Lactic acid and short chain fatty acids (SCFAs) produced by vaginal microbiota have reported antimicrobial and immune modulatory activities indicating their potential as biomarkers of disease and/or disease susceptibility. In asymptomatic women of reproductive-age the vaginal microbiota is comprised of lactic acid-producing bacteria that are primarily responsible for the production of lactic acid present at ~110 mM and acidifying the vaginal milieu to pH ~3.5. In contrast, bacterial vaginosis (BV), a dysbiosis of the vaginal microbiota, is characterized by decreased lactic acid-producing microbiota and increased diverse anaerobic bacteria accompanied by an elevated pH>4.5. BV is also characterized by a dramatic loss of lactic acid and greater concentrations of mixed SCFAs including acetate, propionate, butyrate, and succinate. Notably women with lactic acid-producing microbiota have more favorable reproductive and sexual health outcomes compared to women with BV. Regarding the latter, BV is associated with increased susceptibility to sexually transmitted infections (STIs) including HIV. In vitro studies demonstrate that lactic acid produced by vaginal microbiota has microbicidal and virucidal activities that may protect against STIs and endogenous opportunistic bacteria as well as immune modulatory properties that require further characterization with regard to their effects on the vaginal mucosa. In contrast, BV-associated SCFAs have far less antimicrobial activity with the potential to contribute to a pro-inflammatory vaginal environment. Here we review the composition of lactic acid and SCFAs in respective states of eubiosis (non-BV) or dysbiosis (BV), their effects on susceptibility to bacterial/viral STIs and whether they have inherent microbicidal/virucidal and immune modulatory properties. We also explore their potential as biomarkers for the presence and/or increased susceptibility to STIs.

Munc18-1 and Syntaxin1 are essential proteins for SNARE-mediated neurotransmission. Munc18-1 participates in synaptic vesicle fusion via dual roles: as a docking/chaperone protein by binding closed Syntaxin1, and as a fusion protein that binds SNARE complexes in a Syntaxin1 N-peptide dependent manner. The two roles are associated with a closed-open Syntaxin1 conformational transition. Here, we show that Syntaxin N-peptide binding to Munc18-1 is not highly selective, suggesting that other parts of the SNARE complex are involved in binding to Munc18-1. We also find that Syntaxin1, with an N peptide and a physically anchored C terminus, binds to Munc18-1 and that this complex can participate in SNARE complex formation. We report a Munc18-1-N-peptide crystal structure that, together with other data, reveals how Munc18-1 might transit from a conformation that binds closed Syntaxin1 to one that may be compatible with binding open Syntaxin1 and SNARE complexes. Our results suggest the possibility that structural transitions occur in both Munc18-1 and Syntaxin1 during their binary interaction. We hypothesize that Munc18-1 domain 3a undergoes a conformational change that may allow coiled-coil interactions with SNARE complexes.membrane trafficking | protein-peptide interaction | protein-protein interaction | Sec/Munc protein S ec/Munc (SM) and soluble NSF attachment protein receptor (SNARE) proteins play fundamental roles in regulating membrane traffic (1-3). The cognate interacting partners comprising the SM protein Munc18-1 and the SNARE protein Syntaxin1 (Sx1) are of special importance to human physiology because they regulate synaptic vesicle-mediated neurotransmitter release (4). Two alternate binding modes have been described for this pair of proteins. One mode involves Munc18-1 interacting with "closed" Sx1, in which the SNARE H3 helical motif is sequestered by the three Habc helices of Sx1 to form a four-helix bundle (5, 6) ( Fig. 1). This closed binding mode is consistent with a negative regulatory role for Munc18-1 because the SNARE H3 helix in closed Sx1 is unable to interact with SNARE partners to form the complexes that drive vesicle fusion (7) (Fig. 1A). However, the closed binding mode of Syntaxin is not universal and may be a specialization of regulated exocytosis (8). A second binding mode, which likely underpins a general function of SM proteins, occurs when Sx1 is in an "open" conformation (i.e., when the H3 helix is separated from the Habc helices) in the SNARE ternary complex (9, 10) (Fig. 1A). This binding mode is dependent on the very N-terminal 10 residues of Sx1, the N peptide. This second mode is consistent with a positive regulatory role for Munc18-1, because SNARE ternary complex formation is required for vesicle fusion. The N-peptide interaction has been characterized structurally for the highly homologous protein pair of Munc18-3 and Syntaxin4 (Sx4) (11), which regulate trafficking of the insulin-stimulated glucose transporter GLUT4 in muscle and fat cells (12).Munc18 proteins contribute to s...

Sec1p/Munc18 (SM) proteins are believed to play an integral role in vesicle transport through their interaction with SNAREs. Different SM proteins have been shown to interact with SNAREs via different mechanisms, leading to the conclusion that their function has diverged. To further explore this notion, in this study, we have examined the molecular interactions between Munc18c and its cognate SNAREs as these molecules are ubiquitously expressed in mammals and likely regulate a universal plasma membrane trafficking step. Thus, Munc18c binds to monomeric syntaxin4 and the N-terminal 29 amino acids of syntaxin4 are necessary for this interaction. We identified key residues in Munc18c and syntaxin4 that determine the N-terminal interaction and that are consistent with the N-terminal binding mode of yeast proteins Sly1p and Sed5p. In addition, Munc18c binds to the syntaxin4/SNAP23/ VAMP2 SNARE complex. Pre-assembly of the syntaxin4/ Munc18c dimer accelerates the formation of SNARE complex compared to assembly with syntaxin4 alone. These data suggest that Munc18c interacts with its cognate SNAREs in a manner that resembles the yeast proteins Sly1p and Sed5p rather than the mammalian neuronal proteins Munc18a and syntaxin1a. The Munc18c-SNARE interactions described here imply that Munc18c could play a positive regulatory role in SNARE assembly.

Sec1/Munc18 proteins (SM proteins) bind to soluble NSF attachment protein receptors (SNAREs) and play an essential role in membrane fusion. Divergent modes of regulation have been proposed for different SM proteins indicating that they can either promote or inhibit SNARE assembly. This is in part because of discrete modes of binding that have been described for various SM/SNARE complexes. One mode suggests that SM proteins bind only to Syntaxins (Stx) preventing SNARE assembly, whereas in another they facilitate SNARE assembly and bind to SNARE complexes. The mammalian cell surface SM protein Munc18c binds to an N-peptide in Stx4, and this is compatible with its interaction with SNARE complexes. Here we describe the crystal structure of Munc18c in complex with the Stx4 N-peptide. This structure shows remarkable similarity with a yeast complex indicating that the mode of binding, which can accommodate SNARE complexes, is highly conserved throughout evolution. Modeling reveals the presence of the N-peptide binding mode in most but not all yeast and mammalian SM/Stx pairs, suggesting that it has coevolved to fulfill a specific regulatory function. It is unlikely that the N-peptide interaction alone accounts for the specificity in SM/SNARE binding, implicating other contact surfaces in this function. Together with other data, our results support a sequential two-state model for SM/SNARE binding involving an initial interaction via the Stx N-peptide, which somehow facilitates a second, more comprehensive interaction comprising other contact surfaces in both proteins. crystallography ͉ protein:protein interactions ͉ vesicle trafficking I ntracellular trafficking relies on membrane fusion, a tightly regulated process that requires the formation of a coiled coil complex between helical motifs originating from soluble NSF attachment protein receptor (SNARE) proteins on vesicle and target membranes (1-3). Sec1/Munc18 proteins (SM proteins) play a fundamental role in this process by interacting with SNARE protein(s) and regulating the formation of SNARE complexes (4, 5). SM proteins have been identified in organisms from yeast to humans, and loss-of-function mutants lead to severe impairment in vesicle fusion (6).Despite their obvious importance, the nature of SNARE regulation by SM proteins remains controversial, in that both positive and negative regulatory roles have been reported (6, 7). Possibly the most compelling evidence in support of negative regulation is the observation that the mammalian Syntaxin1a (Stx1a) SNARE protein involved in synaptic neurotransmission exists in both an ''open'' (SNARE complex-compatible) and a ''closed'' (SNARE complexincompatible) conformation (8, 9). The crystal structure of Stx1a in complex with the SM protein Munc18-1 (Munc18a, nSec1) reveals that Munc18-1 embraces a closed conformation preventing Stx1a from forming the SNARE complex required for vesicle fusion (10). On the other hand, deletion of Munc18-1 results in impaired exocytosis in mouse neurons and chromaffin cells (11, 12),...

Eukaryotic secretory proteins exit the endoplasmic reticulum via transport vesicles generated by the essential COPII coat proteins. The outer coat complex, Sec13-Sec31, forms a scaffold that is thought to enforce curvature. By exploiting yeast bypass-of-sec-thirteen (bst) mutants, where Sec13p is dispensable, we probed the relationship between a compromised COPII coat and the cellular context in which it could still function. Genetic and biochemical analyses suggested that Sec13p was required to generate vesicles from membranes that contained asymmetrically distributed cargoes that were likely to confer opposing curvature. Thus Sec13p may rigidify the COPII cage and increase its membrane-bending capacity; this function could be bypassed when a bst mutation renders the membrane more deformable.