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In JoVE (2)
- Analyzing Large Protein Complexes by Structural Mass Spectrometry
- T-wave Ion Mobility-mass Spectrometry: Basic Experimental Procedures for Protein Complex Analysis
Other Publications (14)
- The Journal of Biological Chemistry
- The Journal of Biological Chemistry
- Molecular Pharmacology
- Molecular Pharmacology
- Journal of Molecular Biology
- Pflügers Archiv : European Journal of Physiology
- Pflügers Archiv : European Journal of Physiology
- Biochemistry
- Biochemistry
- The Journal of Biological Chemistry
- Developmental Neurobiology
- Molecular & Cellular Proteomics : MCP
- Science Signaling
- Analytical Chemistry
Articles by Izhak Michaelevski in JoVE
JoVE General
Analyzing Large Protein Complexes by Structural Mass Spectrometry
Department of Biological Chemistry, Weizmann Institute of Science
Mass spectrometry has proven to be a valuable tool for analyzing large protein complexes. This method enables insights into the composition, stoichiometry and overall architecture of multi-subunit assemblies. Here, we describe, step-by-step, how to perform a structural mass spectrometry analysis, and characterize macromolecular structures.
JoVE General
T-wave Ion Mobility-mass Spectrometry: Basic Experimental Procedures for Protein Complex Analysis
Department of Biological Chemistry, Weizmann Institute of Science
Ion mobility-mass spectrometry is an emerging gas-phase technology that separates ions, based on their collision cross-section and mass. The method provides three-dimensional information on the overall topology and shape of protein complexes. Here, we outline a basic procedure for instrument setting and optimization, calibration of drift times, and data interpretation.
Other articles by Izhak Michaelevski on PubMed
Modulation of a Brain Voltage-gated K+ Channel by Syntaxin 1A Requires the Physical Interaction of Gbetagamma with the Channel
Recently we suggested that direct interactions between voltage-gated K(+) channels and proteins of the exocytotic machinery, such as those observed between the Kv1.1/Kvbeta channel, syntaxin 1A, and SNAP-25 may be involved in neurotransmitter release. Furthermore, we demonstrated that the direct interaction with syntaxin 1A enhances the fast inactivation of Kv1.1/Kvbeta1.1 in oocytes. Here we show that G-protein betagamma subunits play a crucial role in the enhancement of inactivation by syntaxin 1A. The effect caused by overexpression of syntaxin 1A is eliminated in the presence of chelators of endogenous betagamma subunits in the whole cell and at the plasma membrane. Conversely, enhancement of inactivation caused by overexpression of beta(1)gamma(2) subunits is eliminated upon knock-down of endogenous syntaxin or its scavenging at the plasma membrane. We further show that the N terminus of Kv1.1 binds brain synaptosomal and recombinant syntaxin 1A and concomitantly binds beta(1)gamma(2); the binding of beta(1)gamma(2) enhances that of syntaxin 1A. Taken together, we suggest a mechanism whereby syntaxin and G protein betagamma subunits interact concomitantly with a Kv channel to regulate its inactivation.
Direct Interaction of Target SNAREs with the Kv2.1 Channel. Modal Regulation of Channel Activation and Inactivation Gating
Previously we suggested that interaction between voltage-gated K+ channels and protein components of the exocytotic machinery regulated transmitter release. This study concerns the interaction between the Kv2.1 channel, the prevalent delayed rectifier K+ channel in neuroendocrine and endocrine cells, and syntaxin 1A and SNAP-25. We recently showed in islet beta-cells that the Kv2.1 K+ current is modulated by syntaxin 1A and SNAP-25. Here we demonstrate, using co-immunoprecipitation and immunocytochemistry analyses, the existence of a physical interaction in neuroendocrine cells between Kv2.1 and syntaxin 1A. Furthermore, using concomitant co-immunoprecipitation from plasma membranes and two-electrode voltage clamp analyses in Xenopus oocytes combined with in vitro binding analysis, we characterized the effects of these interactions on the Kv2.1 channel gating pertaining to the assembly/disassembly of the syntaxin 1A/SNAP-25 (target (t)-SNARE) complex. Syntaxin 1A alone binds strongly to Kv2.1 and shifts both activation and inactivation to hyperpolarized potentials. SNAP-25 alone binds weakly to Kv2.1 and probably has no effect by itself. Expression of SNAP-25 together with syntaxin 1A results in the formation of t-SNARE complexes, with consequent elimination of the effects of syntaxin 1A alone on both activation and inactivation. Moreover, inactivation is shifted to the opposite direction, toward depolarized potentials, and its extent and rate are attenuated. Based on these results we suggest that exocytosis in neuroendocrine cells is tuned by the dynamic coupling of the Kv2.1 channel gating to the assembly status of the t-SNARE complex.
Kv2.1 Channel Activation and Inactivation is Influenced by Physical Interactions of Both Syntaxin 1A and the Syntaxin 1A/soluble N-ethylmaleimide-sensitive Factor-25 (t-SNARE) Complex with the C Terminus of the Channel
Kv2.1, the prevalent delayed-rectifier K(+) channel in neuroendocrine and endocrine cells, was suggested previously by our group to be modulated in islet beta-cells by syntaxin 1A (Syx) and soluble N-ethylmaleimide-sensitive factor attachment protein-25 (SNAP-25). We also demonstrated physical interactions in neuroendocrine cells between Kv2.1, Syx, and SNAP-25, characterized their effects on Kv2.1 activation and inactivation in Xenopus laevis oocytes, and suggested that they pertain to the assembly/disassembly of the Syx/SNAP-25 (t-SNARE) complex. In the present work, we established the existence of a causal relationship between the physical and the functional interactions of Syx with the Kv2.1 channel using three different peptides that compete with the channel for binding of Syx when injected into oocytes already coexpressing Syx with Kv2.1 in the plasma membrane: one peptide corresponding to the Syx-binding region on the N-type Ca(2+) channel, and two peptides corresponding to Syx-binding regions on the Kv2.1 C terminus. All peptides reversed the effects of Syx on Kv2.1, suggesting that the hyperpolarizing shifts of the steady-state inactivation and activation of Kv2.1 caused by Syx result from cell-surface protein-protein interactions and point to participation of the C terminus in such an interaction. In line with these findings, the effects of Syx were dissipated by partial deletions of the C terminus. Furthermore, the t-SNARE complex was shown to bind to the Kv2.1 C terminus, and its effects on the inactivation of Kv2.1 were dissipated by partial deletions of the C terminus. Taken together, these findings suggest that physical interactions of both Syx and the t-SNARE complex with the C terminus of Kv2.1 are involved in channel regulation.
Target Soluble N-ethylmaleimide-sensitive Factor Attachment Protein Receptors (t-SNAREs) Differently Regulate Activation and Inactivation Gating of Kv2.2 and Kv2.1: Implications on Pancreatic Islet Cell Kv Channels
We have hypothesized that the plasma membrane protein components of the exocytotic soluble N-ethylmaleimide-sensitive factor attachment protein (SNAP) receptor (SNARE) complex, syntaxin 1A and SNAP-25, distinctly regulate different voltage-gated K+ (Kv) channels that are differentially distributed. Neuroendocrine islet cells (alpha, beta, delta) uniformly contain both syntaxin 1A and SNAP-25. However, using immunohistochemistry, we show that the different pancreatic islet cells contain distinct dominant Kv channels, including Kv2.1 in beta cells and Kv2.2 in alpha and delta cells, whose interactions with the SNARE proteins would, respectively regulate insulin, glucagon and somatostatin secretion. We therefore examined the regulation by syntaxin 1A and SNAP-25 of these two channels. We have shown that Kv2.1 interacts with syntaxin 1A and SNAP-25 and, based on studies in oocytes, suggested a model of two distinct modes of interaction of syntaxin 1A and the complex syntaxin 1A/SNAP-25 with the C terminus of the channel. Here, we characterized the interactions of syntaxin 1A and SNAP-25 with Kv2.2 which is highly homologous to Kv2.1, except for the C-terminus. Comparative two-electrode voltage clamp analysis in oocytes between Kv2.2 and Kv2.1 shows that Kv2.2 interacts only with syntaxin 1A and, in contrast to Kv2.1, it does not interact with the syntaxin 1A/SNAP-25 complex and hence is not sensitive to the assembly/disassembly state of the complex. The distinct regulation of these closely related channels by SNAREs may be attributed to differences in their C termini. Together with the differential distribution of these channels among islet cells, their distinct regulation suggests that the documented profound down-regulation of islet SNARE levels in diabetes could distort islet cell ion channels and secretory responses in different ways, ultimately contributing to the abnormal glucose homeostasis.
Vimentin Binding to Phosphorylated Erk Sterically Hinders Enzymatic Dephosphorylation of the Kinase
Cleavage fragments of de novo synthesized vimentin were recently reported to interact with phosphorylated Erk1 and Erk2 MAP kinases (pErk) in injured sciatic nerve, thus linking pErk to a signaling complex retrogradely transported on importins and dynein. Here we clarify the structural basis for this interaction, which explains how pErk is protected from dephosphorylation while bound to vimentin. Pull-down and ELISA experiments revealed robust calcium-dependent binding of pErk to the second coiled-coil domain of vimentin, with observed affinities of binding increasing from 180 nM at 0.1 microM calcium to 15 nM at 10 microM calcium. In contrast there was little or no binding of non-phosphorylated Erk to vimentin under these conditions. Geometric and electrostatic complementarity docking generated a number of solutions wherein vimentin binding to pErk occludes the lip containing the phosphorylated residues in the kinase. Binding competition experiments with Erk peptides confirmed a solution in which vimentin covers the phosphorylation lip in pErk, interacting with residues above and below the lip. The same peptides inhibited pErk binding to the dynein complex in sciatic nerve axoplasm, and interfered with protection from phosphatases by vimentin. Thus, a soluble intermediate filament fragment interacts with a signaling kinase and protects it from dephosphorylation by calcium-dependent steric hindrance.
Interaction of Syntaxin with a Single Kv1.1 Channel: a Possible Mechanism for Modulating Neuronal Excitability
Voltage-gated K(+) channels are crucial for intrinsic neuronal plasticity and present a target for modulations by protein-protein interactions, notably, by exocytotic proteins demonstrated by us in several systems. Here, we investigated the interaction of a single Kv1.1 channel with syntaxin 1A. Syntaxin decreased the unitary conductance of all conductance states (two subconductances and a full conductance) and decreased their open probabilities by prolongation of mean closed dwell-times at depolarized potentials. However, at subthreshold potentials syntaxin 1A increased the probabilities of the subconductance states. Consequently, the macroscopic conductance is decreased at potentials above threshold and increased at threshold potentials. Numerical modeling based on steady-state and kinetic analyses suggests: (1) a mechanism whereby syntaxin controls activation gating by forcing the conductance pathway only via a sequence of discrete steps through the subconductance states, possibly via a breakdown of cooperative movements of voltage sensors that exist in Kv1.1; (2) a physiological effect, apparently paradoxical for an agent that reduces K(+) current, of attenuating neuronal firing frequency via an increase in K(+) shunting conductance. Such modulation of the gain of neuronal output in response to different levels of syntaxin is in accord with the suggested role for Kv1.1 in axonal excitability and synaptic efficacy.
VAMP2 Interacts Directly with the N Terminus of Kv2.1 to Enhance Channel Inactivation
Recently, we demonstrated that the Kv2.1 channel plays a role in regulated exocytosis of dense-core vesicles (DCVs) through direct interaction of its C terminus with syntaxin 1A, a plasma membrane soluble NSF attachment receptor (SNARE) component. We report here that Kv2.1 interacts with VAMP2, the vesicular SNARE partner that is also present at high concentration in neuronal plasma membrane. This is the first report of VAMP2 interaction with an ion channel. The interaction was demonstrated in brain membranes and characterized using electrophysiological and biochemical analyses in Xenopus oocytes combined with an in vitro binding analysis and protein modeling. Comparative study performed with wild-type and mutant Kv2.1, wild-type Kv1.5, and chimeric Kv1.5N/Kv2.1 channels revealed that VAMP2 enhanced the inactivation of Kv2.1, but not of Kv1.5, via direct interaction with the T1 domain of the N terminus of Kv2.1. Given the proposed role for surface VAMP2 in the regulation of the vesicle cycle and the important role for the sustained Kv2.1 current in the regulation of dendritic calcium entry during high-frequency stimulation, the interaction of VAMP2 with Kv2.1 N terminus may contribute, alongside with the interaction of syntaxin with Kv2.1 C terminus, to the activity dependence of DCV release.
Formation of the Full SNARE Complex Eliminates Interactions of Its Individual Protein Components with the Kv2.1 Channel
Previously, we have demonstrated physical and functional interactions of the voltage-gated potassium channel Kv2.1 with the plasma membrane protein components of the exocytotic SNARE complex, syntaxin 1A, and the t-SNARE, syntaxin 1A/SNAP-25, complex. Importantly, the physical interaction of Kv2.1 with syntaxin was shown to be involved in the facilitation of secretion from PC12 cells, which was independent of potassium currents. Recently, we showed that also VAMP2, the vesicular SNARE, interacts physically and functionally with Kv2.1. Here, we first set out to test the interaction of the full SNARE, syntaxin/SNAP-25/VAMP2, complex with the channel. Using the interaction of VAMP2 with Kv2.1 in Xenopus oocytes as a probe, we showed that coexpression of the t-SNARE complex with VAMP2 abolished the VAMP2 effect on channel inactivation and reduced the amount of VAMP2 that coprecipitated with Kv2.1. Further, in vitro pull down assays showed that the full SNARE complex failed to interact with Kv2.1 N- and C-termini in tandem, in contrast to the individual SNARE components. This suggests that the interactions of the SNARE components with Kv2.1 are abolished upon their recruitment into a full SNARE complex, which does not interact with the channel. Other important findings arising from the in vitro study are that the t-SNARE complex, in addition to syntaxin, interacts with a specific C-terminal channel domain, C1a, shown to mediate the facilitation of release by Kv2.1 and that the presence of Kv2.1 N-terminus has crucial contribution to these interactions. These findings provide important insights into the understanding of the complex molecular events involved in the novel phenomenon of secretion facilitation in neuroendocrine cells by Kv2.1.
Syntaxin Modulates Kv1.1 Through Dual Action on Channel Surface Expression and Conductance
The Kv1.1 channel that is expressed throughout the central and peripheral nervous system is known to interact with syntaxin 1A, a member of the exocytosis machinery protein complex. This interaction was previously shown to increase the macroscopic currents of the presynaptic Kv1.1 channel when coexpressed in Xenopus oocytes, while it decreased the unitary channel conductance and open probability. This apparent discrepancy has been resolved in this work, using electrophysiological, biochemical, and immunohistochemical analyses in oocytes by overexpression and antisense knockdown of syntaxin. Here, we demonstrate that syntaxin plays a dual role in the modulation of Kv1.1 function: enhancement of the channel's surface expression along with attenuation of single channel ion flux. These findings broaden the scope of channels and transporters that are dually modulated by syntaxin. Although the dual functioning of syntaxin in modulation of Kv1.1 channel activity may seem antagonistic, the combination of the two mechanisms may provide a useful means for fine-tuning axonal excitability and synaptic efficacy.
Rearrangements in the Relative Orientation of Cytoplasmic Domains Induced by a Membrane-anchored Protein Mediate Modulations in Kv Channel Gating
Interdomain interactions between intracellular N and C termini have been described for various K(+) channels, including the voltage-gated Kv2.1, and suggested to affect channel gating. However, no channel regulatory protein directly affecting N/C interactions has been demonstrated. Most Kv2.1 channel interactions with regulatory factors occur at its C terminus. The vesicular SNARE that is also present at a high concentration in the neuronal plasma membrane, VAMP2, is the only protein documented to affect Kv2.1 gating by binding to its N terminus. As its binding target has been mapped near a site implicated in Kv2.1 N/C interactions, we hypothesized that VAMP2 binding to the N terminus requires concomitant conformational changes in the C terminus, which wraps around the N terminus from the outside, to give VAMP2 access. Here, we first determined that the Kv2.1 N terminus, although crucial, is not sufficient to convey functional interaction with VAMP2, and that, concomitant to its binding to the "docking loop" at the Kv2.1 N terminus, VAMP2 binds to the proximal part of the Kv2.1 C terminus, C1a. Next, using computational biology approaches (ab initio modeling, docking, and molecular dynamics simulations) supported by molecular biology, biochemical, electrophysiological, and fluorescence resonance energy transfer analyses, we mapped the interaction sites on both VAMP2 and Kv2.1 and found that this interaction is accompanied by rearrangements in the relative orientation of Kv2.1 cytoplasmic domains. We propose that VAMP2 modulates Kv2.1 inactivation by interfering with the interaction between the docking loop and C1a, a mechanism for gating regulation that may pertain also to other Kv channels.
Axoplasm Isolation from Peripheral Nerve
Localized changes in the composition of axonal cytoplasm (axoplasm) are critical for many biological processes, including axon guidance, responses to injury, neurite outgrowth, and axon-glia interactions. Biochemical and molecular studies of these mechanisms have been heavily focused on in vitro systems because of the difficulty of obtaining subcellular extracts from mammalian tissues in vivo. As in vitro systems might not replicate the in vivo situation, reliable methods of axoplasm extraction from whole nerve would be helpful for mechanistic studies on axons. Here we develop and evaluate a new procedure for preparation of axoplasm from rat peripheral nerve, based on incubation of separated short segements of nerve fascicles in hypotonic medium to separate myelin and lyse nonaxonal structures, followed by extraction of the remaining axon-enriched material. We show that this new procedure reduces serum and glial cell contamination and facilitates proteomic analyses of axonal contents.
Axonal Transport Proteomics Reveals Mobilization of Translation Machinery to the Lesion Site in Injured Sciatic Nerve
Investigations of the molecular mechanisms underlying responses to nerve injury have highlighted the importance of axonal transport systems. To obtain a comprehensive view of the protein ensembles associated with axonal transport in injured axons, we analyzed the protein compositions of axoplasm concentrated at ligatures following crush injury of rat sciatic nerve. LC-MS/MS analyses of iTRAQ-labeled peptides from axoplasm distal and proximal to the ligation sites revealed protein ensembles transported in both anterograde and retrograde directions. Variability of replicates did not allow straightforward assignment of proteins to functional transport categories; hence, we performed principal component analysis and factor analysis with subsequent clustering to determine the most prominent injury-related transported proteins. This strategy circumvented experimental variability and allowed the extraction of biologically meaningful information from the quantitative neuroproteomics experiments. 299 proteins were highlighted by principal component analysis and factor analysis, 145 of which correlate with retrograde and 154 of which correlate with anterograde transport after injury. The analyses reveal extensive changes in both anterograde and retrograde transport proteomes in injured peripheral axons and emphasize the importance of RNA binding and translational machineries in the axonal response to injury.
Signaling to Transcription Networks in the Neuronal Retrograde Injury Response
Retrograde signaling from axon to soma activates intrinsic regeneration mechanisms in lesioned peripheral sensory neurons; however, the links between axonal injury signaling and the cell body response are not well understood. Here, we used phosphoproteomics and microarrays to implicate approximately 900 phosphoproteins in retrograde injury signaling in rat sciatic nerve axons in vivo and approximately 4500 transcripts in the in vivo response to injury in the dorsal root ganglia. Computational analyses of these data sets identified approximately 400 redundant axonal signaling networks connected to 39 transcription factors implicated in the sensory neuron response to axonal injury. Experimental perturbation of individual overrepresented signaling hub proteins, including Abl, AKT, p38, and protein kinase C, affected neurite outgrowth in sensory neurons. Paradoxically, however, combined perturbation of Abl together with other hub proteins had a reduced effect relative to perturbation of individual proteins. Our data indicate that nerve injury responses are controlled by multiple regulatory components, and suggest that network redundancies provide robustness to the injury response.
Gas-phase Compaction and Unfolding of Protein Structures
Ion-mobility mass spectrometry is emerging as a powerful tool for studying the structures of less established protein assemblies. The method provides simultaneous measurement of the mass and size of intact protein assemblies, providing information not only on the subunit composition and network of interactions but also on the overall topology and shape of protein complexes. However, how the experimental parameters affect the measured collision cross-sections remains elusive. Here, we present an extensive systematic study on a range of proteins and protein complexes with differing sizes, structures, and oligomerization states. Our results indicate that the experimental parameters, T-wave height and velocity, influence the determined collision cross-section independently and in opposite directions. Increasing the T-wave height leads to compaction of the protein structures, while higher T-wave velocities lead to their expansion. These different effects are attributed to differences in energy transmission and dissipation rates. Moreover, by analyzing proteins in their native and denatured states, we could identify the lower and upper boundaries of the collision cross-section, which reflect the "maximally packed" and "ultimately unfolded" states. Together, our results provide grounds for selecting optimal experimental parameters that will enable preservation of the nativelike conformation, providing structural information on uncharacterized protein assemblies.
