Update on the Kelch-like (KLHL) gene family

The Kelch-like (KLHL) gene family encodes a group of proteins that generally possess a BTB/POZ domain, a BACK domain, and five to six Kelch motifs. BTB domains facilitate protein binding and dimerization. The BACK domain has no known function yet is of functional importance since mutations in this domain are associated with disease. Kelch domains form a tertiary structure of β-propellers that have a role in extracellular functions, morphology, and binding to other proteins. Presently, 42 KLHL genes have been classified by the HUGO Gene Nomenclature Committee (HGNC), and they are found across multiple human chromosomes. The KLHL family is conserved throughout evolution. Phylogenetic analysis of KLHL family members suggests that it can be subdivided into three subgroups with KLHL11 as the oldest member and KLHL9 as the youngest. Several KLHL proteins bind to the E3 ligase cullin 3 and are known to be involved in ubiquitination. KLHL genes are responsible for several Mendelian diseases and have been associated with cancer. Further investigation of this family of proteins will likely provide valuable insights into basic biology and human disease.     

The ApoE gene of Alzheimer's disease (AD)

The ApoE gene responsible for the Alzheimer's disease has been examined to identify functional consequences of single-nucleotide polymorphisms (SNPs). Eighty-eight SNPs have been identified in the ApoE gene in which 31 are found to be nonsynonymous, 8 of them are coding synonymous, 33 are found to be in intron, and 3 are in untranslated region. The SNPs found in the untranslated region consisted of two SNPs from 5′ and one SNP from the 3′. Twenty-nine percent of the identified nsSNPs have been reported as damaging. In the analysis of SNPs in the UTR regions, it has been recognized that rs72654467 from 5′ and rs71673244 from 5′ and 3′ are responsible for the alteration in levels of expression. Both native and mutant protein structures were analyzed along with the stabilization residues. It has been concluded that among all SNPs of ApoE, the mutation in rs11542041 (R132S) has the most significant effect on functional variation.     

Discrete analyses of protein dynamics

Abstract

Protein structures are highly dynamic macromolecules. This dynamics is often analysed through experimental and/or computational methods only for an isolated or a limited number of proteins. Here, we explore large-scale protein dynamics simulation to observe dynamics of local protein conformations using different perspectives. We analysed molecular dynamics to investigate protein flexibility locally, using classical approaches such as RMSf, solvent accessibility, but also innovative approaches such as local entropy. First, we focussed on classical secondary structures and analysed specifically how β-strand, β–turns, and bends evolve during molecular simulations. We underlined interesting specific bias between β–turns and bends, which are considered as the same category, while their dynamics show differences. Second, we used a structural alphabet that is able to approximate every part of the protein structures conformations, namely protein blocks (PBs) to analyse (i) how each initial local protein conformations evolve during dynamics and (ii) if some exchange can exist among these PBs. Interestingly, the results are largely complex than simple regular/rigid and coil/flexible exchange. Abbreviations N_eq number of equivalent

PB Protein Blocks

PDB Protein DataBank

RMSf root mean square fluctuations

Communicated by Ramaswamy H. Sarma     

Influence of different seed priming techniques on oxidative and antioxidative responses during the germination of Oryza sativa varieties

Physiology and Molecular Biology of Plants - NaCl and PEG stresses have negative impacts on seed germination and early seedling establishment in Oryza sativa. The present study was designed to...     

Morphological and seed protein characterization of the cultivated and the wild taxa of Sesamum L. (Pedaliaceae)

Seed protein analysis and morphological characterization were carried out in one cultivated and one wild species of Sesamum, Sesamum indicum L. and S. occidentale Regel and Heer. Data on 13 quantitative and 33 qualitative characters of the cultivated species and seven accessions of the wild taxa were analyzed. The genetic diversity of the taxa was assessed using UPGMA dendrogram and one-way ANOVA (p?<?0.05). Principal component analysis (PCA) was executed to identify the significant characters to delimit the taxa. Seed protein analysis showed diverse bands, ranging from 16 to 88?kDa. A dendrogram based on UPGMA analysis of seed protein suggested intraspecific relationships of the wild taxa as evidenced from the morphological characterization.     

Huntingtin-interacting protein 14, a palmitoyl transferase required for exocytosis and targeting of CSP to synaptic vesicles

Posttranslational modification through palmitoylation regulates protein localization and function. In this study, we identify a role for the Drosophila melanogaster palmitoyl transferase Huntingtin-interacting protein 14 (HIP14) in neurotransmitter release. hip14 mutants show exocytic defects at low frequency stimulation and a nearly complete loss of synaptic transmission at higher temperature. Interestingly, two exocytic components known to be palmitoylated, cysteine string protein (CSP) and SNAP25, are severely mislocalized at hip14 mutant synapses. Complementary DNA rescue and localization experiments indicate that HIP14 is required solely in the nervous system and is essential for presynaptic function. Biochemical studies indicate that HIP14 palmitoylates CSP and that CSP is not palmitoylated in hip14 mutants. Furthermore, the hip14 exocytic defects can be suppressed by targeting CSP to synaptic vesicles using a chimeric protein approach. Our data indicate that HIP14 controls neurotransmitter release by regulating the trafficking of CSP to synapses.     

Studies on crystallization and cross-linking of lipase for biocatalysis

The development of robust biocatalysts with increased stability and activity is a major challenge to industry. A major breakthrough in this field was the development of cross-linked enzyme crystals with high specificity and stability. A method is described to produce micro crystals of CLEC lipase, which is thermostable and solvent stable. Lipase from Burkholderia cepacia was crystallized using ammonium sulfate and cross-linked with glutaraldehyde to produce catalytically active enzyme. The maximum yield of CLEC was obtained with 70% ammonium sulfate and cross-linked with 5% (v/v) glutaraldehyde. SEM studies showed small hexagonal-shaped crystals of 2–5 μm size. CLEC lipase had improved thermal and reuse stability. It is versatile, having good activity in both polar and nonpolar organic solvents. CLEC lipase was coated using β cyclodextrin for improving the storage and reuse stability. CLEC was successfully used for esterification of Ibuprofen and synthesis of ethyl butyrate.     

Rumi is a CAP10 domain glycosyltransferase that modifies Notch and is required for Notch signaling

Notch signaling is broadly used to regulate cell-fate decisions. We have identified a gene, rumi, with a temperature-sensitive Notch phenotype. At 28 degrees C-30 degrees C, rumi clones exhibit a full-blown loss of Notch signaling in all tissues tested. However, at 18 degrees C only a mild Notch phenotype is evident. In vivo analyses reveal that the target of Rumi is the extracellular domain of Notch. Notch accumulates intracellularly and at the cell membrane of rumi cells but fails to be properly cleaved, despite normal binding to Delta. Rumi is an endoplasmic reticulum-retained protein with a highly conserved CAP10 domain. Our studies show that Rumi is a protein O-glucosyltransferase, capable of adding glucose to serine residues in Notch EGF repeats with the consensus C1-X-S-X-P-C2 sequence. These data indicate that by O-glucosylating Notch in the ER, Rumi regulates its folding and/or trafficking and allows signaling at the cell membrane.     

Studies on the biosynthesis of the carane skeleton

Measurements of isotope ratios in car-3-ene biosynthesized in Pinus sylvestris from (3RS)-mevalonate-[2-¹⁴C,2R-³H₁], and [2-¹⁴C,4R-³H₁] and the corresponding S-epimers and also from geraniol- [¹⁴C,1-³H₂] and nerol-[¹⁴ C,1-³H₂] have shown that the carane skeleton is constructed from its presumed monocyclic precursor with migration of an olefinic bond, together with an unexpected 1,2-shift of a proton to the site of the original double bond. The detailed stereochemistry of the processes allows a two-step mechanism to be inferred for the cyclization in which a bonded intermediate is involved. The conversion of geraniol into nerol (en route to car-3-ene) probably is a redox process with the intermediacy of the corresponding aldehydes. The present results eliminate a possible mechanism for this isomerization wherein cyclopropane derivatives occur as intermediates.     

Identification of restriction-fragment-length polymorphisms in genomic DNA of the lesser snow goose (Anser caerulescens caerulescens)

A genomic library of partially EcoRI-digested DNA from the lesser snow goose, Anser caerulescens caerulescens, was constructed in the phage vector Charon 4. Phage containing only unique sequences were identified by screening plaques with 32P-labeled genomic DNA. Restriction-fragment- length polymorphisms (RFLPs) were identified by probing DNA from 11-13 male birds from the breeding colony at La Perouse Bay. Of the 17 probes examined, all detected RFLPs with at least one of EcoRi, HindIII, Msp1, and Taq1. Several of them identified highly variable regions with multiple alleles. These RFLPs are valuable DNA markers that can be used for (1) the examination of DNA variation, relatedness, and genetic distance and (2) assessing paternity and maternity. These data suggest that there are higher levels of variation of DNA sequence in birds than had previously been thought to exist.