Unravelling Molecular and Cellular Disease Mechanisms in Infantile Neuronal Ceroid Lipofuscinosis (INCL)

Studying neurodegeneration provides an opportunity to gain insights into normal cell physiology, and not just pathophysiology. In this thesis work the focus is on Infantile Neuronal Ceroid Lipofuscinosis (INCL). It is a recessively inherited lysosomal storage disorder. The disease belongs to the neuronal ceroid lipofuscinoses (NCLs), a group of common progressive neurodegenerative diseases of the childhood. Characteristic accumulation of autofluorescent storage material is seen in most tissues but only neurons of the central nervous system are damaged and eventually lost during the course of the disease leaving most other cell types unaffected. The disease is caused by mutations in the CLN1 gene, but the physiological function of the corresponding protein the palmitoyl protein thioesterase (PPT1) has remained elusive. The aim of this thesis work was to shed light on the molecular and cell biological mechanisms behind INCL. This study pinpointed the localization of PPT1 in axonal presynapses of neurons. It also established the role of PPT1 in early neuronal maturation as well as importance in mature neuronal synapses. This study revealed an endocytic defect in INCL patient cells manifesting itself as delayed trafficking of receptor and non-receptor mediated endocytic markers. Furthermore, this study was the first to connect the INCL storage proteins the sphingolipid activator proteins (SAPs) A and D to pathological events on the cellular level. Abnormal endocytic processing and intracellular re-localization was demonstrated in patient cells and disease model knock-out mouse neurons. To identify early affected cellular and metabolic pathways in INCL, knock-out mouse neurons were studied by global transcript profiling and functional analysis. The gene expression analysis revealed changes in neuronal maturation and cell communication strongly associated with the regulated secretory system. Furthermore, cholesterol metabolic pathways were found to be affected. Functional studies with the knock-out mouse model revealed abnormalities in neuronal maturation as well as key neuronal functions including abnormalities in intracellular calcium homeostasis and cholesterol metabolism. Together the findings, introduced in this thesis work, support the essential role of PPT1 in developing neurons as well as synaptic sites of mature neurons. Results of this thesis also elucidate early events in INCL pathogenesis revealing defective pathways ultimately leading to the neurodegenerative process. These results contribute to the understanding of the vital physiological function of PPT1 and broader knowledge of common cellular mechanisms behind neurodegeneration. These results add to the knowledge of these severe diseases offering basis for new approaches in treatment strategies.

Molecular organization of the gene encoding Xenopus laevis transforming growth factor-beta 5

Transforming growth factor-beta s (TGF-beta 5) are multifunctional polypeptides, known to influence proliferation and differentiation of many cell types. TGF-beta 5 cDNA was cloned from Xenopus laevis and this isoform is unique to the amphibians. Here, we report the isolation and characterization of the TGF-beta 5 genomic clones to determine the structure of TGF-beta 5 gene. The gene consists of seven exons and all intron-exon boundaries follow the GT-AG consensus. The organization of TGF-beta 5 gene was identical to that of the mammalian TGF-beta isoforms, with the exception of position of the first splice junction. We determined the size of TGF-beta 5 gene to be approximately 20 kb.

THE LEGACY OF GN RAMACHANDRAN AND THE DEVELOPMENT OF STRUCTURAL BIOLOGY IN INDIA

G.N. Ramachandran is among the founding fathers of structural molecular biology. He made pioneering contributions in computational biology, modelling and what we now call bioinformatics. The triple helical coiled coil structure of collagen proposed by him forms the basis of much of collagen research at the molecular level. The Ramachandran map remains the simplest descriptor and tool for validation of protein structures. He has left his imprint on almost all aspects of biomolecular conformation. His contributions in the area of theoretical crystallography have been outstanding. His legacy has provided inspiration for the further development of structural biology in India. After a pause, computational biology and bioinformatics are in a resurgent phase. One of the two schools established by Ramachandran pioneered the development of macromolecular crystallography, which has now grown into an important component of modern biological research in India. Macromolecular NMR studies in the country are presently gathering momentum. Structural biology in India is now poised to again approach heights of the kind that Ramachandran conquered more than a generation ago.

Molecular and Functional Characterization of RecD, a Novel Member of the SF1 Family of Helicases, from Mycobacterium tuberculosis

Background: The heterotrimeric M. tuberculosis RecBCD complex, or each of its individual subunits, remains uncharacterized. Results: MtRecD exists as a homodimer in solution, catalyzes ssDNA-dependent ATP hydrolysis, unwinding of DNA replication/recombination intermediates, and interacts with RecA. Conclusion: MtRecD possesses strong 5 3- and weak 3 5-helicase activities. Significance: These findings provide insights into the mechanism underlying DSB repair and homologous recombination in mycobacteria. The annotated whole-genome sequence of Mycobacterium tuberculosis revealed the presence of a putative recD gene; however, the biochemical characteristics of its encoded protein product (MtRecD) remain largely unknown. Here, we show that MtRecD exists in solution as a stable homodimer. Protein-DNA binding assays revealed that MtRecD binds efficiently to single-stranded DNA and linear duplexes containing 5 overhangs relative to the 3 overhangs but not to blunt-ended duplex. Furthermore, MtRecD bound more robustly to a variety of Y-shaped DNA structures having 18-nucleotide overhangs but not to a similar substrate containing 5-nucleotide overhangs. MtRecD formed more salt-tolerant complexes with Y-shaped structures compared with linear duplex having 3 overhangs. The intrinsic ATPase activity of MtRecD was stimulated by single-stranded DNA. Site-specific mutagenesis of Lys-179 in motif I abolished the ATPase activity of MtRecD. Interestingly, although MtRecD-catalyzed unwinding showed a markedly higher preference for duplex substrates with 5 overhangs, it could also catalyze significant unwinding of substrates containing 3 overhangs. These results support the notion that MtRecD is a bipolar helicase with strong 5 3 and weak 3 5 unwinding activities. The extent of unwinding of Y-shaped DNA structures was approximate to 3-fold lower compared with duplexes with 5 overhangs. Notably, direct interaction between MtRecD and its cognate RecA led to inhibition of DNA strand exchange promoted by RecA. Altogether, these studies provide the first detailed characterization of MtRecD and present important insights into the type of DNA structure the enzyme is likely to act upon during the processes of DNA repair or homologous recombination.

The legacy of G. N. Ramachandran and the development of structural biology in India

G. N. Ramachandran is among the founding fathers of structural molecular biology. He made pioneering contributions in computational biology, modelling and what we now call bioinformatics. The triple helical coiled coil structure of collagen proposed by him forms the basis of much of collagen research at the molecular level. The Ramachandran map remains the simplest descriptor and tool for validation of protein structures. He has left his imprint on almost all aspects of biomolecular conformation. His contributions in the area of theoretical crystallography have been outstanding. His legacy has provided inspiration for the further development of structural biology in India. After a pause, computational biology and bioinformatics are in a resurgent phase. One of the two schools established by Ramachandran pioneered the development of macromolecular crystallography, which has now grown into an important component of modern biological research in India. Macromolecular NMR studies in the country are presently gathering momentum. Structural biology in India is now poised to again approach heights of the kind that Ramachandran conquered more than a generation ago.

Molecular evolution of microcephalin, a gene determining human brain size

Microcephalin gene is one of the major players in regulating human brain development. It was reported that truncated mutations in this gene can cause primary microcephaly in humans with a brain size comparable with that of early hominids. We studied the m

Molecular evolution of CXCR1, a G protein-coupled receptor involved in signal transduction of neutrophils

Human neutrophils are a type of white blood cell, which forms an early line of defense against bacterial infections. Neutrophils are highly responsive to the chemokine, interleukin-8 (IL-8) due to the abundant distribution of CXCR1, one of the IL-8 receptors on the neutrophil cell surface. As a member of the GPCR family, CXCR1 plays a crucial role in the IL-8 signal transduction pathway in neutrophils. We sequenced the complete coding region of the CXCR1 gene in worldwide human populations and five representative nonhuman primate species. Our results indicate accelerated protein evolution in the human lineage, which was likely caused by Darwinian positive selection. The sliding window analysis and the codon-based neutrality test identified signatures of positive selection at the N-terminal ligand/receptor recognition domain of human CXCR1.

Molecular evolution of a primate-specific microRNA family

Lineage-specific microRNA (miRNA) families may contribute to developmental novelties during evolution. However, little is known about the origin and evolution of new miRNA families. We report evidence of an Alu-mediated rapid expansion of miRNA genes in a

A NOVEL PLASMINOGEN-ACTIVATOR FROM SNAKE-VENOM - PURIFICATION, CHARACTERIZATION, AND MOLECULAR-CLONING

A novel plasminogen activator from Trimeresurus stejnegeri venom (TSV-PA) has been identified and purified to homogeneity. It is a single chain glycoprotein with an apparent molecular weight of 33,000 and an isoelectric point of pH 5.2. It specifically activates plasminogen through an enzymatic reaction. The activation of human native GIu-plasminogen by TSV-PA is due to a single cleavage of the molecule at the peptide bond Arg(561)-Val-(562). Purified TSV-PA, which catalyzes the hydrolysis of several tripeptide p-nitroanilide substrates, does not activate nor degrade prothrombin, factor X, or protein C and does not clot fibrinogen nor show fibrino(geno)lytic activity in the absence of plasminogen. The activity of TSV-PA was readily inhibited by phenylmethanesulfonyl fluoride and by p-nitrophenyl-p-guanidinobenzoate. Oligonucleotide primers designed on the basis of the N-terminal and the internal peptide sequences of TSV-PA were used for the amplification of cDNA fragments by polymerase chain reaction. This allowed the cloning of a full-length cDNA encoding TSV-PA from a cDNA library prepared from the venom glands. The deduced complete amino acid sequence of TSV-PA indicates that the mature TSV-PA protein is composed of 234 amino acids and contains a single potential N-gIycosylation site at Asn(1G1). The sequence of TSV-PA exhibits a high degree of sequence identity with other snake venom proteases: 66% with the protein C activator from Aghistrodon contortrix contortrix venom, 63% with batroxobin, and 60% with the factor V activator from Russell's viper venom. On the other hand, TSV-PA shows only 21-23% sequence similarity with the catalytic domains of u-PA and t-PA. Furthermore, TSV-PA lacks the sequence site that has been demonstrated to be responsible for the interaction of t-PA (KHRR) and u-PA (RRHR) with plasminogen activator inhibitor type 1.

Trimeresurus stejnegeri snake venom plasminogen activator - Site-directed mutagenesis and molecular modeling

The specific plasminogen activator from Trimeresurus stejnegeri venom (TSV-PA) is a serine proteinase presenting 23% sequence identity with the proteinase domain of tissue type plasminogen activator, and 63% with batroxobin, a fibrinogen clotting enzyme from Bothrops atrox venom that does not activate plasminogen. TSV-PA contains six disulfide bonds and has been successfully overexpressed in Escherichia coli (Zhang, Y., Wisner, A., Xiong, Y. L,, and Bon, C, (1995) J. Biol. Chem. 270, 10246-10255), To identify the functional domains of TSV-PA, we focused on three short peptide fragments of TSV-PA showing important sequence differences with batroxobin and other venom serine proteinases. Molecular modeling shows that these sequences are located in surface loop regions, one of which is next to the catalytic site, When these sequences were replaced in TSV-PA by the equivalent batroxobin residues none generated either fibrinogen-clotting or direct fibrinogenolytic activity, Two of the replacements had little effect in general and are not critical to the specificity of TSV-PA for plasminogen. Nevertheless, the third replacement, produced by the conversion of the sequence DDE 96a-98 to NVI, significantly increased the K-m for some tripeptide chromogenic substrates and resulted in undetectable plasminogen activation, indicating the key role that the sequence plays in substrate recognition by the enzyme.

Molecular dynamics research of G249 and S249 substitutions of p53 protein.

,The molecular dynamics research of the core domain of p53 protein crystal structure shows that besides the stability in biochemistry this domain also shows a high stability in molecular mechanics. Based on that work, the residue R249 was substituted with amino acids Gly and Ser respectively, and molecular dynamics researches were performed separately. The results show that these substitutions cause a relax tendency between loop2 and 3 domains, leading to an alteration of the whole conformation of p53 core domain and ruining its stability. The results visually explains the mechanism of p53 changes in immunological and biochemical reactions, which are caused by 249 residue substitutions from 3-D structure variations.

Molecular characterization of Trimeresurus stejnegeri venom L-amino acid oxidase with potential anti-HIV activity

A novel L-amino acid oxidase, named TSV-LAO, has been purified and cloned from the snake Trimeresurus stejnegeri. Fifty percentage cytotoxic concentrations (CC50) of TSV-LAO on C8166 cells were 24 and 390 nM in the absence or presence of catalase (400nM), respectively. However, at concentrations that showed little effect on cell viability, TSV-LAO displayed dose dependent inhibition on HIV-1 infection and replication. The antiviral selectivity indexes (CC50/EC50) were 16 and 6, respectively, corresponding to the measurements of syncytium formation and HIV-1 p24 antigen expression. Interestingly, the presence of catalase resulted in an increase of its antiviral selectivity to 52 and 38. Under the same conditions, no anti-HIV-1 activity was observed by exogenous addition of H2O2. The complete amino acid sequence of TSV-LAO, as deduced from its cDNA, exhibits a high degree of sequence identity with other snake venom LAOs. (C) 2003 Elsevier Inc. All rights reserved.

Molecular systematics of pikas (genus Ochotona) inferred from mitochondrial DNA sequences

The phylogenetic relationships among worldwide species of genus Ochotona were investigated by sequencing mitochondrial cytochrome b and ND4 genes. Parsimony and neighbor-joining analyses of the sequence data yielded congruent results that strongly indicated three major clusters: the shrub-steppe group, the northern group, and the mountain group. The subgeneric classification of Ochotona species needs to be revised because each of the two subgenera in the present classification contains species from the mountain group. To solve this taxonomic problem so that each taxon is monophyletic, i.e., represents a natural clade, Ochotona could be divided into three subgenera, one for the shrub-steppe species, a second for the northern species, and a third for the mountain species. The inferred tree suggests that the differentiation of this genus in the Palearctic Region was closely related to the gradual uplifting of the Tibet (Qinghai-Xizang) Plateau, as hypothesized previously, and that vicariance might have played a major role in the differentiation of this genus on the Plateau, On the other hand, the North American species, O. princeps, is most likely a dispersal event, which might have happened during the Pliocene through the opening of the Bering Strait. The phylogenetic relationships within the shrub-steppe group are worth noting in that instead of a monophyletic shrub-dwelling group, shrub dwellers and steppe dwellers are intermingled with each other. Moreover, the sequence divergence within the sister tars of one steppe? dweller and one shrub dweller is very low. These findings support the hypothesis that pikes have entered the steppe environment several times and that morphological similarities within steppe dwellers were due to convergent evolution. (C) 2000 Academic Press.

Molecular systematics of Xenocyprinae (Teleostei : Cyprinidae): Taxonomy, biogeography, and coevolution of a special group restricted in east Asia

We surveyed mitochondrial DNA (mtDNA) sequence variation in the subfamily Xenocyprinae from China and used these data to estimate intraspecific, interspecific, and intergeneric phylogeny and assess biogeographic scenarios underlying the geographic structu