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Annexin proteins function as Ca-dependent regulators of membrane trafficking and repair that may also modulate membrane curvature. Here, using high-resolution confocal imaging, we report that the intestine-specific annexin A13 (ANX A13) localizes to the tips of intestinal microvilli and determined the crystal structure of the ANX A13a isoform to 2.6 Å resolution. The structure revealed that the N terminus exhibits an alternative fold that converts the first two helices and the associated helix-loop-helix motif into a continuous α-helix, as stabilized by a domain-swapped dimer. We also found that the dimer is present in solution and partially occludes the membrane-binding surfaces of annexin, suggesting that dimerization may function as a means for regulating membrane binding. Accordingly, as revealed by binding and cellular localization assays, ANX A13a variants that favor a monomeric state exhibited increased membrane association relative to variants that favor the dimeric form. Together, our findings support a mechanism for how the association of the ANX A13a isoform with the membrane is regulated.
© 2019 McCulloch et al.
Porphyromonas gingivalis is a keystone bacterium in the oral microbial communities that elicits a dysbiosis between the microbiota and the host. Therefore, inhibition of this organism in dental plaques has been one of the strategies for preventing and treating chronic periodontitis. We previously identified a Streptococcal ArcA derived Anti-P gingivalils Peptide (SAPP) that in vitro, is capable of repressing the expression of several virulence genes in the organism. This leads to a significant reduction in P gingivalis virulence potential, including its ability to colonize on the surface of Streptococcus gordonii, to invade human oral epithelial cells, and to produce gingipains. In this study, we showed that SAPP had minimal cytotoxicity to human oral keratinocytes and gingival fibroblasts. We observed that SAPP directly bound to the cell surface of P gingivalis, and that alterations in the sequence at the N-terminus of SAPP diminished its abilities to interact with P gingivalis cells and repressed the expression of virulence genes. Most strikingly, we demonstrated using an ex-vivo assay that besides its inhibitory activity against P gingivalis colonization, SAPP could also reduce the levels of several other oral Gram-negative bacteria strongly associated with periodontitis in multispecies biofilms. Our results provide a platform for the development of SAPP-targeted therapeutics against chronic periodontitis.
© 2018 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.
Ca/calmodulin-dependent protein kinase II (CaMKII) and metabotropic glutamate receptor 5 (mGlu) are critical signaling molecules in synaptic plasticity and learning/memory. Here, we demonstrate that mGlu is present in CaMKII complexes isolated from mouse forebrain. Further in vitro characterization showed that the membrane-proximal region of the C-terminal domain (CTD) of mGlu directly interacts with purified Thr286-autophosphorylated (activated) CaMKII However, the binding of CaMKII to this CTD fragment is reduced by the addition of excess Ca/calmodulin or by additional CaMKII autophosphorylation at non-Thr286 sites. Furthermore, in vitro binding of CaMKII is dependent on a tribasic residue motif Lys-Arg-Arg (KRR) at residues 866-868 of the mGlu-CTD, and mutation of this motif decreases the coimmunoprecipitation of CaMKII with full-length mGlu expressed in heterologous cells by about 50%. The KRR motif is required for two novel functional effects of coexpressing constitutively active CaMKII with mGlu in heterologous cells. First, cell-surface biotinylation studies showed that CaMKII increases the surface expression of mGlu Second, using Ca fluorimetry and single-cell Ca imaging, we found that CaMKII reduces the initial peak of mGlu-mediated Ca mobilization by about 25% while doubling the relative duration of the Ca signal. These findings provide new insights into the physical and functional coupling of these key regulators of postsynaptic signaling.
Copyright © 2018 by The American Society for Pharmacology and Experimental Therapeutics.
Computationally designed transmembrane α-helical peptides (CHAMP) have been used to compete for helix-helix interactions within the membrane, enabling the ability to probe the activation of the integrins αIIbβ3 and αvβ3. Here, this method is extended towards the design of CHAMP peptides that inhibit the association of the α5β1 transmembrane (TM) domains, targeting the Ala-X3-Gly motif within α5. Our previous design algorithm was performed alongside a new workflow implemented within the widely used Rosetta molecular modeling suite. Peptides from each computational approach activated integrin α5β1 but not αVβ3 in human endothelial cells. Two CHAMP peptides were shown to directly associate with an α5 TM domain peptide in detergent micelles to a similar degree as a β1 TM peptide does. By solution-state nuclear magnetic resonance, one of these CHAMP peptides was shown to bind primarily the integrin β1 TM domain, which itself has a Gly-X3-Gly motif. The second peptide associated modestly with both α5 and β1 constructs, with slight preference for α5. Although the design goal was not fully realized, this work characterizes novel CHAMP peptides activating α5β1 that can serve as useful reagents for probing integrin biology.
Proper morphogenesis of dendrites plays a fundamental role in the establishment of neural circuits. The molecular mechanism by which dendrites grow highly complex branches is not well understood. Here, using the Caenorhabditis elegans PVD neuron, we demonstrate that high-order dendritic branching requires actin polymerization driven by coordinated interactions between two membrane proteins, DMA-1 and HPO-30, with their cytoplasmic interactors, the RacGEF TIAM-1 and the actin nucleation promotion factor WAVE regulatory complex (WRC). The dendrite branching receptor DMA-1 directly binds to the PDZ domain of TIAM-1, while the claudin-like protein HPO-30 directly interacts with the WRC. On dendrites, DMA-1 and HPO-30 form a receptor-associated signaling complex to bring TIAM-1 and the WRC to close proximity, leading to elevated assembly of F-actin needed to drive high-order dendrite branching. The synergistic activation of F-actin assembly by scaffolding distinct actin regulators might represent a general mechanism in promoting complex dendrite arborization.
Copyright © 2018. Published by Elsevier Inc.
We recently reported the case of a young patient with multisystem failure carrying a de novo mutation in SLC12A2, the gene encoding the Na-K-2Cl cotransporter-1 (NKCC1). Heterologous expression studies in nonepithelial cells failed to demonstrate dominant-negative effects. In this study, we examined expression of the mutant cotransporter in epithelial cells. Using Madin-Darby canine kidney (MDCK) cells grown on glass coverslips, permeabilized support, and Matrigel, we show that the fluorescently tagged mutant cotransporter is expressed in cytoplasm and at the apical membrane and affects epithelium integrity. Expression of the mutant transporter at the apical membrane also results in the mislocalization of some of the wild-type transporter to the apical membrane. This mistargeting is specific to NKCC1 as the Na-K-ATPase remains localized on the basolateral membrane. To assess transporter localization in vivo, we created a mouse model using CRISPR/cas9 that reproduces the 11 bp deletion in exon 22 of Slc12a2. Although the mice do not display an overt phenotype, we show that the colon and salivary gland expresses wild-type NKCC1 abundantly at the apical pole, confirming the data obtained in cultured epithelial cells. Enough cotransporter must remain, however, on the basolateral membrane to participate in saliva secretion, as no significant decrease in saliva production was observed in the mutant mice.
UNC-8 and MEC-4 are two members of the degenerin/epithelial Na channel (DEG/ENaC) family of voltage-independent Na channels that share a high degree of sequence homology and functional similarity. For example, both can be hyperactivated by genetic mutations [UNC-8(d) and MEC-4(d)] that induce neuronal death by necrosis. Both depend in vivo on chaperone protein MEC-6 for function, as demonstrated by the finding that neuronal death induced by hyperactive UNC-8 and MEC-4 channels is prevented by null mutations in mec-6. UNC-8 and MEC-4 differ functionally in three major ways: 1) MEC-4 is calcium permeable, whereas UNC-8 is not; 2) UNC-8, but not MEC-4, is blocked by extracellular calcium and magnesium in the micromolar range; and 3) MEC-6 increases the number of MEC-4 channels at the cell surface in oocytes but does not have this effect on UNC-8. We previously reported that Capermeability of MEC-4 is conferred by the second transmembrane domain. We show here that the extracellular "finger" domain of UNC-8 is sufficient to mediate inhibition by divalent cations and that regulation by MEC-6 also depends on this region. Thus, our work confirms that the finger domain houses residues involved in gating of this channel class and shows for the first time that the finger domain also mediates regulation by chaperone protein MEC-6. Given that the finger domain is the most divergent region across the DEG/ENaC family, we speculate that it influences channel trafficking and function in a unique manner depending on the channel subunit.
Two genes encode the Na -K -2Cl cotransporters, NKCC1 and NKCC2, that mediate the tightly coupled movement of 1Na , 1K , and 2Cl across the plasma membrane of cells. Na -K -2Cl cotransport is driven by the chemical gradient of the three ionic species across the membrane, two of them maintained by the action of the Na /K pump. In many cells, NKCC1 accumulates Cl above its electrochemical potential equilibrium, thereby facilitating Cl channel-mediated membrane depolarization. In smooth muscle cells, this depolarization facilitates the opening of voltage-sensitive Ca channels, leading to Ca influx, and cell contraction. In immature neurons, the depolarization due to a GABA-mediated Cl conductance produces an excitatory rather than inhibitory response. In many cell types that have lost water, NKCC is activated to help the cells recover their volume. This is specially the case if the cells have also lost Cl . In combination with the Na /K pump, the NKCC's move ions across various specialized epithelia. NKCC1 is involved in Cl -driven fluid secretion in many exocrine glands, such as sweat, lacrimal, salivary, stomach, pancreas, and intestine. NKCC1 is also involved in K -driven fluid secretion in inner ear, and possibly in Na -driven fluid secretion in choroid plexus. In the thick ascending limb of Henle, NKCC2 activity in combination with the Na /K pump participates in reabsorbing 30% of the glomerular-filtered Na . Overall, many critical physiological functions are maintained by the activity of the two Na -K -2Cl cotransporters. In this overview article, we focus on the functional roles of the cotransporters in nonpolarized cells and in epithelia. © 2018 American Physiological Society. Compr Physiol 8:871-901, 2018.
Copyright © 2018 American Physiological Society. All rights reserved.
Highly effective HIV-1-neutralizing antibodies could have utility in the prevention or treatment of HIV-1 infection. To improve the potency of 10E8, an antibody capable of near pan-HIV-1 neutralization, we engineered 10E8-surface mutants and screened for improved neutralization. Variants with the largest functional enhancements involved the addition of hydrophobic or positively charged residues, which were positioned to interact with viral membrane lipids or viral glycan-sialic acids, respectively. In both cases, the site of improvement was spatially separated from the region of antibody mediating molecular contact with the protein component of the antigen, thereby improving peripheral semi-specific interactions while maintaining unmodified dominant contacts responsible for broad recognition. The optimized 10E8 antibody, with mutations to phenylalanine and arginine, retained the extraordinary breadth of 10E8 but with ∼10-fold increased potency. We propose surface-matrix screening as a general method to improve antibodies, with improved semi-specific interactions between antibody and antigen enabling increased potency without compromising breadth.
Published by Elsevier Inc.
Poly(lactic-co-glycolic acid) (PLGA) is widely used as a vehicle for delivery of pharmaceutically relevant payloads. PLGA is readily fabricated as a nano- or microparticle (MP) matrix to load both hydrophobic and hydrophilic small molecular drugs as well as biomacromolecules such as nucleic acids and proteins. However, targeting such payloads to the cell cytosol is often limited by MP entrapment and degradation within acidic endolysosomes. Poly(propylacrylic acid) (PPAA) is a polyelectrolyte polymer with the membrane disruptive capability triggered at low pH. PPAA has been previously formulated in various carrier configurations to enable cytosolic payload delivery, but requires sophisticated carrier design. Taking advantage of PPAA functionality, we have incorporated PPAA into PLGA MPs as a simple polymer mixture to enhance cytosolic delivery of PLGA-encapsulated payloads. Rhodamine loaded PLGA and PPAA/PLGA blend MPs were prepared by a modified nanoprecipitation method. Incorporation of PPAA into PLGA MPs had little to no effect on the size, shape, or loading efficiency, and evidenced no toxicity in Chinese hamster ovary epithelial cells. Notably, incorporation of PPAA into PLGA MPs enabled pH-dependent membrane disruption in a hemolysis assay, and a three-fold increased endosomal escape and cytosolic delivery in dendritic cells after 2 h of MP uptake. These results demonstrate that a simple PLGA/PPAA polymer blend is readily fabricated into composite MPs, enabling cytosolic delivery of an encapsulated payload. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 1022-1033, 2018.
© 2017 Wiley Periodicals, Inc.