USP10 Targeted Self-Deliverable siRNA to Prevent Scarring in the Cornea.

Ocular scarring after surgery, trauma, or infection leads to vision loss. The transparent cornea is an excellent model system to test anti-scarring therapies. Cholesterol-conjugated fully modified asymmetric small interfering RNAs (siRNAs) (self-deliverable siRNAs [sdRNAs]) are a novel modality for in vivo gene knockdown, transfecting cells and tissues without any additional formulations. Myofibroblasts are a main contributor to scarring and fibrosis. αv integrins play a central role in myofibroblast pathological adhesion, overcontraction, and transforming growth factor ß (TGF-ß) activation. Previously, we demonstrated that αv integrins are protected from intracellular degradation after wounding by upregulation of the deubiquitinase (DUB) ubiquitin-specific protease 10 (USP10), leading to integrin cell surface accumulation. In this study, we tested whether knockdown of USP10 with a USP10-targeting sdRNA (termed US09) will reduce scarring after wounding a rabbit cornea in vivo. The wounded corneal stroma was treated once with US09 or non-targeting control (NTC) sdRNA. At 6 weeks US09 treatment resulted in faster wound closure, limited scarring, and suppression of fibrotic markers and immune response. Specifically, fibronectin-extra domain A (EDA), collagen III, and a-smooth muscle actin (p < 0.05), CD45+ cell infiltration (p < 0.01), and apoptosis at 24 (p < 0.01) and 48 h (p < 0.05) were reduced post-wounding. Corneal thickness and cell proliferation were restored to unwounded parameters. Targeting the DUB, USP10 is a novel strategy to reduce scarring. This study indicates that ubiquitin-mediated pathways should be considered in the pathogenesis of fibrotic healing.

Rapid, directed transport of DC-SIGN clusters in the plasma membrane.

C-type lectins, including dendritic cell-specific intercellular adhesion molecule-3-grabbing nonintegrin (DC-SIGN), are all-purpose pathogen receptors that exist in nanoclusters in plasma membranes of dendritic cells. A small fraction of these clusters, obvious from the videos, can undergo rapid, directed transport in the plane of the plasma membrane at average speeds of more than 1 µm/s in both dendritic cells and MX DC-SIGN murine fibroblasts ectopically expressing DC-SIGN. Surprisingly, instantaneous speeds can be considerably greater. In MX DC-SIGN cells, many cluster trajectories are colinear with microtubules that reside close to the ventral membrane, and the microtubule-depolymerizing drug, nocodazole, markedly reduced the areal density of directed movement trajectories, suggesting a microtubule motor-driven transport mechanism; by contrast, latrunculin A, which affects the actin network, did not depress this movement. Rapid, retrograde movement of DC-SIGN may be an efficient mechanism for bringing bound pathogen on the leading edge and projections of dendritic cells to the perinuclear region for internalization and processing. Dengue virus bound to DC-SIGN on dendritic projections was rapidly transported toward the cell center. The existence of this movement within the plasma membrane points to an unexpected lateral transport mechanism in mammalian cells and challenges our current concepts of cortex-membrane interactions.

Beyond attachment: Roles of DC-SIGN in dengue virus infection.

Dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN), a C-type lectin expressed on the plasma membrane by human immature dendritic cells, is a receptor for numerous viruses including Ebola, SARS and dengue. A controversial question has been whether DC-SIGN functions as a complete receptor for both binding and internalization of dengue virus (DENV) or whether it is solely a cell surface attachment factor, requiring either hand-off to another receptor or a co-receptor for internalization. To examine this question, we used 4 cell types: human immature dendritic cells and NIH3T3 cells expressing either wild-type DC-SIGN or 2 internalization-deficient DC-SIGN mutants, in which either the 3 cytoplasmic internalization motifs are silenced by alanine substitutions or the cytoplasmic region is truncated. Using confocal and super-resolution imaging and high content single particle tracking, we investigated DENV binding, DC-SIGN surface transport, endocytosis, as well as cell infectivity. DC-SIGN was found colocalized with DENV inside cells suggesting hand-off at the plasma membrane to another receptor did not occur. Moreover, all 3 DC-SIGN molecules on NIH3T3 cells supported cell infection. These results imply the involvement of a co-receptor because cells expressing the internalization-deficient mutants could still be infected.

Identification of the dimer interface of a bacterial Ca(2+)/H(+) antiporter.

Members of the calcium/cation antiporter superfamily, including the cardiac sodium/calcium exchangers, are secondary active transporters that play an essential role in cellular Ca(2+) homeostasis. A notable feature of this group of transporters is the high levels of sequence similarity in relatively short sequences constituting the functionally important α-1 and α-2 regions in contrast to relatively lower degrees of similarity in the extended adjoining sequences. This suggests a similar structure and function of core transport machinery but possible differences in topology and/or oligomerization, a topic that has not been adequately addressed. Here we present the first example of purification of a bacterial member of this superfamily (CAX(CK31)) and analyze its quaternary structure. Purification of CAX(CK31) required the presence of a choline headgroup-containing detergent or lipid to yield stable preparations of the monomeric transporter. H(+)-driven Ca(2+) transport was demonstrated by reconstituting purified CAX(CK31) into liposomes. Dimeric CAX(CK31) could be isolated by manipulation of detergent micelles. Dimer formation was shown to be dependent on micelle composition as well as protein concentration. Furthermore, we establish that CAX(CK31) forms dimers in the membrane by analysis of cross-linked proteins. Using a dimeric homology model derived from the monomeric structure of the archaeal NCX homologue (Protein Data Bank entry 3V5U ), we introduced cysteine residues and through cross-linking experiments established the role of transmembrane helices 2 and 6 in the putative dimer interface.

Restrained expression, a method to overproduce toxic membrane proteins by exploiting operator-repressor interactions.

A major rate-limiting step in determining structures of membrane proteins is heterologous protein production. Toxicity often associated with rapid overexpression results in reduced biomass along with low yields of target protein. Mitigation of toxic effects was achieved using a method we call "restrained expression," a controlled reduction in the frequency of transcription initiation by exploiting the infrequent transitions of Lac repressor to a free state from its complex with the lac-operator site within a T7lac promoter that occur in the absence of the inducer isopropyl ß-D-1-thiogalactopyranoside. In addition, production of the T7 RNA polymerase that drives transcription of the target is limited using the tightly regulated arabinose promoter in Escherichia coli strain BL21-AI. Using this approach, we can achieve a 200-fold range of green fluorescent protein expression levels. Application to members of a family of ion pumps results in 5- to 25-fold increases in expression over the benchmark BL21(DE3) host strain. A viral ion channel highly toxic to E. coli can also be overexpressed. In comparative analyses, restrained expression outperforms commonly used E. coli expression strategies. The mechanism underlying improved target protein yield arises from minimization of protein aggregation and proteolysis that reduce membrane integrity and cell viability. This study establishes a method to overexpress toxic proteins.

Structure of Galphaq-p63RhoGEF-RhoA complex reveals a pathway for the activation of RhoA by GPCRs.

The guanine nucleotide exchange factor p63RhoGEF is an effector of the heterotrimeric guanine nucleotide-binding protein (G protein) Galphaq and thereby links Galphaq-coupled receptors (GPCRs) to the activation of the small-molecular-weight G protein RhoA. We determined the crystal structure of the Galphaq-p63RhoGEF-RhoA complex, detailing the interactions of Galphaq with the Dbl and pleckstrin homology (DH and PH) domains of p63RhoGEF. These interactions involve the effector-binding site and the C-terminal region of Galphaq and appear to relieve autoinhibition of the catalytic DH domain by the PH domain. Trio, Duet, and p63RhoGEF are shown to constitute a family of Galphaq effectors that appear to activate RhoA both in vitro and in intact cells. We propose that this structure represents the crux of an ancient signal transduction pathway that is expected to be important in an array of physiological processes.