TCL/RhoJ Plasma Membrane Localization and Nucleotide Exchange Is Coordinately Regulated by Amino Acids within the N Terminus and a Distal Loop Region.

TCL/RhoJ is a Cdc42-related Rho GTPase with reported activities in endothelial cell biology and angiogenesis, metastatic melanoma, and corneal epithelial cells; however, less is known about how it is inherently regulated in comparison to its closest homologues TC10 and Cdc42. TCL has an N-terminal extension of 18 amino acids in comparison to Cdc42, but the function of this amino acid sequence has not been elucidated. A truncation mutant lacking the N terminus (ΔN) was found to alter TCL plasma membrane localization and nucleotide binding, and additional truncation and point mutants mapped the alterations of TCL biochemistry to amino acids 17-20. Interestingly, whereas the TCL ΔN mutant clearly influenced nucleotide exchange, deletion of the N terminus from its closest homologue, TC10, did not have a similar effect. Chimeras of TCL and TC10 revealed amino acids 121-129 of TCL contributed to the differences in nucleotide loading. Together, these results identify amino acids within the N terminus and a loop region distal to the nucleotide binding pocket of TCL capable of allosterically regulating nucleotide exchange and thus influence membrane association of the protein.

Dedicator of cytokinesis 8 interacts with talin and Wiskott-Aldrich syndrome protein to regulate NK cell cytotoxicity.

Recently, patients with mutations in DOCK8 have been reported to have a combined immunodeficiency characterized by cutaneous viral infections and allergies. NK cells represent a first-line defense against viral infections, suggesting that DOCK8 might participate in NK cell function. In this study, we demonstrate that DOCK8-suppressed human NK cells showed defects in natural cytotoxicity as well as specific activating receptor-mediated NK cytotoxicity. Additionally, compared with control NK cells, NK cells depleted of DOCK8 showed defective conjugate formation, along with decreased polarization of LFA-1, F-actin, and cytolytic granules toward the cytotoxic synapse. Using a proteomic approach, we found that DOCK8 exists in a macromolecular complex with the Wiskott-Aldrich syndrome protein, an actin nucleation-promoting factor activated by CDC42, as well as talin, which is required for integrin-mediated adhesion. Taken together, our results demonstrate an important role for DOCK8 in NK cell effector function and provide important new mechanistic insight into how DOCK8 regulates F-actin and integrin-mediated adhesion in immune cells.

Detection of Rho GEF and GAP activity through a sensitive split luciferase assay system.

Rho GTPases regulate the assembly of cellular actin structures and are activated by GEFs (guanine-nucleotide-exchange factors) and rendered inactive by GAPs (GTPase-activating proteins). Using the Rho GTPases Cdc42, Rac1 and RhoA, and the GTPase-binding portions of the effector proteins p21-activated kinase and Rhophilin1, we have developed split luciferase assays for detecting both GEF and GAP regulation of these GTPases. The system relies on purifying split luciferase fusion proteins of the GTPases and effectors from bacteria, and our results show that the assays replicate GEF and GAP specificities at nanomolar concentrations for several previously characterized Rho family GEFs (Dbl, Vav2, Trio and Asef) and GAPs [p190, Cdc42 GAP and PTPL1-associated RhoGAP]. The assay detected activities associated with purified recombinant GEFs and GAPs, cell lysates expressing exogenous proteins, and immunoprecipitates of endogenous Vav1 and p190. The results demonstrate that the split luciferase system provides an effective sensitive alternative to radioactivity-based assays for detecting GTPase regulatory protein activities and is adaptable to a variety of assay conditions.

Effects of on-Table Extubation after Pediatric Cardiac Surgery.

Background: Enhanced recovery after surgery (ERAS) protocols are utilizing a multidisciplinary approach, reassessing physiology to improve clinical outcomes, reducing length of hospital stay (LOS) stay, resulting in cost reduction. Since its introduction in colorectal surgery. the concept has been utilized in various fields and benefits have been recognized also in adult cardiac surgery. However, ERAS concepts in pediatric cardiac surgery are not yet widely established. Therefore, the aim of the present study was to assess the effects of on-table extubation (OTE) after pediatric cardiac surgery compared to the standard approach of delayed extubation (DET) during intensive care treatment. Study Design and Methods: We performed a retrospective analysis of all pediatric cardiac surgery cases performed in children below the age of two years using cardiopulmonary bypass at our institution in 2021. Exclusion criteria were emergency and off pump surgeries as well as children already ventilated preoperatively. Results: OTE children were older (267.3 days vs. 126.7 days, p < 0.001), had a higher body weight (7.0 ± 1.6 kg vs. 4.9 ± 1.9 kg, p < 0.001), showed significantly reduced duration of ICU treatment (75.9 ± 56.8 h vs. 217.2 ± 211.4 h, p < 0.001) and LOS (11.1 ± 10.2 days vs. 20.1 ± 23.4 days; p = 0.001) compared to DET group. Furthermore, OTE children had significantly fewer catecholamine dependencies at 12-, 24-, 48-, and 72-h post-surgery, while DET children showed a significantly increased intrafluid shift relative to body weight (109.1 ± 82.0 mL/kg body weight vs. 63.0 ± 63.0 mL/kg body weight, p < 0.001). After propensity score matching considering age, weight, bypass duration, Society of Thoracic Surgeons-European Association for Cardio-Thoracic Surgery Mortality (STATS)-Score, and the outcome variables, including duration of ICU treatment, catecholamine dependencies, and hospital LOS, findings significantly favored the OTE group. Conclusion: Our results suggest that on-table extubation after pediatric cardiac surgery is feasible and in our cohort was associated with a favorable postoperative course.

Asef2 functions as a Cdc42 exchange factor and is stimulated by the release of an autoinhibitory module from a concealed C-terminal activation element.

Asef (herein called Asef1) was identified as a Rac1-specific exchange factor stimulated by adenomatous polyposis coli (APC), contributing to colorectal cancer cell metastasis. We investigated Asef2, an Asef1 homologue having a similar N-terminal APC binding region (ABR) and Src-homology 3 (SH3) domain. Contrary to previous reports, we found that Asef1 and Asef2 exchange activity is Cdc42 specific. Moreover, the ABR of Asef2 did not function independently but acted in tandem with the SH3 domain to bind APC. The ABRSH3 also bound the C-terminal tail of Asef2, allowing it to function as an autoinhibitory module within the protein. Deletion of the C-terminal tail did not constitutively activate Asef2 as predicted; rather, a conserved C-terminal segment was required for augmented Cdc42 GDP/GTP exchange. Thus, Asef2 activation involves APC releasing the ABRSH3 from the C-terminal tail, resulting in Cdc42 exchange. These results highlight a novel exchange factor regulatory mechanism and establish Asef1 and Asef2 as Cdc42 exchange factors, providing a more appropriate context for understanding the contribution of APC in establishing cell polarity and migration.

Unraveling a model of TCL/RhoJ allosterism using TC10 reverse chimeras.

In addition to the classical regulation of GTPase activity by effector proteins, investigating the variations in the amino acid sequence and structures of GTPases often provides insights into regulatory mechanisms that are more GTPase-specific. TCL/RhoJ is a Rho GTPase most closely related to Cdc42 and TC10; however, its nucleotide exchange activity is distinctly influenced by N-terminal amino acids 17-20 and the more distal amino acids 121-129. In this short study, we have further explored the differences between TCL and its homolog TC10 and show that its unique mode of allosteric regulation requires broader diversification of its amino acid sequence than previously appreciated.

Generating realistic scaled complex networks.

Research on generative models plays a central role in the emerging field of network science, studying how statistical patterns found in real networks could be generated by formal rules. Output from these generative models is then the basis for designing and evaluating computational methods on networks including verification and simulation studies. During the last two decades, a variety of models has been proposed with an ultimate goal of achieving comprehensive realism for the generated networks. In this study, we (a) introduce a new generator, termed ReCoN; (b) explore how ReCoN and some existing models can be fitted to an original network to produce a structurally similar replica, (c) use ReCoN to produce networks much larger than the original exemplar, and finally (d) discuss open problems and promising research directions. In a comparative experimental study, we find that ReCoN is often superior to many other state-of-the-art network generation methods. We argue that ReCoN is a scalable and effective tool for modeling a given network while preserving important properties at both micro- and macroscopic scales, and for scaling the exemplar data by orders of magnitude in size.

Formation of disulfide bond in p53 correlates with inhibition of DNA binding and tetramerization.

The p53 tumor suppressor protein is susceptible to oxidation, which prevents it from binding to its DNA response element. The goal of the current research was to determine the nature of the cysteine residue thiol oxidation that prevents p53 from binding its DNA target and its effect on p53 structure. Recombinant p53, purified in the presence of the reducing agent dithiothreitol (DTT), contains five free thiol groups on the surface of the protein. In the absence of DTT, p53 contains only four thiol groups, indicating that an average of one surface thiol group is readily susceptible to oxidation. Sulfite-mediated disulfide bond cleavage followed by reaction with 2-nitro-5-thiosulfobenzoate showed that oxidized p53 contains a single disulfide bond per monomer. By atomic force microscopy, we determined that reduced p53 binds to a double-stranded DNA containing the p53 promoter element of the MDM2 gene. The DNA-bound reduced p53 has an average cross-sectional diameter of 8.61 nm and a height of 4.12 nm. The amount of oxidized p53 that bound to the promoter element was ninefold lower, and it has an 18% larger average cross-sectional diameter. Electromobility shift assays showed that binding of oxidized p53 to DNA was enhanced upon addition of DTT, indicating that oxidation is reversible. The possibility that oxidized p53 contained significant amounts of sulfenic (-SOH), sulfinic (-SO2H), or sulfonic acid (-SO3H) was ruled out. Gel filtration chromatography indicated that oxidation increases the percentage of p53 monomers and high-molecular-weight oligomers (>1,000 kDa) relative to tetrameric p53. Protein modeling studies suggest that a mixed disulfide glutathione adduct on Cys182 could account for the observed stoichiometry of oxidized thiols and structural changes. The glutathione adduct may prevent proper helix-helix interaction within the DNA binding domain and contribute to tetramer dissociation.

Vav links the T cell antigen receptor to the actin cytoskeleton and T cell activation independently of intrinsic Guanine nucleotide exchange activity.

BACKGROUND: T cell receptor (TCR) engagement leads to formation of signaling microclusters and induction of rapid and dynamic changes in the actin cytoskeleton, although the exact mechanism by which the TCR initiates actin polymerization is incompletely understood. The Vav family of guanine nucleotide exchange factors (GEF) has been implicated in generation of TCR signals and immune synapse formation, however, it is currently not known if Vav's GEF activity is required in T cell activation by the TCR in general, and in actin polymerization downstream of the TCR in particular. METHODOLOGY/PRINCIPAL FINDINGS: Here, we report that Vav1 assembles into signaling microclusters at TCR contact sites and is critical for TCR-initiated actin polymerization. Surprisingly, Vav1 functions in TCR signaling and Ca(++) mobilization via a mechanism that does not appear to strictly depend on the intrinsic GEF activity. CONCLUSIONS/SIGNIFICANCE: We propose here a model in which Vav functions primarily as a tyrosine phosphorylated linker-protein for TCR activation of T cells. Our results indicate that, contrary to expectations based on previously published studies including from our own laboratory, pharmacological inhibition of Vav1's intrinsic GEF activity may not be an effective strategy for T cell-directed immunosuppressive therapy.

Vav1 acidic region tyrosine 174 is required for the formation of T cell receptor-induced microclusters and is essential in T cell development and activation.

Vav proteins are multidomain signaling molecules critical for mediating signals downstream of several surface receptors, including the antigen receptors of T and B lymphocytes. The catalytic guanine nucleotide exchange factor (GEF) activity of the Vav Dbl homology (DH) domain is thought to be controlled by an intramolecular autoinhibitory mechanism involving an N-terminal extension and phosphorylation of tyrosine residues in the acidic region (AC). Here, we report that the sequences surrounding the Vav1 AC: Tyr(142), Tyr(160), and Tyr(174) are evolutionarily conserved, conform to consensus SH2 domain binding motifs, and bind several proteins implicated in TCR signaling, including Lck, PI3K p85alpha, and PLCgamma1, through direct interactions with their SH2 domains. In addition, the AC tyrosines regulate tyrosine phosphorylation of Vav1. We also show that Tyr(174) is required for the maintenance of TCR-signaling microclusters and for normal T cell development and activation. In this regard, our data demonstrate that while Vav1 Tyr(174) is essential for maintaining the inhibitory constraint of the DH domain in both developing and mature T cells, constitutively activated Vav GEF disrupts TCR-signaling microclusters and leads to defective T cell development and proliferation.

Dynamin 2 regulates T cell activation by controlling actin polymerization at the immunological synapse.

Actin reorganization at the immunological synapse is required for the amplification and generation of a functional immune response. Using small interfering RNA, we show here that dynamin 2 (Dyn2), a large GTPase involved in receptor-mediated internalization, did not alter antibody-mediated T cell receptor internalization but considerably affected T cell receptor-stimulated T cell activation by regulating multiple biochemical signaling pathways and the accumulation of F-actin at the immunological synapse. Moreover, Dyn2 interacted directly with the Rho family guanine nucleotide exchange factor Vav1, and this interaction was required for T cell activation. These data identify a functionally important interaction between Dyn2 and Vav1 that regulates actin reorganization and multiple signaling pathways in T lymphocytes.

Irreversible thiol oxidation in carbonic anhydrase III: protection by S-glutathiolation and detection in aging rats.

Proteins with reactive sulfhydryls are central to many important metabolic reactions and also contribute to a variety of signal transduction systems. In this report, we examine the mechanisms of oxidative damage to the two reactive sulfhydryls of carbonic anhydrase III. Hydrogen peroxide (H2O2), peroxy radicals, or hypochlorous acid (HOCl) produced irreversibly oxidized forms, primarily cysteine sulfinic acid or cysteic acid, of carbonic anhydrase III if glutathione (GSH) was not present. When GSH was approximately equimolar to protein thiols, irreversible oxidation was prevented. H202 and peroxyl radicals both generated S-glutathiolated carbonic anhydrase III via partially oxidized protein sulfhydryl intermediates, while HOCl did not cause S-glutathiolation. Thus, oxidative damage from H202 or AAPH was prevented by protein S-glutathiolation, while a direct reaction between GSH and oxidant likely prevents HOCl-mediated protein damage. In cultured rat hepatocytes, carbonic anhydrase III was rapidly S-glutathiolated by menadione. When hepatocyte glutathione was depleted, menadione instead caused irreversible oxidation. We hypothesized that normal depletion of glutathione in aged animals might also lead to an increase in irreversible oxidation. Indeed, both total protein extracts and carbonic anhydrase III contained significantly more cysteine sulfinic acid in older rats compared to young animals. These experiments show that, in the absence of sufficient GSH, oxidation reactions lead to irreversible protein sulfhydryl damage in purified proteins, cellular systems, and whole animals.