Herpes simplex virus type 1 (HSV-1) mutants defective for envelope glycoprotein C (gC) and gB are highly impaired in the ability to attach to cell surface heparan sulfate (HS) moieties of proteoglycans, the initial virus receptor. Here we report studies aimed at defining the HS binding element of HSV-1 (strain KOS) gB and determining whether this structure is functionally independent of gB’s role in extracellular virus penetration or intercellular virus spread. A mutant form of gB deleted for a putative HS binding lysine-rich (pK) sequence (residues 68 to 76) was transiently expressed in Vero cells and shown to be processed normally, leading to exposure on the cell surface. Solubilized gBpK− also had substantially lower affinity for heparin-acrylic beads than did wild-type gB, confirming that the HS binding domain had been inactivated. The gBpK− gene was used to rescue a KOS gB null mutant virus to produce the replication-competent mutant KgBpK−. Compared with wild-type virus, KgBpK− showed reduced binding to mouse L cells (ca. 20%), while a gC null mutant virus in which the gC coding sequence was replaced by the lacZ gene (KCZ) was substantially more impaired (ca. 65%-reduced binding), indicating that the contribution of gC to HS binding was greater than that of gB. The effect of combining both mutations into a single virus (KgBpK−gC−) was additive (ca. 80%-reduced binding to HS) and displayed a binding activity similar to that observed for KOS virus attachment to sog9 cells, a glycosaminoglycan-deficient L-cell line. Cell-adsorbed individual and double HS mutant viruses exhibited a lower rate of virus entry following attachment, suggesting that HS binding plays a role in the process of virus penetration. Moreover, the KgBpK− mutant virus produced small plaques on Vero cells in the presence of neutralizing antibody where plaque formation depended on cell-to-cell virus spread. These studies permitted the following conclusions: (i) the pK sequence is not essential for gB processing or function in virus infection, (ii) the lysine-rich sequence of gB is responsible for HS binding, and (iii) binding to HS is cooperatively linked to the process of efficient virus entry and lateral spread but is not absolutely required for virus infectivity.
Neurotransmitter release from the presynaptic terminal is under very precise spatial and temporal control. Following neurotransmitter release, synaptic vesicles are recycled by endocytosis and refilled with neurotransmitter. During the exocytosis event leading to release, SNARE proteins provide most of the mechanical force for membrane fusion. Here, we show one of these proteins, Syntaxin1A, is SUMOylated near its C-terminal transmembrane domain in an activity-dependent manner. Preventing SUMOylation of Syntaxin1A reduces its interaction with other SNARE proteins and disrupts the balance of synaptic vesicle endo/exocytosis, resulting in an increase in endocytosis. These results indicate that SUMOylation regulates the emerging role of Syntaxin1A in vesicle endocytosis, which in turn, modulates neurotransmitter release and synaptic function.
The covalent posttranslational modifications of proteins are critical events in signaling cascades that enable cells to efficiently, rapidly and reversibly respond to extracellular stimuli. This is especially important in the CNS where the processes affecting synaptic communication between neurons are highly complex and very tightly regulated. Sumoylation regulates the function and fate of a diverse array of proteins and participates in the complex cell signaling pathways required for cell survival. One of the most complex signaling pathways is synaptic transmission.Correct synaptic function is critical to the working of the brain and its alteration through synaptic plasticity mediates learning, mental disorders and stroke. The investigation of neuronal sumoylation is a new and exciting field and the functional and pathophysiological implications are far-reaching. Sumoylation has already been implicated in a diverse array of neurological disorders. Here we provide an overview of current literature highlighting recent insights into the role of sumoylation in neurodegeneration. In addition we present a brief assessment of drug discovery in the analogous ubiquitin system and extrapolate on the potential for development of novel therapies that might target SUMO-associated mechanisms of neurodegenerative disease.
SUMMARYSmall ubiquitin-related modifier (SUMO) proteins are ~11 kDa proteins that can be covalently conjugated to lysine residues in defined target proteins. The resultant post-translational modification, SUMOylation, is vital for the viability of mammalian cells and regulates, among other things, a range of essential nuclear processes. It has become increasingly apparent in recent years that SUMOylation also serves multiple functions outside the nucleus and that it plays a critical role in the regulation of neuronal integrity and synaptic function. In particular, dysfunction of the SUMOylation pathway has been implicated in the molecular and cellular dysfunction associated with neurodegenerative and psychiatric disorders. Here, we outline current knowledge of the SUMO pathway and discuss the growing evidence for its involvement in multiple neurodegenerative disorders, with a view to highlighting the potential of the SUMO pathway as a putative drug target.SUMO (small ubiquitin-related modifier) proteins are ~11 kDa proteins that can be covalently conjugated to lysine residues in target proteins, altering the biochemical and/or functional properties of the modified protein. SUMO conjugation occurs via an enzymatic cascade analogous to that of ubiquitin, another well-characterized protein modifier. 1 SUMO was first described in 1996 as a protein involved in the partitioning of the nuclear Ras-related GTPase-activating protein (RanGAP) between the cytosol and nuclear pore complex. 2,3 In the decade following its discovery, numerous studies reported the SUMO modification of other nuclear proteins, suggesting that SUMOylation is a predominantly nuclear phenomenon. However, in recent years, multiple cytosolic and plasma membrane SUMO targets have been described, many of which are essential for neuronal function. In addition, an increasing number of SUMO targets have been linked to various neuropathological conditions. This review provides a summary of recent findings related to SUMOylation and neurological disease, with a view to the potential for modulation of protein SUMOylation in the brain as a means for therapeutic intervention. SUMO ISOFORMSYeast contain only one SUMO protein, ubiquitin-like protein Smt3, whereas humans possess four SUMO isoforms, designated SUMO1 to SUMO4. 4,5 In their conjugatable forms, Copyright © 2009 Europe PMC Funders Author ManuscriptsEurope PMC Funders Author Manuscripts SUMO2 and SUMO3 differ only in three N-terminal residues and have yet to be functionally distinguished (hence they are usually collectively referred to as SUMO2/3). SUMO1 and SUMO2/3 are evolutionarily conserved, and SUMO1 shares 18% homology with ubiquitin ( Fig. 1). However, despite the relatively low sequence homology, the proteins possess highly similar three-dimensional structures. 6 SUMO1 and SUMO2/3 are conjugated to substrate proteins by the same enzymatic machinery 7 and while some targets are exclusively modified by one family member, 8 many target proteins can be modified by both SUMO1 and SUMO2/3. 9,10 To...
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