Identification of human proteins that modify misfolding and proteotoxicity of pathogenic ataxin-1

Proteins with long, pathogenic polyglutamine (polyQ) sequences have an enhanced propensity to spontaneously misfold and self-assemble into insoluble protein aggregates. Here, we have identified 21 human proteins that influence polyQ-induced ataxin-1 misfolding and proteotoxicity in cell model systems. By analyzing the protein sequences of these modifiers, we discovered a recurrent presence of coiled-coil (CC) domains in ataxin-1 toxicity enhancers, while such domains were not present in suppressors. This suggests that CC domains contribute to the aggregation- and toxicity-promoting effects of modifiers in mammalian cells. We found that the ataxin-1-interacting protein MED15, computationally predicted to possess an N-terminal CC domain, enhances spontaneous ataxin-1 aggregation in cell-based assays, while no such effect was observed with the truncated protein MED15ΔCC, lacking such a domain. Studies with recombinant proteins confirmed these results and demonstrated that the N-terminal CC domain of MED15 (MED15CC) per se is sufficient to promote spontaneous ataxin-1 aggregation in vitro. Moreover, we observed that a hybrid Pum1 protein harboring the MED15CC domain promotes ataxin-1 aggregation in cell model systems. In strong contrast, wild-type Pum1 lacking a CC domain did not stimulate ataxin-1 polymerization. These results suggest that proteins with CC domains are potent enhancers of polyQ-mediated protein misfolding and aggregation in vitro and in vivo.     

Identification of high affinity polo-like kinase 1 (Plk1) polo-box domain binding peptides using oxime-based diversification

In an effort to develop improved binding antagonists of the polo-like kinase 1 (Plk1) polo-box domain (PBD), we optimized interactions of the known high affinity 5-mer peptide PLHSpT using oxime-based post solid-phase peptide diversification of the N-terminal Pro residue. This allowed us to achieve up to two orders of magnitude potency enhancement. An X-ray crystal structure of the highest affinity analogue in complex with Plk1 PBD revealed new binding interactions in a hydrophobic channel that had been occluded in X-ray structures of the unliganded protein. This study represents an important example where amino acid modification by post solid-phase oxime ligation can facilitate the development of protein-protein interaction inhibitors by identifying new binding pockets that would not otherwise be accessible to coded amino acid residues.     

Demographics and economic burden of un-owned cats and dogs in the UK: results of a 2010 census

ABSTRACT: BACKGROUND: The population of dogs and cats passing through rescue shelters may be subject to compromised welfare and increased susceptibility to disease. Little information exists to describe this population, its dynamics and associated management practices. The aim of this study was to carry out a census of un-owned cats and dogs in the UK in 2010, and to document the origins, destinations, husbandry and costs associated with the care of these animals. RESULTS: A sampling frame was constructed by searching the databases of publicly registered charities for England, Scotland and Wales, registers of breed rescues, and by internet searches of animal welfare websites. Overall, 2,352 contacts for 1,380 organisations were identified. All were sent a postal questionnaire asking for data on the number of dogs and cats housed, their origins and eventual outcomes, and details of husbandry between January 1st and December 31st 2010. For those which were registered charities (595), financial records were also obtained. A response rate of 38.8 % was obtained. Overall, in 2010, 89,571 dogs and 156,826 cats entered the care of the participating organisations. Approximately half of these animals were relinquished by their owners. Other origins included being found as strays or confiscated for welfare purposes. Seventy-five per cent of dogs and 77.1 % of cats were rehomed. The next most common outcome was euthanasia, accounting for 10.4 % of dogs and 13.2 % cats. For dogs and cats, 44.3 % and 62 % of participants respectively reported having a waiting list, which frequently exceeded the actual capacity of the facility. Over 19,000 people were involved in the care of these animals, on a paid or voluntary basis. Financial records were available for 519/595 (87.2 %) of the registered charities, and their total expenditure in 2010 was GBP340 million. CONCLUSIONS: This study showed that a large number of animals become un-owned each year, which could have considerable implications for their welfare. Despite the resources expended, demand still exceeds capacity for many organisations, and a substantial number of both cats and dogs are euthanased, suggesting that further understanding of how and why these animals become un-owned is essential in order to target interventions.     

Mobilization of Regulatory T Cells in Response to Carotid Injury Does Not Influence Subsequent Neointima Formation

Aim

T cells have been attributed an important role in modulating repair responses following vascular injury. The aim of this study was to investigate the role of different T cell subsets in this context.

Methods and Results

A non-obstructive collar was introduced to inflict carotid artery injury in mice and subsequent activation of immune cells in draining lymph nodes and spleen were studied by flow cytometry. Carotid artery injury of wild type mice was associated with mobilization of both Th1 type CD4⁺IFNγ⁺ and regulatory CD4⁺CD25⁺FoxP3⁺ T cells in draining lymph nodes. Studies using FoxP3-green fluorescent protein (GFP) transgenic C57/Bl6 mice demonstrated scattered presence of regulatory T cells in the adventitial tissue of injured arteries as well as a massive emigration of regulatory T cells from the spleen in response to carotid injury. However, deletion of antigen presentation to CD4+ T cells (H2⁰ mice), as well as deletion of regulatory T cells (through treatment with blocking anti-CD25 antibodies), did not affect neointima formation. Also deletion of antigen presentation to CD8⁺ T cells (Tap1⁰ mice) was without effect on carotid collar-induced neointima formation.

Conclusion

The results demonstrate that carotid artery injury is associated with mobilization of regulatory T cells. Depletion of regulatory T cells does not, however, influence the subsequent repair processes leading to the formation of a neointima. The results also demonstrate that lack of CD8⁺ T cells does not influence neointima formation in presence of functional CD4⁺ T cells and B cells.     

Quantifying size distributions of nanolipoprotein particles with single-particle analysis and molecular dynamic simulations

Self-assembly of purified apolipoproteins and phospholipids results in the formation of nanometer-sized lipoprotein complexes, referred to as nanolipoprotein particles (NLPs). These bilayer constructs are fully soluble in aqueous environments and hold great promise as a model system to aid in solubilizing membrane proteins. Size variability in the self-assembly process has been recognized for some time, yet limited studies have been conducted to examine this phenomenon. Understanding the source of this heterogeneity may lead to methods to mitigate heterogeneity or to control NLP size, which may be important for tailoring NLPs for specific membrane proteins. Here, we have used atomic force microscopy, ion mobility spectrometry, and transmission electron microscopy to quantify NLP size distributions on the single-particle scale, specifically focusing on assemblies with 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and a recombinant apolipoprotein E variant containing the N-terminal 22 kDa fragment (E422k). Four discrete sizes of E422k/DMPC NLPs were identified by all three techniques, with diameters centered at approximately 14.5, 19, 23.5, and 28 nm. Computer simulations suggest that these sizes are related to the structure and number of E422k lipoproteins surrounding the NLPs and particles with an odd number of lipoproteins are consistent with the double-belt model, in which at least one lipoprotein adopts a hairpin structure.     

Dual functions of Bruton's tyrosine kinase and Tec kinase during Fcgamma receptor-induced signaling and phagocytosis

Tec family nonreceptor tyrosine kinases are expressed by hematopoietic cells, activate phospholipase C (PLC)gamma, and regulate cytoskeletal rearrangement, yet their role in FcgammaR-induced signaling and phagocytosis remains unknown. We demonstrate in this study that Bruton's tyrosine kinase (Btk) and Tec, the only Tec kinases expressed by RAW 264.7 cells, are activated throughout phagocytosis. Activated Btk and Tec kinase accumulate at an early stage at the base of phagocytic cups and inhibition of their activity by the specific inhibitor LFM-A13 or expression by small interfering RNA significantly inhibited FcgammaR-induced phagocytosis. Similarly, a significant role for these kinases in phagocytosis was found in primary macrophages. FcgammaR-induced activation of Mac-1, which is required for optimal phagocytosis, was markedly inhibited and our findings suggest that the roles of kinases Btk and Tec in Mac-1 activation account for their functions in the early stages of phagocytosis. Initial activation of PLCgamma2, the predominant PLC isoform in RAW 264.7 cells, is dependent on Syk. In contrast, a late and prolonged activation of PLCgamma2 was dependent on Btk and Tec. We found accumulation of diacylglycerol (DAG), a PLCgamma product, in phagosome membranes, and activated Btk, but not Tec, colocalized with phagosomal DAG. Inhibition of Tec family kinase activity increased the level of DAG in phagosomes, suggesting a negative regulatory role for Btk. Tec, in contrast, clustered at sites near phagosome formation. In summary, we elucidated that Tec family kinases participate in at least two stages of FcgammaR-mediated phagocytosis: activation of Mac-1 during ingestion, and after phagosome formation, during which Btk and Tec potentially have distinct roles.     

Filamin A stabilizes Fc gamma RI surface expression and prevents its lysosomal routing

Filamin A, or actin-binding protein 280, is a ubiquitously expressed cytosolic protein that interacts with intracellular domains of multiple receptors to control their subcellular distribution, and signaling capacity. In this study, we document interaction between FcgammaRI, a high-affinity IgG receptor, and filamin A by yeast two-hybrid techniques and coimmunoprecipitation. Both proteins colocalized at the plasma membrane in monocytes, but dissociated upon FcgammaRI triggering. The filamin-deficient cell line M2 and a filamin-reconstituted M2 subclone (A7), were used to further study FcgammaRI-filamin interactions. FcgammaRI transfection in A7 cells with filamin resulted in high plasma membrane expression levels. In filamin-deficient M2 cells and in filamin RNA-interference studies, FcgammaRI surface expression was consistently reduced. FcgammaRI localized to LAMP-1-positive vesicles in the absence of filamin as shown by confocal microscopy indicative for lysosomal localization. Mouse IgG2a capture experiments suggested a transient membrane expression of FcgammaRI before being transported to the lysosomes. These data support a pivotal role for filamin in FcgammaRI surface expression via retention of FcgammaRI from a default lysosomal pathway.     

Antibody profiling by proteome microarray reveals the immunogenicity of the attenuated smallpox vaccine modified vaccinia virus ankara is comparable to that of Dryvax

Modified vaccinia virus Ankara (MVA) is a highly attenuated vaccinia virus that is under consideration as an alternative to the conventional smallpox vaccine Dryvax. MVA was attenuated by extensive passage of vaccinia virus Ankara in chicken embryo fibroblasts. Several immunomodulatory genes and genes that influence host range are deleted or mutated, and replication is aborted in the late stage of infection in most nonavian cells. The effect of these mutations on immunogenicity is not well understood. Since the structural genes appear to be intact in MVA, it is hypothesized that critical targets for antibody neutralization have been retained. To test this, we probed microarrays of the Western Reserve (WR) proteome with sera from humans and macaques after MVA and Dryvax vaccination. As most protein sequences of MVA are 97 to 99% identical to those of other vaccinia virus strains, extensive binding cross-reactivity is expected, except for those deleted or truncated. Despite different hosts and immunization regimens, the MVA and Dryvax antibody profiles were broadly similar, with antibodies against membrane and core proteins being the best conserved. The responses to nonstructural proteins were less well conserved, although these are not expected to influence virus neutralization. The broadest antibody response was obtained for hyperimmune rabbits with WR, which is pathogenic in rabbits. These data indicate that, despite the mutations and deletions in MVA, its overall immunogenicity is broadly comparable to that of Dryvax, particularly at the level of antibodies to membrane proteins. The work supports other information suggesting that MVA may be a useful alternative to Dryvax.     

ATP binding by monarch-1/NLRP12 is critical for its inhibitory function

The recently discovered nucleotide binding domain-leucine rich repeat (NLR) gene family is conserved from plants to mammals, and several members are associated with human autoinflammatory or immunodeficiency disorders. This family is defined by a central nucleotide binding domain that contains the highly conserved Walker A and Walker B motifs. Although the nucleotide binding domain is a defining feature of this family, it has not been extensively studied in its purified form. In this report, we show that purified Monarch-1/NLRP12, an NLR protein that negatively regulates NF-κB signaling, specifically binds ATP and exhibits ATP hydrolysis activity. Intact Walker A/B motifs are required for this activity. These motifs are also required for Monarch-1 to undergo self-oligomerization, Toll-like receptor- or CD40L-activated association with NF-κB-inducing kinase (NIK) and interleukin-1 receptor-associated kinase 1 (IRAK-1), degradation of NIK, and inhibition of IRAK-1 phosphorylation. The stable expression of a Walker A/B mutant in THP-1 monocytes results in increased production of proinflammatory cytokines and chemokines to an extent comparable to that in cells in which Monarch-1 is silenced via short hairpin RNA. The results of this study are consistent with a model wherein ATP binding regulates the anti-inflammatory activity of Monarch-1.