Functional defects of SKAP-55-deficient T cells identify a regulatory role for the adaptor in LFA-1 adhesion

The ADAP-SKAP-55 module regulates T-cell receptor (TCR)-induced integrin clustering and adhesion in T cells. However, it has been unclear whether ADAP and/or SKAP-55 is an effector of the response. ADAP controls SKAP-55 expression such that ADAP(-/-) T cells are also deficient in SKAP-55 expression. In this study, we report the phenotype of the SKAP-55-deficient mouse. SKAP-55(-/-) T cells retain ADAP expression yet show defects in beta1 and beta2 integrin adhesion, leukocyte function-associated antigen 1 (LFA-1) clustering, production of the cytokines interleukin-2 and gamma interferon, and proliferation. This dependency was also reflected in more-transient conjugation times in response to the superantigen staphylococcal enterotoxin A on dendritic cells and a reduced number of cells with TCR/CD3 microcluster localization at the immunological synapse. SKAP-55(-/-) T cells showed the same general impairment of function as ADAP(-/-) T cells, indicating that SKAP-55 is an effector of the ADAP-SKAP-55 module. At the same time, the requirement for ADAP and SKAP-55 was not absolute, since a subset of peripheral T cells adhered with loss of expression of either adaptor. Further, dependency on SKAP-55 or ADAP differed with the strength of the TCR signal. As with the ADAP(-/-) mouse, SKAP-55-deficient mice showed no major effects on lymphoid development or the appearance of peripheral T cells, B cells, and NK cells. Our findings identify a clear effector role for SKAP-55 in LFA-1 adhesion in peripheral T cells and demonstrate that dependency on SKAP-55 and ADAP differs among T cells and differs with the strength of the TCR signal.     

Reactions of alkyl and aryl sulfides with a monocarbon platinacarbaborane anion

The reaction of the monocarbon carbaborane complex Na[Pt(PEt₃)₂(η⁵-CB₁₀H₁₁)] with some diaryl- and dialkyl disulfides has been investigated. With Ph₂S₂, two new cage substituted products are formed, [Pt(SPh)(PEt₃)(η⁵-9-SPh-7-CB₁₀H₁₀)] (1) and [Pt(SPh)(PEt₃)(η⁵-8-SPh-11-SPh-7-CB₁₀H₉)] (2), whereas with S₂ the main product is the metal substituted complex, [Pt(SBu^t)(PEt₃)(η⁵-7-CB₁₀H₁₁)] (4). All three new molecules have been identified spectroscopically (¹H, ¹³C, ³¹P, ¹¹B NMR) and through single crystal X-ray diffraction.     

Next-Generation Sequencing of Duplication CNVs Reveals that Most Are Tandem and Some Create Fusion Genes at Breakpoints

Interpreting the genomic and phenotypic consequences of copy-number variation (CNV) is essential to understanding the etiology of genetic disorders. Whereas deletion CNVs lead obviously to haploinsufficiency, duplications might cause disease through triplosensitivity, gene disruption, or gene fusion at breakpoints. The mutational spectrum of duplications has been studied at certain loci, and in some cases these copy-number gains are complex chromosome rearrangements involving triplications and/or inversions. However, the organization of clinically relevant duplications throughout the genome has yet to be investigated on a large scale. Here we fine-mapped 184 germline duplications (14.7 kb–25.3 Mb; median 532 kb) ascertained from individuals referred for diagnostic cytogenetics testing. We performed next-generation sequencing (NGS) and whole-genome sequencing (WGS) to sequence 130 breakpoints from 112 subjects with 119 CNVs and found that most (83%) were tandem duplications in direct orientation. The remainder were triplications embedded within duplications (8.4%), adjacent duplications (4.2%), insertional translocations (2.5%), or other complex rearrangements (1.7%). Moreover, we predicted six in-frame fusion genes at sequenced duplication breakpoints; four gene fusions were formed by tandem duplications, one by two interconnected duplications, and one by duplication inserted at another locus. These unique fusion genes could be related to clinical phenotypes and warrant further study. Although most duplications are positioned head-to-tail adjacent to the original locus, those that are inverted, triplicated, or inserted can disrupt or fuse genes in a manner that might not be predicted by conventional copy-number assays. Therefore, interpreting the genetic consequences of duplication CNVs requires breakpoint-level analysis.     

Gene expression changes in GABA(A) receptors and cognition following chronic ketamine administration in mice

Ketamine is a well-known anesthetic agent and a drug of abuse. Despite its widespread use and abuse, little is known about its long-term effects on the central nervous system. The present study was designed to evaluate the effect of long-term (1- and 3-month) ketamine administration on learning and memory and associated gene expression levels in the brain. The Morris water maze was used to assess spatial memory and gene expression changes were assayed using Affymetrix Genechips; a focus on the expression of GABA(A) receptors that mediate a tonic inhibition in the brain, was confirmed by quantitative real-time PCR and western blot. Compared with saline controls, there was a decline in learning and memory performance in the ketamine-treated mice. Genechip results showed that 110 genes were up-regulated and 136 genes were down-regulated. An ontology analysis revealed the most significant effects of ketamine were on GABA(A) receptors. In particular, there was a significant up-regulation of both mRNA and protein levels of the alpha 5 subunit (Gabra5) of the GABA(A) receptors in the prefrontal cortex. In conclusion, chronic exposure to ketamine impairs working memory in mice, which may be explained at least partly by up-regulation of Gabra5 subunits in the prefrontal cortex.     

Estimates of outcomes up to ten years after stroke: analysis from the prospective South London Stroke Register

Background

Although stroke is acknowledged as a long-term condition, population estimates of outcomes longer term are lacking. Such estimates would be useful for planning health services and developing research that might ultimately improve outcomes. This burden of disease study provides population-based estimates of outcomes with a focus on disability, cognition, and psychological outcomes up to 10 y after initial stroke event in a multi-ethnic European population.

Methods and Findings

Data were collected from the population-based South London Stroke Register, a prospective population-based register documenting all first in a lifetime strokes since 1 January 1995 in a multi-ethnic inner city population. The outcomes assessed are reported as estimates of need and included disability (Barthel Index <15), inactivity (Frenchay Activities Index <15), cognitive impairment (Abbreviated Mental Test < 8 or Mini-Mental State Exam <24), anxiety and depression (Hospital Anxiety and Depression Scale >10), and mental and physical domain scores of the Medical Outcomes Study 12-item short form (SF-12) health survey. Estimates were stratified by age, gender, and ethnicity, and age-adjusted using the standard European population. Plots of outcome estimates over time were constructed to examine temporal trends and sociodemographic differences. Between 1995 and 2006, 3,373 first-ever strokes were registered: 20%–30% of survivors had a poor outcome over 10 y of follow-up. The highest rate of disability was observed 7 d after stroke and remained at around 110 per 1,000 stroke survivors from 3 mo to 10 y. Rates of inactivity and cognitive impairment both declined up to 1 y (280/1,000 and 180/1,000 survivors, respectively); thereafter rates of inactivity remained stable till year eight, then increased, whereas rates of cognitive impairment fluctuated till year eight, then increased. Anxiety and depression showed some fluctuation over time, with a rate of 350 and 310 per 1,000 stroke survivors, respectively. SF-12 scores showed little variation from 3 mo to 10 y after stroke. Inactivity was higher in males at all time points, and in white compared to black stroke survivors, although black survivors reported better outcomes in the SF-12 physical domain. No other major differences were observed by gender or ethnicity. Increased age was associated with higher rates of disability, inactivity, and cognitive impairment.

Conclusions

Between 20% and 30% of stroke survivors have a poor range of outcomes up to 10 y after stroke. Such epidemiological data demonstrate the sociodemographic groups that are most affected longer term and should be used to develop longer term management strategies that reduce the significant poor outcomes of this group, for whom effective interventions are currently elusive. Please see later in the article for the Editors'' Summary     

The identification and characterization of xenoantigenic nonhuman carbohydrate sequences in membrane proteins from porcine kidney

The immunogenic nonhuman carbohydrate sequences in membrane proteins from porcine kidney were identified and characterized using MALDI-TOF MS and ESI-QTOF-MS. The MALDI profile, investigated by incubation with exoglycosidases, showed a series of about 40 carbohydrates that were identified as high mannose glycans (Man(3-9)GlcNAc2) and complex bi-, tri-, and tetra-antennary glycans with and without core fucose. The antennae of many of the complex glycans were terminated with alpha-galactose residues, with the numbers of these residues ranging from one up to the number of antennae. Negative ion ESI-MS/MS spectra confirmed the location of the alpha-galactose residues on the ends of the antennae. This total glycan profile of the membrane proteins from porcine kidney will thus provide important information for the study of molecular interactions between antigenic carbohydrates and proteins in xenotransplantation.     

Sialylation of urinary prothrombin fragment 1 is implicated as a contributory factor in the risk of calcium oxalate kidney stone formation

Urinary glycoproteins are important inhibitors of calcium oxalate crystallization and adhesion of crystals to renal cells, both of which are key mechanisms in kidney stone formation. This has been attributed to glycosylation of the proteins. In South Africa, the black population rarely form stones (incidence < 1%) compared with the white population (incidence 12-15%). A previous study involving urinary prothrombin fragment 1 from both populations demonstrated superior inhibitory activity associated with the protein from the black group. In the present study, we compared N-linked and O-linked oligosaccharides released from urinary prothrombin fragment 1 isolated from the urine of healthy and stone-forming subjects in both populations to elucidate the relationship between glycosylation and calcium oxalate stone pathogenesis. The O-glycans of both control groups and the N-glycans of the black control samples were significantly more sialylated than those of the white stone-formers. This demonstrates a possible association between low-percentage sialylation and kidney stone disease and provides a potential diagnostic method for a predisposition to kidney stones that could lead to the implementation of a preventative regimen. These results indicate that sialylated glycoforms of urinary prothrombin fragment 1 afford protection against calcium oxalate stone formation, possibly by coating the surface of calcium oxalate crystals. This provides a rationale for the established roles of urinary prothrombin fragment 1, namely reducing the potential for crystal aggregation and inhibiting crystal-cell adhesion by masking the interaction of the calcium ions on the crystal surface with the renal cell surface along the nephron.     

Late Domain-Independent Rescue of a Release-Deficient Moloney Murine Leukemia Virus by the Ubiquitin Ligase Itch

Moloney murine leukemia virus (MoMLV) Gag utilizes its late (L) domain motif PPPY to bind members of the Nedd4-like ubiquitin ligase family. These interactions recruit components of the cell''s budding machinery that are critical for virus release. MoMLV Gag contains two additional L domains, PSAP and LYPAL, that are believed to drive residual MoMLV release via interactions with cellular proteins Tsg101 and Alix, respectively. We found that overexpression of Tsg101 or Alix failed to rescue the release of PPPY-deficient MoMLV via these other L domains. However, low-level expression of the ubiquitin ligase Itch potently rescued the release and infectivity of MoMLV lacking PPPY function. In contrast, other ubiquitin ligases such as WWP1, Nedd4.1, Nedd4.2, and Nedd4.2s did not rescue this release-deficient virus. Efficient rescue required the ubiquitin ligase activity of Itch and an intact C2 domain but not presence of the endophilin-binding site. Additionally, we found Itch to immunoprecipitate with MoMLV Gag lacking the PPPY motif and to be incorporated into rescued MoMLV particles. The PSAP and LYPAL motifs were dispensable for Itch-mediated virus rescue, and their absence did not affect the incorporation of Itch into the rescued particles. Itch-mediated rescue of release-defective MoMLV was sensitive to inhibition by dominant-negative versions of ESCRT-III components and the VPS4 AAA ATPase, indicating that Itch-mediated correction of MoMLV release defects requires the integrity of the host vacuolar sorting protein pathway. RNA interference knockdown of Itch suppressed the residual release of the MoMLV lacking the PPPY motif. Interestingly, Itch stimulation of the PPPY-deficient MoMLV release was accompanied by the enhancement of Gag ubiquitination and the appearance of new ubiquitinated Gag proteins in virions. Together, these results suggest that Itch can facilitate MoMLV release in an L domain-independent manner via a mechanism that requires the host budding machinery and involves Gag ubiquitination.Retroviruses require access to the host budding machinery to exit the cell (5, 13, 40). To this end, retroviral Gag polyproteins use short sequences called late (L) domains to promote virus release by recruiting members of the host vacuolar protein sorting (vps) machinery. In the cell, vps proteins are involved in membrane dynamics that facilitate the separation of daughter cells at the completion of cytokinesis (9, 39) and the budding of vesicles into endosomal compartments or multivesicular bodies (MVB) (2, 23), a process topologically similar to virus budding (57). Class E vps proteins are organized into three heteromeric endosomal complexes (called endosomal sorting complexes) required for transport, namely, ESCRT-I, -II, and -III (2). In the current model for budding, sequential recruitment of ESCRT components on the cytoplasmic face of the membrane facilitates vesicle invagination into MVB compartments and viral egress from the cell (2). The disassembly of ESCRT-III components is catalyzed by the activity of VPS4 AAA-type ATPase, which in turn is presumed to trigger membrane fission events (3, 50). Any disruption in this sequence, such as mutations in L domain motifs or dominant-negative interference with the function of ESCRT-III members or the VPS4 ATPase, adversely affects virus release. This indicates that Gag interactions with the ESCRT machinery are necessary for virus budding and separation from the cell (19, 21, 34, 49, 57).Currently, three types of L domain motifs have been identified: PT/SAP, LYPX_nL, and PPPY. All retroviral Gag molecules contain at least one of these motifs, as multiple L domains are believed to synergistically function to ensure efficient viral release. Moloney murine leukemia virus (MoMLV) Gag carries all three L domain motifs, PSAP, LYPAL, and PPPY, which bind the vps protein Tsg101, the ESCRT-associated protein Alix (46), and members of the Nedd4-ubiquitin ligase family (33), respectively. In HIV-1, the PTAP motif in the p6 region of Gag binds Tsg101 (16, 56), which functions in viral budding (16, 35) as a member of ESCRT-I (16, 36, 57). The LYPX_nL motif is also located in p6 and is the binding site for Alix (49, 57), a protein that also interacts with the nucleocapsid domain of HIV-1 Gag (14, 43) and links Gag to components of ESCRT-III (14). Similarly, the human T-cell leukemia virus (HTLV-I) Gag carries PPPY and PTAP L domains, which both contribute to efficient HTLV-1 release (6, 7, 21). The PPPY L domain motif, which is found in numerous retroviral Gag polyproteins (6, 7, 19, 21, 27, 28, 61, 62), plays a critical role in MoMLV release, as mutations disrupting its sequence lead to significant decreases in virus budding and release (33, 62). PSAP and LYPAL, the additional L domain motifs, are believed to serve little to no role in the release of MoMLV Gag virus-like particles (45, 46).The role of Nedd4-like ubiquitin ligases in budding events was initially established by data obtained with the yeast Nedd4-like ligase Rsp5, an enzyme that ubiquitinates surface proteins, thus signaling their incorporation into the MVB pathway (26). From retroviral budding studies, multiple findings support the notion that Nedd4-like ubiquitin ligases link PPPY-containing Gag proteins to the host ESCRT machinery. For example, mutations in the PPPY motif or expression of dominant-negative versions of Nedd4-like ligases resulted in budding defects similar to those seen upon interference with the function of ESCRT-III members (7, 21, 27, 28, 33, 62). Overexpression of Nedd4-like ligases WWP1 and Itch corrected the budding defects of a MoMLV PPPY mutant that retained residual binding to both ligases (33). Also, when transplanted to a heterologous retroviral Gag, the PPPY L domain creates a requirement for Nedd4-like ubiqutin ligase activity to facilitate viral release that is dependent on the presence of a functional ESCRT pathway (63). Collectively, these observations support the notion that Nedd4-like ubiquitin ligases link retroviral Gag polyproteins to components of the ESCRT pathway necessary for budding.Both endosomal and viral budding require the ubiquitin conjugation properties of Nedd4-like ligases, indicating that ubiquitin transfer to a key protein(s) is necessary to promote budding. A role for Gag ubiquitination in viral budding has been suggested (8, 20, 22, 48). In fact, ubiquitin attachment to equine infectious anemia virus (EIAV) Gag can substitute for the lack of L domains and rescue viral budding (25), suggesting that ubiquitin molecules conjugated to Gag can signal the recruitment of the host ESCRT machinery. For feline immunodeficiency virus, efficient budding seems to require L domain-dependent ubiquitination of Gag proteins (8) that is independent of the L domain ability to directly recruit Nedd4-like ubiquitin ligases (i.e., by means of the PT/SAP L domain motif) (8). Similarly, ubiquitination of HTLV-1 Gag was also shown to play a significant role in viral release (22). Conversely, data arguing in favor of a role for the ubiquitination of transacting factors, but not Gag, in the facilitation of viral budding have also been reported (10, 63). Thus Gag polyproteins recruit, in a PPPY-dependent or -independent manner, enzymatically active Nedd4-like ubiquitin ligases that conjugate ubiquitin molecules to Gag or to Gag-binding host factors. Such interactions, whether direct or indirect, are believed to link the viral protein to the host ESCRT pathway and facilitate release.In addition to the well-characterized cellular proteins that bind primary L domain motifs, retroviral Gag can recruit other host factors, either via secondary L domains or independently of L domains (10, 24, 29, 55, 59). These cellular factors are believed to promote virus production by facilitating Gag protein trafficking to the plasma membrane and/or providing additional L domain-independent links to the host vps pathway. Examples of these parallel pathways are illustrated in the rescue of a budding-defective HIV-1 lacking the PTAP domain by overexpression of Alix (15, 54) and in the remarkably potent rescue of HIV-1 lacking all known L domains by the overexpression of Nedd4.2s, a Nedd4.2 isoform that belongs to the Nedd4-like ubiquitin ligase family (10, 55). In this study, we sought to identify host cell factors that rescue budding defects of the MoMLV mutant lacking the PPPY motif (MoMLV AAAY mutant). Our studies provide evidence that Itch overexpression rescued budding and infectivity defects of the MoMLV AAAY mutant virus, indicating that Gag can recruit the ubiquitin ligase Itch in an L domain-independent manner to facilitate MoMLV release via a mechanism that involves Gag ubiquitination.