Pelizaeus-Merzbacher-like disease caused by AIMP1/p43 homozygous mutation

Pelizaeus-Merzbacher disease is an X-linked hypomyelinating leukodystrophy caused by PLP1 mutations. A similar autosomal-recessive phenotype, Pelizaeus-Merzbacher-like disease (PMLD), has been shown to be caused by homozygous mutations in GJC2 or HSPD1. We report a consanguineous Israeli Bedouin kindred with clinical and radiological findings compatible with PMLD in which linkage to PLP1, GJC2, and HSPD1 was excluded. Through genome-wide homozygosity mapping and mutation analysis, we demonstrated in all affected individuals a homozygous frameshift mutation that fully abrogates the main active domain of AIMP1, encoding ARS-interacting multifunctional protein 1. The mutation fully segregates with the disease-associated phenotype and was not found in 250 Bedouin controls. Our findings are in line with the previously demonstrated inability of mutant mice lacking the AIMP1/p43 ortholog to maintain axon integrity in the central and peripheral neural system.     

Mutations disrupting selenocysteine formation cause progressive cerebello-cerebral atrophy

The essential micronutrient selenium is found in proteins as selenocysteine (Sec), the only genetically encoded amino acid whose biosynthesis occurs on its cognate tRNA in humans. In the final step of selenocysteine formation, the essential enzyme SepSecS catalyzes the conversion of Sep-tRNA to Sec-tRNA. We demonstrate that SepSecS mutations cause autosomal-recessive progressive cerebellocerebral atrophy (PCCA) in Jews of Iraqi and Moroccan ancestry. Both founder mutations, common in these two populations, disrupt the sole route to the biosynthesis of the 21st amino acid, Sec, and thus to the generation of selenoproteins in humans.     

Killing of Avian and Swine Influenza Virus by Natural Killer Cells

Today, global attention is focused on two influenza virus strains: the current pandemic strain, swine origin influenza virus (H1N1-2009), and the highly pathogenic avian influenza virus, H5N1. At present, the infection caused by the H1N1-2009 is moderate, with mortality rates of less <1%. In contrast, infection with the H5N1 virus resulted in high mortality rates, and ca. 60% of the infected patients succumb to the infection. Thus, one of the world greatest concerns is that the H5N1 virus will evolve to allow an efficient human infection and human-to-human transmission. Natural killer (NK) cells are one of the innate immune components playing an important role in fighting against influenza viruses. One of the major NK activating receptors involved in NK cell cytotoxicity is NKp46. We previously demonstrated that NKp46 recognizes the hemagglutinin proteins of B and A influenza virus strains. Whether NKp46 could also interact with H1N1-2009 virus or with the avian influenza virus is still unknown. We analyzed the immunological properties of both the avian and the H1N1-2009 influenza viruses. We show that NKp46 recognizes the hemagglutinins of H1N1-2009 and H5 and that this recognition leads to virus killing both in vitro and in vivo. However, importantly, while the swine H1-NKp46 interactions lead to the direct killing of the infected cells, the H5-NKp46 interactions were unable to elicit direct killing, probably because the NKp46 binding sites for these two viruses are different.Natural killer (NK) cells, which comprise 5 to 15% of peripheral blood lymphocytes, are a key frontline defense against a number of pathogens, including intracellular bacteria, parasites, and most importantly with respect to the present study, viruses (6, 40). The antiviral mechanisms by which NK cells operate include both cytotoxic activity and cytokine/chemokine secretion (21). The NK killing activity is executed by numerous receptors, including NKG2D, NKp80, CD16, and the natural cytotoxic receptors (NCRs): NKp30, NKp44, and NKp46 (7, 10, 25).Although the cellular ligands for NKG2D were identified (31, 38), the identity of several of the cellular ligands for the human NCRs is still unknown, except for BAT3 and B7-H6, which are ligands for NKp30 (8, 30). In contrast, viral ligands were identified for the NCRs, and we demonstrated that pp65 of HCMV interacts with NKp30 (3) and that various influenza virus hemagglutinins (HAs) are ligands for the NKp44 and NKp46 receptors (5, 22). Supporting these observations, it was recently shown that the HA-neuraminidase of Newcastle disease virus could also interact with NKp46 and NKp44 but not with NKp30 (17). Furthermore, we have shown in vivo that in the absence of NCR1 (the mouse homologue of NKp46), A/PR8 influenza virus infection is lethal (14).Human influenza virus (H1 and H3 subtype) infections pose a major threat to the entire population, as exemplified by the three major influenza pandemics that occurred during the 20th century. The Asian (A/H2N2) in 1957 to 1958 and the Hong Kong (A/H3N2) pandemics in 1968 to 1969 resulted in the deaths of 1 to 2 million people and the 1918 “Spanish flu” (A/H1N1) pandemic killed around 50 million people (18). At present, the worldwide concern regarding influenza pandemics concentrates mainly on two viruses: the A/H1N1 swine origin influenza virus (H1N1-2009), which currently causes only a moderate pandemic (the mortality rates are ca. 1%) but is more pathogenic than a regular seasonal influenza virus (19, 26, 27), and the avian influenza virus carrying the unique H5 HA (20). The avian influenza virus is quite deadly and, although it remains a zoonotic infection, ca. 60% of infected humans died due to the infection (28).The unique properties of the H5 protein of the avian influenza virus are one of the main reasons for the virulence of the virus. The H5 of the avian influenza virus binds to cell surface glycoproteins or glycolipids containing terminal sialyl-galactosyl residues linked by 2-3-linkage [Neu5Ac(α2-3)Gal] that are found in the human conjunctiva and ciliated portion of the respiratory columnar epithelium (33). In contrast, human viruses (including all three strains that caused the pandemics described above and the H1N1-2009) bind to receptors that mostly contain terminal 2-6-linked sialyl-galactosyl moieties [Neu5Ac(α2-6)Gal]. Such glycosylations are predominant on epithelial cells in the nasal mucosa, paranasal sinuses, pharynx, trachea, and bronchi (33, 37). It has been suggested that the lack of human-to-human transmission of avian influenza viruses is due to their α2,3-SA receptor binding preference, and the concern is that genetic changes in H5 might alter its preference from α2,3-SA to α2,6-SA, allowing human-to-human transmission.In our previous studies (4, 22) we showed that the interaction between NKp46 and influenza virus HAs depends on the sialylation of the NKp46 receptor. We further demonstrated that the sialic acid residues, which are linked via α2,6 to the threonine 225 residue of NKp46, are crucial for the NKp46 interactions with the various influenza virus HAs (4).We show that, both in vitro and in vivo, the killing of H1N1-2009-infected cells is correlated with the degree of NKp46 binding. Surprisingly, we observed that although NKp46 efficiently recognized the avian H5 HA, such interactions were unable to elicit the direct killing of the infected cells. By using mutagenesis analysis experiments and killing assays we demonstrate that NKp46 interacts with H1 and H5 at distinct sites, since we show that the sugar carrying residue at position 225 is crucial for the NKp46-H1N1-2009 interactions, whereas the interaction of H5 with NKp46 depends on both residues 216 and 225.     

Interleukin-1β Regulates Fat-Liver Crosstalk in Obesity by Auto-Paracrine Modulation of Adipose Tissue Inflammation and Expandability

The inflammasome has been recently implicated in obesity-associated dys-metabolism. However, of its products, the specific role of IL-1β was clinically demonstrated to mediate only the pancreatic beta-cell demise, and in mice mainly the intra-hepatic manifestations of obesity. Yet, it remains largely unknown if IL-1β, a cytokine believed to mainly function locally, could regulate dysfunctional inter-organ crosstalk in obesity. Here we show that High-fat-fed (HFF) mice exhibited a preferential increase of IL-1β in portal compared to systemic blood. Moreover, portally-drained mesenteric fat transplantation from IL-1βKO donors resulted in lower pyruvate-glucose flux compared to mice receiving wild-type (WT) transplant. These results raised a putative endocrine function for visceral fat-derived IL-1β in regulating hepatic gluconeogenic flux. IL-1βKO mice on HFF exhibited only a minor or no increase in adipose expression of pro-inflammatory genes (including macrophage M1 markers), Mac2-positive crown-like structures and CD11b-F4/80-double-positive macrophages, all of which were markedly increased in WT-HFF mice. Further consistent with autocrine/paracrine functions of IL-1β within adipose tissue, adipose tissue macrophage lipid content was increased in WT-HFF mice, but significantly less in IL-1βKO mice. Ex-vivo, adipose explants co-cultured with primary hepatocytes from WT or IL-1-receptor (IL-1RI)-KO mice suggested only a minor direct effect of adipose-derived IL-1β on hepatocyte insulin resistance. Importantly, although IL-1βKOs gained weight similarly to WT-HFF, they had larger fat depots with similar degree of adipocyte hypertrophy. Furthermore, adipogenesis genes and markers (pparg, cepba, fabp4, glut4) that were decreased by HFF in WT, were paradoxically elevated in IL-1βKO-HFF mice. These local alterations in adipose tissue inflammation and expansion correlated with a lower liver size, less hepatic steatosis, and preserved insulin sensitivity. Collectively, we demonstrate that by promoting adipose inflammation and limiting fat tissue expandability, IL-1β supports ectopic fat accumulation in hepatocytes and adipose-tissue macrophages, contributing to impaired fat-liver crosstalk in nutritional obesity.     

Recognition and killing of human and murine pancreatic beta cells by the NK receptor NKp46

Type 1 diabetes is an incurable disease that is currently treated by insulin injections or in rare cases by islet transplantation. We have recently shown that NKp46, a major killer receptor expressed by NK cells, recognizes an unknown ligand expressed by β cells and that in the absence of NKp46, or when its activity is blocked, diabetes development is inhibited. In this study, we investigate whether NKp46 is involved in the killing of human β cells that are intended to be used for transplantation, and we also thoroughly characterize the interaction between NKp46 and its human and mouse β cell ligands. We show that human β cells express an unknown ligand for NKp46 and are killed in an NKp46-dependent manner. We further demonstrate that the expression of the NKp46 ligand is detected on human β cells already at the embryonic stage and that it appears on murine β cells only following birth. Because the NKp46 ligand is detected on healthy β cells, we wondered why type 1 diabetes does not develop in all individuals and show that NK cells are absent from the vicinity of islets of healthy mice and are detected in situ in proximity with β cells in NOD mice. We also investigate the molecular mechanisms controlling NKp46 interactions with its β cell ligand and demonstrate that the recognition is confined to the membrane proximal domain and stalk region of NKp46 and that two glycosylated residues of NKp46, Thr(125) and Asn(216), are critical for this recognition.     

Rubisco mutagenesis provides new insight into limitations on photosynthesis and growth in Synechocystis PCC6803

Orthophosphate (Pi) stimulates the activation of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) while paradoxically inhibiting its catalysis. Of three Pi-binding sites, the roles of the 5P- and latch sites have been documented, whereas that of the 1P-site remained unclear. Conserved residues at the 1P-site of Rubisco from the cyanobacterium Synechocystis PCC6803 were substituted and the kinetic properties of the enzyme derivatives and effects on cell photosynthesis and growth were examined. While Pi-stimulated Rubisco activation diminished for enzyme mutants T65A/S and G404A, inhibition of catalysis by Pi remained unchanged. Together with previous studies, the results suggest that all three Pi-binding sites are involved in stimulation of Rubisco activation, whereas only the 5P-site is involved in inhibition of catalysis. While all the mutations reduced the catalytic turnover of Rubisco (K(cat)) between 6- and 20-fold, the photosynthesis and growth rates under saturating irradiance and inorganic carbon (Ci) concentrations were only reduced 40-50% (in the T65A/S mutants) or not at all (G404A mutant). Analysis of the mutant cells revealed a 3-fold increase in Rubisco content that partially compensated for the reduced K(cat) so that the carboxylation rate per chlorophyll was one-third of that in the wild type. Correlation between the kinetic properties of Rubisco and the photosynthetic rate (P(max)) under saturating irradiance and Ci concentrations indicate that a >60% reduction in K(cat) can be tolerated before P(max) in Synechocystsis PCC6803 is affected. These results indicate that the limitation of Rubisco activity on the rate of photosynthesis in Synechocystis is low. Determination of Calvin cycle metabolites revealed that unlike in higher plants, cyanobacterial photosynthesis is constrained by phosphoglycerate reduction probably due to limitation of ATP or NADPH.     

X inactivation testing for identifying a non-syndromic X-linked mental retardation gene

The purpose of this study was to identify a gene causing non-syndromic X-linked mental retardation in an extended family, taking advantage of the X chromosome inactivation status of the females in order to determine their carrier state. X inactivation in the females was determined with the androgen receptor methylation assay; thereafter, the X chromosome was screened with evenly spaced polymorphic markers. Once initial linkage was identified, the region of interest was saturated with additional markers and the males were added to the analysis. Candidate genes were sequenced. Ten females showed skewed inactivation, while six revealed a normal inactivation pattern. A maximal lod score of 5.54 at θ?=?0.00 was obtained with the marker DXS10151. Recombination events mapped the disease gene to a 17.4-Mb interval between the markers DXS10153 and DXS10157. Three candidate genes in the region were sequenced and a previously described missense mutation (P375L) was identified in the ACSL4/FACL4 gene. On the basis of the female X inactivation status, we have mapped and identified the causative mutation in a gene causing non-syndromic X-linked mental retardation.     

The Tomato yellow leaf curl virus (TYLCV) V2 protein interacts with the host papain-like cysteine protease CYP1

The V2 protein of Tomato yellow leaf curl geminivirus (TYLCV) is an RNA-silencing suppressor that counteracts the innate immune response of the host plant. However, this anti-host defense function of V2 may include targeting of other defensive mechanisms of the plant. Specifically, we show that V2 recognizes and directly binds the tomato CYP1 protein, a member of the family of papain-like cysteine proteases which are involved in plant defense against diverse pathogens. This binding occurred both in vitro and in vivo, within living plant cells. The V2 binding site within mCYP1 was identified in the direct proximity to the papain-like cysteine protease active site.