Structure-based identification of catalytic residues

The identification of catalytic residues is an essential step in functional characterization of enzymes. We present a purely structural approach to this problem, which is motivated by the difficulty of evolution-based methods to annotate structural genomics targets that have few or no homologs in the databases. Our approach combines a state-of-the-art support vector machine (SVM) classifier with novel structural features that augment structural clues by spatial averaging and Z scoring. Special attention is paid to the class imbalance problem that stems from the overwhelming number of non-catalytic residues in enzymes compared to catalytic residues. This problem is tackled by: (1) optimizing the classifier to maximize a performance criterion that considers both Type I and Type II errors in the classification of catalytic and non-catalytic residues; (2) under-sampling non-catalytic residues before SVM training; and (3) during SVM training, penalizing errors in learning catalytic residues more than errors in learning non-catalytic residues. Tested on four enzyme datasets, one specifically designed by us to mimic the structural genomics scenario and three previously evaluated datasets, our structure-based classifier is never inferior to similar structure-based classifiers and comparable to classifiers that use both structural and evolutionary features. In addition to the evaluation of the performance of catalytic residue identification, we also present detailed case studies on three proteins. This analysis suggests that many false positive predictions may correspond to binding sites and other functional residues. A web server that implements the method, our own-designed database, and the source code of the programs are publicly available at http://www.cs.bgu.ac.il/～meshi/functionPrediction.     

Grape berry position affects the diurnal dynamics of its metabolic profile

Solar irradiance and air temperature are characterized by dramatic circadian fluctuations and are known to significantly modulate fruit composition. To date, it remains unclear whether the abrupt, yet predictive, diurnal changes in radiation and temperature prompt direct metabolic turn‐over in the fruit. We assessed the role of fruit insolation, air temperature, and source‐tissue CO₂ assimilation in the diurnal compositional changes in ripening grape berries. This was performed by comparing the diurnal changes in metabolite profiles of berries positioned such that they experienced (a) contrasting diurnal solar irradiance patterns, and (b) similar irradiance but contrasting diurnal CO₂ assimilation patterns of adjacent leaves. Grape carbon levels increased during the morning and decreased thereafter. Sucrose levels decreased throughout the day and were correlated with air temperature, but not with the diurnal pattern of leaf CO₂ assimilation. Tight correlation between sucrose and glucose‐6‐phosphate indicated the involvement of photorespiration/glycolysis in sucrose depletion. Amino acids, polyamines, and phenylpropanoids fluctuated diurnally, and were highly responsive to the diurnal insolation pattern of the fruit. Our results fill the knowledge gap regarding the circadian pattern of source‐sink assimilate‐translocation in grapevine. In addition, they suggest that short‐term direct solar exposure of the fruit impacts both its diurnal and nocturnal metabolism.     

Enrichment of NPC1-deficient cells with the lipid LBPA stimulates autophagy,improves lysosomal function,and reduces cholesterol storage

Niemann–Pick C (NPC) is an autosomal recessive disorder characterized by mutations in the NPC1 or NPC2 genes encoding endolysosomal lipid transport proteins, leading to cholesterol accumulation and autophagy dysfunction. We have previously shown that enrichment of NPC1-deficient cells with the anionic lipid lysobisphosphatidic acid (LBPA; also called bis(monoacylglycerol)phosphate) via treatment with its precursor phosphatidylglycerol (PG) results in a dramatic decrease in cholesterol storage. However, the mechanisms underlying this reduction are unknown. In the present study, we showed using biochemical and imaging approaches in both NPC1-deficient cellular models and an NPC1 mouse model that PG incubation/LBPA enrichment significantly improved the compromised autophagic flux associated with NPC1 disease, providing a route for NPC1-independent endolysosomal cholesterol mobilization. PG/LBPA enrichment specifically enhanced the late stages of autophagy, and effects were mediated by activation of the lysosomal enzyme acid sphingomyelinase. PG incubation also led to robust and specific increases in LBPA species with polyunsaturated acyl chains, potentially increasing the propensity for membrane fusion events, which are critical for late-stage autophagy progression. Finally, we demonstrated that PG/LBPA treatment efficiently cleared cholesterol and toxic protein aggregates in Purkinje neurons of the NPC1^I1061T mouse model. Collectively, these findings provide a mechanistic basis supporting cellular LBPA as a potential new target for therapeutic intervention in NPC disease.     

Human receptor for activated protein kinase C1 associates with polyglutamine aggregates and modulates polyglutamine toxicity

Formation of SDS-insoluble protein aggregates in affected neurons is a cellular pathological feature of polyglutamine (polyQ) disease. We identified a multi-WD-domain protein, receptor for activated protein kinase C1 (RACK1), as a novel polyQ aggregate component from a Drosophila transgenic polyQ disease model. We showed that WD domains were crucial determinants for the recruitment of RACK1 to polyQ aggregates. Over-expression of the human RACK1 protein suppressed polyQ-induced neurodegeneration in vivo. This is the first report to demonstrate the involvement of WD-domain proteins in polyQ pathogenesis, and the proteomic approach described here can be applied to the investigation of other protein aggregation disorders including Alzheimer’s and Parkinson’s diseases.     

A cholesterol-lowering gene maps to chromosome 13q

A cholesterol-lowering gene has been postulated from familial hypercholesterolemia (FH) families having heterozygous persons with normal LDL levels and homozygous individuals with LDL levels similar to those in persons with heterozygous FH. We studied such a family with FH that also had members without FH and with lower-than-normal LDL levels. We performed linkage analyses and identified a locus at 13q, defined by markers D13S156 and D13S158. FASTLINK and GENEHUNTER yielded LOD scores >5 and >4, respectively, whereas an affected-sib-pair analysis gave a peak multipoint LOD score of 4.8, corresponding to a P value of 1.26x10-6. A multipoint quantitative-trait-locus (QTL) linkage analysis with maximum-likelihood binomial QTL verified this locus as a QTL for LDL levels. To test the relevance of this QTL in an independent normal population, we studied MZ and DZ twin subjects. An MZ-DZ comparison confirmed genetic variance with regard to lipid concentrations. We then performed an identity-by-descent linkage analysis on the DZ twins, with markers at the 13q locus. We found strong evidence for linkage at this locus with LDL (P<.0002), HDL (P<.004), total cholesterol (P<.0002), and body-mass index (P<.0001). These data provide support for the existence of a new gene influencing lipid concentrations in humans.     

Morphometry of the adult human earlobe: a study of 547 subjects and clinical application

The purpose of this study was to define the factors that influence earlobe length and to establish a standard for adult earlobe length by sex and age. The study sample consisted of 547 adult subjects older than 20 years of age. A randomized, prospective design was used. Patients with malignancies, previous surgery or trauma to the earlobe, or congenital earlobe anomalies were excluded. The following variables were studied: sex; age; ethnic origin; skin complexion; height, weight, and body mass index; and piercing. Pearson's correlation, analysis of variance, t test, and multiple regression analysis were used for the statistical analysis. There were 383 women (70 percent) and 164 men (30 percent) aged 20 to 80 years. The average length of the left earlobe was 1.97 cm (SD, 0.42 cm), and that of the right earlobe, 2.01 cm (SD, 0.42 cm) (p < 0.0001). A post hoc test revealed a statistically significant difference among the three age groups (20 to 40 years, 40 to 60 years, and >60 years) in both men and women. Pendulous earlobes were significantly longer and less symmetrical than nonpendulous ones by t test. In men, nonpierced left earlobes were longer than pierced lobes; in women, there was no significant difference between pierced and nonpierced ears. Pearson's correlation tests for weight, height, and body mass index showed that only weight had a significant effect on earlobe length, and only in women. Analysis of variance for ethnic origin and skin color revealed a longer left earlobe in Ashkenazi and Sephardic Jews compared with Ethiopian, Asian, and American Jews and Arabs and a short earlobe in blacks compared with dark and fair-skinned people. On multiple regression analysis, sex and age were the only factors that contributed to earlobe length. A table of average earlobe length by age was formulated on the basis of the authors' findings. These data, together with the knowledge that earlobe length changes little in women over 40, that earlobes are not symmetrical, and that right and left nonpendulous earlobes are symmetrical in individual patients and shorter than pendulous earlobes, can assist the plastic surgeon in deciding on the proper time for loboplasty. The preferable technique is creating a nonpendulous earlobe to minimize the chances of further elongation with time.     

Premature termination codon at the sterol 27-hydroxylase gene causes cerebrotendinous xanthomatosis in a French family

Cerebrotendinous xanthomatosis (CTX) is an autosomal recessive lipid-storage disease caused by mutations in the sterol 27-hydroxylase gene (CYP27). So far several mutations causing CTX have been identified and characterized. A new mutation creating an insertion of cytosine at position 6 in the cDNA, which is expected to result in a frameshift and a premature termination codon at codon 179, has been identified in a French family. The mutation creates a new site for the restriction endonuclease HaeIII.