. The structural information about the CaM-Munc13 peptide complexes will facilitate the design of Munc13 variants with altered CaM affinity and thereby advance the detailed functional analysis of the role of Munc13 proteins in synaptic transmission and plasticity.
Regulation of Rap2A by the Ubiquitin Ligase Nedd4-1 Controls Neurite Development
Nedd4-1 is a "neuronal precursor cell expressed and developmentally downregulated protein" and among the most abundant E3 ubiquitin ligases in mammalian neurons. In analyses of conventional and conditional Nedd4-1-deficient mice, we found that Nedd4-1 plays a critical role in dendrite formation. Nedd4-1, the serine/threonine kinase TNIK, and Rap2A form a complex that controls Nedd4-1-mediated ubiquitination of Rap2A. Ubiquitination by Nedd4-1 inhibits Rap2A function, which reduces the activity of Rap2 effector kinases of the TNIK family and promotes dendrite growth. We conclude that a Nedd4-1/Rap2A/TNIK signaling pathway regulates neurite growth and arborization in mammalian neurons.
Modular Architecture of Munc13/calmodulin Complexes: Dual Regulation by Ca2+ and Possible Function in Short-term Synaptic Plasticity
Ca(2+) signalling in neurons through calmodulin (CaM) has a prominent function in regulating synaptic vesicle trafficking, transport, and fusion. Importantly, Ca(2+)-CaM binds a conserved region in the priming proteins Munc13-1 and ubMunc13-2 and thus regulates synaptic neurotransmitter release in neurons in response to residual Ca(2+) signals. We solved the structure of Ca(2+)(4)-CaM in complex with the CaM-binding domain of Munc13-1, which features a novel 1-5-8-26 CaM-binding motif with two separated mobile structural modules, each involving a CaM domain. Photoaffinity labelling data reveal the same modular architecture in the complex with the ubMunc13-2 isoform. The N-module can be dissociated with EGTA to form the half-loaded Munc13/Ca(2+)(2)-CaM complex. The Ca(2+) regulation of these Munc13 isoforms can therefore be explained by the modular nature of the Munc13/Ca(2+)-CaM interactions, where the C-module provides a high-affinity interaction activated at nanomolar [Ca(2+)](i), whereas the N-module acts as a sensor at micromolar [Ca(2+)](i). This Ca(2+)/CaM-binding mode of Munc13 likely constitutes a key molecular correlate of the characteristic Ca(2+)-dependent modulation of short-term synaptic plasticity.
