Development of a high-throughput screen targeting caspase-8-mediated cleavage of the amyloid precursor protein

Caspases, effectors of apoptosis, are key mediators of neuronal death in several neurodegenerative diseases. Caspase-8 and caspase-6 have been implicated in the pathogenesis of amyotrophic lateral sclerosis, multiple sclerosis, Parkinson’s disease, and Alzheimer’s disease (AD). ß-Amyloid precursor protein (APP) is cleaved at Asp664 in its intracellular domain by caspase-8. We and other laboratories recently showed that obliteration of the caspase cleavage site on APP alleviates functional AD-like deficits in a mouse model. Therefore, caspase cleavage of APP constitutes a potential novel target for therapeutic intervention. To identify chemical inhibitors of caspase-8 cleavage, we screened a subset of the chemical library at the Harvard NeuroDiscovery Center’s Laboratory for Drug Discovery in Neurodegeneration. We show that caspase-8, but not caspase-1, -3, or -9, cleaves a biotinylated peptide derived from APP at Asp664, and we report the development of a sensitive high-throughput assay for caspase-8 cleavage of APP and the use of that assay for the identification of specific small molecule “hit” compounds that potently inhibit Asp664 cleavage of APP. Furthermore, we demonstrate that one of these compounds (LDN-0021835) inhibits the cleavage of APP at Asp664 in cell-based assays.     

Apolipoprotein A-V dependent modulation of plasma triacylglycerol: a puzzlement

The discovery of apolipoprotein A-V (apoA-V) in 2001 has raised a number of intriguing questions about its role in lipid transport and triglyceride (TG) homeostasis. Genome wide association studies (GWAS) have consistently identified APOA5 as a contributor to plasma TG levels. Single nucleotide polymorphisms (SNP) within the APOA5 gene locus have been shown to correlate with elevated plasma TG. Furthermore, transgenic and knockout mouse models support the view that apoA-V plays a critical role in maintenance of plasma TG levels. The present review describes recent concepts pertaining to apoA-V SNP analysis and their association with elevated plasma TG. The interaction of apoA-V with glycosylphosphatidylinositol-anchored high-density lipoprotein binding protein 1 (GPIHBP1) is discussed relative to its postulated role in TG-rich lipoprotein catabolism. The potential role of intracellular apoA-V in regulation of TG homeostasis, as a function of its ability to associate with cytosolic lipid droplets, is reviewed. While some answers are emerging, numerous mysteries remain with regard to this low abundance, yet potent, modulator of TG homeostasis. Given the strong correlation between elevated plasma TG and heart disease, there is great scientific and public interest in deciphering the numerous biological riddles presented by apoA-V. This article is part of a Special Issue entitled Triglyceride Metabolism and Disease.     

Conserved Ser/Arg-rich motif in PPZ orthologs from fungi is important for its role in cation tolerance

PPZ1 orthologs, novel members of a phosphoprotein phosphatase family of phosphatases, are found only in fungi. They regulate diverse physiological processes in fungi e.g. ion homeostasis, cell size, cell integrity, etc. Although they are an important determinant of salt tolerance in fungi, their physiological role remained unexplored in any halotolerant species. In this context we report here molecular and functional characterization of DhPPZ1 from Debaryomyces hansenii, which is one of the most halotolerant and osmotolerant species of yeast. Our results showed that DhPPZ1 knock-out strain displayed higher tolerance to toxic cations, and unlike in Saccharomyces cerevisiae, Na(+)/H(+) antiporter appeared to have an important role in this process. Besides salt tolerance, DhPPZ1 also had role in cell wall integrity and growth in D. hansenii. We have also identified a short, serine-arginine-rich sequence motif in DhPpz1p that is essential for its role in salt tolerance but not in other physiological processes. Taken together, these results underscore a distinct role of DhPpz1p in D. hansenii and illustrate an example of how organisms utilize the same molecular tool box differently to garner adaptive fitness for their respective ecological niches.     

In silico proteomic characterization of human epidermal growth factor receptor 2 (HER-2) for the mapping of high affinity antigenic determinants against breast cancer

Multiple different approaches are being used to activate the immune system against breast cancer. Vaccine therapy in general follows the principle that injections of various substances ultimately result in the presentation of tumor peptides to the patient’s immune system. We proposed a potential in silico DNA vaccine against breast cancer by integrating high affinity T cell (MHC-I and MHC-II) and B cell (continuous and discontinuous) epitopes. The matching of the HLA haplotype and antigen was performed to provide the appropriate peptide epitope suitable for majority of the patients. The immunogenic nature of the antigenic construct was also enhanced by the administration of consensus epitopes. The potency of DNA vaccines depends on the efficient expression and presentation of the encoded antigen of interest and the chances of efficient expression of our antigenic construct in host organism was also verified by in silico approaches. An attempt was made to overcome the limited potency of the DNA vaccine by targeting DNA to professional antigen-presenting cells (APCs). A higher immune response theoretically corresponds to a higher survival rate of patients. Therefore, optimization studies were also employed to enhance the immunogenicity of proposed in silico DNA vaccine.     

Modulation of Leishmania major aquaglyceroporin activity by a mitogen-activated protein kinase

Leishmania major aquaglyceroporin (LmjAQP1) adventitiously facilitates the uptake of antimonite [Sb(III)], an active form of Pentostam® or Glucantime®, which are the first line of defence against all forms of leishmaniasis. The present paper shows that LmjAQP1 activity is modulated by the mitogen‐activated protein kinase, LmjMPK2. Leishmania parasites coexpressing LmjAQP1 and LmjMPK2 show increased Sb(III) uptake and increased Sb(III) sensitivity. When subjected to a hypo‐osmotic stress, these cells show faster volume recovery than cells expressing LmjAQP1 alone. LmjAQP1 is phosphorylated in vivo at Thr‐197 and this phosphorylation requires LmjMPK2 activity. Lys‐42 of LmjMPK2 is critical for its kinase activity. Cells expressing altered T197A LmjAQP1 or K42A LmjMPK2 showed decreased Sb(III) influx and a slower volume recovery than cells expressing wild‐type proteins. Phosphorylation of LmjAQP1 led to a decrease in its turnover rate affecting LmjAQP1 activity. Although LmjAQP1 is localized to the flagellum of promastigotes, upon phosphorylation, it is relocalized to the entire surface of the parasite. Leishmania mexicana promastigotes with an MPK2 deletion showed reduced Sb(III) uptake and slower volume recovery than wild‐type cells. This is the first report where a parasite aquaglyceroporin activity is post‐translationally modulated by a mitogen‐activated protein kinase.     

Deoxycholic acid impairs glycosylation and fucosylation processes in esophageal epithelial cells

It is generally accepted that esophageal adenocarcinoma arises from a Barrett's metaplastic lesion. Altered glycoprotein expression has been demonstrated in tissue from patients with Barrett's esophagus and esophageal cancer but the mechanisms regarding such changes are unknown. The bile acid deoxycholic acid (DCA) alters many cell signaling pathways and is implicated in esophageal cancer progression. We have demonstrated that DCA disrupts Golgi structure and affects protein secretion and glycosylation processes in cell lines derived from normal squamous epithelium (HET-1A) and Barrett's metaplastic epithelium (QH). Cell surface expression of glycans was identified using carbohydrate-specific probes (wheat germ agglutinate, conconavalin A, peanut agglutinin, lithocholic acid and Ulex europaeus agglutinin) that monitored N-glycosylation, O-glycosylation and core fucosylation in resting and DCA-treated cells. DCA altered intracellular localization and reduced cell surface expression of N-acetyl-D-glucosamine, α-methyl-mannopyranoside (Man/Glc) and fucose in both cell lines. Furthermore, DCA reduced the expression of epithelial growth factor receptor and E-cadherin in a manner analogous to treatment of cells with the N-glycan biosynthesis inhibitor tunicamycin. This is the first study to identify an altered Golgi structure and glycomic profile in response to DCA in esophageal epithelial cells, a process which could potentially contribute to metaplasia, dysplasia and cancer of the esophagus.     

Calreticulin transacetylase: a novel enzyme-mediated protein acetylation by acetoxy derivatives of 3-alkyl-4-methylcoumarins

Our earlier investigations culminated in the discovery of a unique membrane-bound enzyme Calreticulin transacetylase (CRTAase) in mammalian cells catalyzing the transfer of acetyl group from polyphenolic acetates (PAs) to certain functional proteins viz. Glutathione S-transferase (GST), NADPH Cytochrome c reductase and Nitric oxide synthase (NOS) resulting in the modulation of their biological activities. In order to develop SAR study, herein, we studied the influence of alkyl group at C-3 position of acetoxy coumarins on the CRTAase activity. The alkylated acetoxy coumarins lead to inhibition of catalytic activity of GST, and ADP induced platelet aggregation by the way of activation of platelet Nitric oxide synthase (NOS). Furthermore, the increase in size of the coumarin C-3 alkyl group was found to decrease the CRTAase activity.     

Biotransformation of Acetamide to Acetohydroxamic Acid at Bench Scale Using Acyl Transferase Activity of Amidase of <Emphasis Type="Italic">Geobacillus pallidus</Emphasis> BTP-5x MTCC 9225

The bioprocess employing acyl transferase activity of intracellular amidase of Geobacillus pallidus BTP-5x MTCC 9225 was harnessed for the synthesis of pharmaceutically important acetohydroxamic acid. G. pallidus BTP-5x exhibited highest acyl transferase activity with acetamide: hydroxylamine in ratio of 1:5 in 0.1 M NaH₂PO₄/Na₂HPO₄ buffer (pH 7.5) at 65°C. In one liter fed-batch reaction containing 1:5 ratio of two substrates total of eight feedings of 0.05 M/20 min of acetamide were made and it was found that maximum acetohydroxamic production was achieved at 3:5 ratios of substrate and cosubstrate. In 1 l bench scale batch reaction containing 0.3 M acetamide, 0.5 M hydroxylamine in 0.1 M NaH₂PO₄/Na₂HPO₄ buffer (pH 7.5, 50°C, 400 rpm) and 0.5 mg/ml (dry cell weight) of whole cells of G. pallidus BTP-5x (as biocatalyst) resulted in an yield of 0.28 M of acetohydroxamic acid after 20 min reaction time at 50°C. The acetamide bioconversion rate was 90–95% (mol mol⁻¹) and 51 g powder containing 40% (w/w) acetohydroxamic acid was recovered after lyophilization.     

Phylogenetic footprinting: a boost for microbial regulatory genomics

Phylogenetic footprinting is a method for the discovery of regulatory elements in a set of homologous regulatory regions, usually collected from multiple species. It does so by identifying the best conserved motifs in those homologous regions. There are two popular sets of methods-alignment-based and motif-based, which are generally employed for phylogenetic methods. However, serious efforts have lacked to develop a tool exclusively for phylogenetic footprinting, based on either of these methods. Nevertheless, a number of software and tools exist that can be applied for prediction of phylogenetic footprinting with variable degree of success. The output from these tools may get affected by a number of factors associated with current state of knowledge, techniques and other resources available. We here present a critical apprehension of various phylogenetic approaches with reference to prokaryotes outlining the available resources and also discussing various factors affecting footprinting in order to make a clear idea about the proper use of this approach on prokaryotes.