Calcium as an extracellular signalling molecule: perspectives on the Calcium Sensing Receptor in the brain

Calcium acts as a universal signal that is responsible for controlling a spectrum of cellular processes ranging from fertilization to apoptosis. For a long time, calcium was regarded solely as an intracellular second messenger. However, the discovery that calcium can also act as an external ligand together with the molecular cloning of its cell surface receptor, the Calcium Sensing Receptor (CaSR), demonstrated that calcium also acts as an important extracellular or first messenger. Here, we give an overview of the main structural, pharmacological and physiological features of the CaSR and provide an assessment of its functions and cellular and molecular mechanisms of action. In addition, we propose possible avenues for future research into the trafficking of CaSR and the role(s) of this receptor in the central nervous system.     

AMP-activated protein kinase beta subunit tethers alpha and gamma subunits via its C-terminal sequence (186-270)

AMP-activated protein kinase (AMPK) is an important metabolic stress-sensing protein kinase responsible for regulating metabolism in response to changing energy demand and nutrient supply. Mammalian AMPK is a stable alphabetagamma heterotrimer comprising a catalytic alpha and two non-catalytic subunits, beta and gamma. The beta subunit targets AMPK to membranes via an N-terminal myristoyl group and to glycogen via a mid-molecule glycogen-binding domain. Here we find that the conserved C-terminal 85-residue sequence of the beta subunit, beta1-(186-270), is sufficient to form an active AMP-dependent heterotrimer alpha1beta1-(186-270)-gamma1, whereas the 25-residue beta1 C-terminal (246-270) sequence is sufficient to bind gamma1, gamma2, or gamma3 but not the alpha subunit. Deletion of the beta C-terminal Ile-270 precludes betagamma association in the absence of the alpha subunit, but the presence of the alpha subunit or substitution of Ile-270 with Ala or Glu restores betagamma binding. Truncation of the alpha subunit reveals that beta1 binding requires the alpha1-(313-473) sequence. The conserved C-terminal 85-residue sequence of the beta subunit (90% between beta1 and beta2) is the primary alphagamma binding sequence responsible for the formation of the AMPK alphabetagamma heterotrimer.     

Homo- and hetero-oligomerization of beta-arrestins in living cells

Arrestins are important proteins, which regulate the function of serpentine heptahelical receptors and contribute to multiple signaling pathways downstream of receptors. The ubiquitous beta-arrestins are believed to function exclusively as monomers, although self-association is assumed to control the activity of visual arrestin in the retina, where this isoform is particularly abundant. Here the oligomerization status of beta-arrestins was investigated using different approaches, including co-immunoprecipitation of epitope-tagged beta-arrestins and resonance energy transfer (BRET and FRET) in living cells. At steady state and at physiological concentrations, beta-arrestins constitutively form both homo- and hetero-oligomers. Co-expression of beta-arrestin2 and beta-arrestin1 prevented beta-arrestin1 accumulation into the nucleus, suggesting that hetero-oligomerization may have functional consequences. Our data clearly indicate that beta-arrestins can exist as homo- and hetero-oligomers in living cells and raise the hypothesis that the oligomeric state may regulate their subcellular distribution and functions.     

Identification of zebrafish insertional mutants with defects in visual system development and function

Genetic analysis in zebrafish has been instrumental in identifying genes necessary for visual system development and function. Recently, a large-scale retroviral insertional mutagenesis screen, in which 315 different genes were mutated, that resulted in obvious phenotypic defects by 5 days postfertilization was completed. That the disrupted gene has been identified in each of these mutants provides unique resource through which the formation, function, or physiology of individual organ systems can be studied. To that end, a screen for visual system mutants was performed on 250 of the mutants in this collection, examining each of them histologically for morphological defects in the eye and behaviorally for overall visual system function. Forty loci whose disruption resulted in defects in eye development and/or visual function were identified. The mutants have been divided into the following phenotypic classes that show defects in: (1) morphogenesis, (2) growth and central retinal development, (3) the peripheral marginal zone, (4) retinal lamination, (5) the photoreceptor cell layer, (6) the retinal pigment epithelium, (7) the lens, (8) retinal containment, and (9) behavior. The affected genes in these mutants highlight a diverse set of proteins necessary for the development, maintenance, and function of the vertebrate visual system.     

Proteomic analysis of mouse growth plate cartilage

Cartilage is a highly specialized load-bearing tissue with a small number of cells and a high proportion of extracellular matrix (ECM). The abundance of heavily sulfated proteoglycans and a poorly soluble collagenous ECM presents a major technical challenge to 2-DE. Here we report proteomic analysis of mouse growth plate cartilage using novel methodology for tissue dissection and sample prefractionation. We have successfully resolved cartilage tissue extracts by 2-DE for the first time and identified cartilage ECM proteins by Western blotting and MS/MS.     

Early Selection in Gag by Protective HLA Alleles Contributes to Reduced HIV-1 Replication Capacity That May Be Largely Compensated for in Chronic Infection

Mutations that allow escape from CD8 T-cell responses are common in HIV-1 and may attenuate pathogenesis by reducing viral fitness. While this has been demonstrated for individual cases, a systematic investigation of the consequence of HLA class I-mediated selection on HIV-1 in vitro replication capacity (RC) has not been undertaken. We examined this question by generating recombinant viruses expressing plasma HIV-1 RNA-derived Gag-Protease sequences from 66 acute/early and 803 chronic untreated subtype B-infected individuals in an NL4-3 background and measuring their RCs using a green fluorescent protein (GFP) reporter CD4 T-cell assay. In acute/early infection, viruses derived from individuals expressing the protective alleles HLA-B*57, -B*5801, and/or -B*13 displayed significantly lower RCs than did viruses from individuals lacking these alleles (P < 0.05). Furthermore, acute/early RC inversely correlated with the presence of HLA-B-associated Gag polymorphisms (R = −0.27; P = 0.03), suggesting a cumulative effect of primary escape mutations on fitness during the first months of infection. At the chronic stage of infection, no strong correlations were observed between RC and protective HLA-B alleles or with the presence of HLA-B-associated polymorphisms restricted by protective alleles despite increased statistical power to detect these associations. However, RC correlated positively with the presence of known compensatory mutations in chronic viruses from B*57-expressing individuals harboring the Gag T242N mutation (n = 50; R = 0.36; P = 0.01), suggesting that the rescue of fitness defects occurred through mutations at secondary sites. Additional mutations in Gag that may modulate the impact of the T242N mutation on RC were identified. A modest inverse correlation was observed between RC and CD4 cell count in chronic infection (R = −0.17; P < 0.0001), suggesting that Gag-Protease RC could increase over the disease course. Notably, this association was stronger for individuals who expressed B*57, B*58, or B*13 (R = −0.27; P = 0.004). Taken together, these data indicate that certain protective HLA alleles contribute to early defects in HIV-1 fitness through the selection of detrimental mutations in Gag; however, these effects wane as compensatory mutations accumulate in chronic infection. The long-term control of HIV-1 in some persons who express protective alleles suggests that early fitness hits may provide lasting benefits.The host immune response elicited by CD8⁺ cytotoxic T lymphocytes (CTLs) is a major contributor to viral control following human immunodeficiency virus type 1 (HIV-1) infection (6, 39), but antiviral pressure exerted by CTLs is diminished by the selection of escape mutations in targeted regions throughout the viral proteome (7, 18, 29, 35, 41, 45, 57). A comprehensive identification of HLA-associated viral polymorphisms has recently been achieved through population-based analyses of HIV-1 sequences and HLA class I types from different cohorts worldwide (3, 8, 13-15, 34, 43, 50, 56, 63). However, despite improved characterization of the sites and pathways of immune escape, effective ways to incorporate these findings into immunogen design remain an area of debate. A better understanding of the impact of escape mutations on viral fitness may provide novel directions for HIV-1 vaccines that are designed to attenuate pathogenesis.The development of innovative vaccine strategies that can overcome the extreme diversity of HIV is a key priority (4). One proposed approach is to target the most conserved T-cell epitopes, which presumably cannot escape from CTL pressure easily due to structural or functional constraints on the viral protein (55). Complementary approaches include the design of polyvalent and/or mosaic immunogens that incorporate commonly observed viral diversity (4, 38) or the specific targeting of vulnerable regions of the viral proteome that do escape but only at a substantial cost to viral replication capacity (RC) (1, 40). A chief target of such vaccine approaches is the major HIV-1 structural protein Gag, which is known to be highly immunogenic and to elicit CTL responses that correlate with the natural control of infection (22, 36, 66). Indeed, several lines of evidence support a relationship between the selection of CTL escape mutations and reduced HIV-1 fitness. These include the reversion of escape mutations following transmission to an HLA-mismatched recipient who cannot target the epitope (19, 24, 41) as well as reduced plasma viral load (pVL) set point following the transmission of certain escape variants from donors who expressed protective HLA alleles (17, 27). Notably, these in vivo observations have been made most often for variations within Gag that are attributed to CTL responses restricted by the protective alleles HLA-B*57 and -B*5801 (17, 19, 27, 41). Most recently, reduced in vitro RCs of clinical isolates and/or engineered strains encoding single or multiple escape mutations in Gag selected in the context of certain protective HLA alleles, including B*57, B*5801, B*27, and B*13, have been demonstrated (9, 10, 42, 53, 59, 62). Despite these efforts, the goal of a T-cell vaccine that targets highly conserved and attenuation-inducing sites is hampered by a lack of knowledge concerning the contribution of most escape mutations to HIV-1 fitness as well as a poor understanding of the relative influence of HLA on the viral RC at different stages of infection.The mutability of HIV-1 permits the generation of progeny viruses encoding compensatory mutations that restore normal protein function and/or viral fitness. Detailed studies have demonstrated that the in vitro RC of escape variants in human and primate immunodeficiency viruses can be enhanced by the addition of secondary mutations outside the targeted epitope (10, 20, 52, 59, 65). Thus, vaccine strategies aimed at attenuating HIV-1 must also consider, among other factors, the frequency, time course, and extent to which compensation might overcome attenuation mediated by CTL-induced escape. Despite its anticipated utility for HIV-1 vaccine design, systematic studies to examine the consequences of naturally occurring CTL escape and compensatory mutations on viral RC have not been undertaken.We have described previously an in vitro recombinant viral assay to examine the impact of Gag-Protease mutations on HIV-1 RC (47, 49). Gag and protease have been included in each virus to minimize the impact of sequence polymorphisms at Gag cleavage sites, which coevolve with changes in protease (5, 37). Using this approach, we have demonstrated that viruses derived from HIV-1 controllers replicated significantly less well than those derived from noncontrollers and that these differences were detectable at both the acute/early (49) and chronic (47) stages. Escape mutations in Gag associated with the protective HLA-B*57 allele, as well as putative compensatory mutations outside known CTL epitopes, contributed to this difference in RC (47). However, substantial variability was observed for viruses from controllers and noncontrollers, indicating that additional factors were likely to be involved. Benefits of this assay include its relatively high-throughput capacity as well as the fact that clinically derived HIV-1 sequences are used in their entirety. Thus, it is possible to examine a large number of “real-world” Gag-Protease sequences, to define an RC value for each one, and to identify sequences within the population of recombinant strains that are responsible for RC differences.Here, we use this recombinant virus approach to examine the contribution of HLA-associated immune pressure on Gag-Protease RC during acute/early (n = 66) and chronic (n = 803) infections in the context of naturally occurring HIV-1 subtype B isolates from untreated individuals. In a recent report (64), we employed this system to examine the Gag-Protease RC in a similar cohort of chronic HIV-1 subtype C-infected individuals. The results of these studies provide important insights into the roles of immune pressure and fitness constraints on HIV-1 evolution that may contribute to the rational design of an effective vaccine.     

Abeta42-driven cerebral amyloidosis in transgenic mice reveals early and robust pathology

We have generated a novel transgenic mouse model on a C57BL/6J genetic background that coexpresses KM670/671NL mutated amyloid precursor protein and L166P mutated presenilin 1 under the control of a neuron-specific Thy1 promoter element (APPPS1 mice). Cerebral amyloidosis starts at 6-8 weeks and the ratio of human amyloid (A)beta42 to Abeta40 is 1.5 and 5 in pre-depositing and amyloid-depositing mice, respectively. Consistent with this ratio, extensive congophilic parenchymal amyloid but minimal amyloid angiopathy is observed. Amyloid-associated pathologies include dystrophic synaptic boutons, hyperphosphorylated tau-positive neuritic structures and robust gliosis, with neocortical microglia number increasing threefold from 1 to 8 months of age. Global neocortical neuron loss is not apparent up to 8 months of age, but local neuron loss in the dentate gyrus is observed. Because of the early onset of amyloid lesions, the defined genetic background of the model and the facile breeding characteristics, APPPS1 mice are well suited for studying therapeutic strategies and the pathomechanism of amyloidosis by cross-breeding to other genetically engineered mouse models.     

Analysis of clinical ostreid herpesvirus 1 (Malacoherpesviridae) specimens by sequencing amplified fragments from three virus genome areas

Although there are a number of ostreid herpesvirus 1 (OsHV-1) variants, it is expected that the true diversity of this virus will be known only after the analysis of significantly more data. To this end, we analyzed 72 OsHV-1 "specimens" collected mainly in France over an 18-year period, from 1993 to 2010. Additional samples were also collected in Ireland, the United States, China, Japan, and New Zealand. Three virus genome regions (open reading frame 4 [ORF4], ORF35, -36, -37, and -38, and ORF42 and -43) were selected for PCR analysis and sequencing. Although ORF4 appeared to be the most polymorphic genome area, distinguishing several genogroups, ORF35, -36, -37, and -38 and ORF42 and -43 also showed variations useful in grouping subpopulations of this virus.