Molekulare Pinzetten mit zusätzlichen Bindungsstellen gegen Protein Aggregation

My doctoral thesis aims at the development of molecular tweezers with increased affinity and specificity for amyloidogenic proteins. A particular focus lies on the amyloid-β protein, a major pathological hallmark of Alzheimer’s disease. In Klärner group, a molecular tweezer called the phosphate tweezer was developed which selectively binds to the amino acid lysine and arginine both in peptides and proteins, and thereby inhibits the aggregation and toxicity of amyloid proteins. However, the symmetrical prototype which carries two phosphate anions at its periphery, does not distinguish between well-accessible lysines on a protein surface. In my thesis I try to develop symmetrical and unsymmetrical molecular tweezers with new recognition sites, which become selective for a given protein/epitope. In the first part of the work, I explored the chemical and biological effects of replacing the phosphate anions by other anionic groups of biological relevance. For this purpose, I synthesized the sulfate tweezer and carried out comparative binding studies among the phosphate, phosphonate, sulfate and methyl carboxylate tweezers with basic amino acids and relevant peptide guests. Using NMR, fluorescence, ITC, and molecular simulation, I observed substantial differences in the binding characteristics of all the four tweezer types toward their peptide guests. In collaboration, we studied the inhibition potential of the phosphate tweezer against the pathologic aggregation of the islet amyloid polypeptide (IAPP), a protein involved in the type II diabetes. For more structural insight, I carried out an investigation about complexes between the tweezer and IAPP fragments by NMR, fluorescence and ITC experiments. Biological experiments and QM/MM calculations were performed in collaborations. In the second part of my work, I synthesized several unsymmetrical molecular tweezers that carry a phosphate group on one side and several different linkers on the other side of tweezer’s central benzene ring. These tweezers with attached linker units display varying affinities for lysine and arginine derivatives from milimolar to micromolar. Biological experiments of these unsymmetrical tweezers are currently underway in Prof. Bitan’s lab (neurology, UCLA). Lastly, I developed an even more general and potent synthetic route for the synthesis of unsymmetrical molecular tweezers which keep both the phosphate groups on the tweezer. One of the two phosphate groups is substituted with an alkyne or an ester linker unit. The alkyne linker tweezer can be coupled with an azide containing recognition site via alkyne-azide click reaction, whereas, the ester linker tweezer can be coupled with an amine containing recognition site to synthesize more powerful unsymmetrical molecular tweezer

Cathepsin B cysteine protease gene is upregulated during leaf senescence and exhibits differential expression behavior in response to phytohormones in Picrorhiza kurrooa Royle ex Benth.

AbstractMedicinal importance of Picrorhiza (Picrorhiza kurrooa Royle ex Benth — an herb of western Himalayan region) and its endangered status in Red Data Book presses an urgent need for intensive R&D interventions towards ensuring its availability for the medicinal use, its sustainability and improvement. The present study was conducted on cathepsin B cysteine protease in Picrorhiza. Cathepsin B cysteine protease has been reported to function in diverse processes such as senescence, abscission, programmed cell death, fruit ripening and in response to pathogen and pest attacks. A full-length cDNA-Pk-cbcp encoding cathepsin B-like cysteine protease was cloned from Picrorhiza. The full length Pk-cbcp cDNA consisted of 1369bp with an open reading frame of 1080bp, 80bp 5′ untranslated region and 209bp 3′ untranslated region. The deduced Pk-cbcp protein contained 359 amino acids with a molecular weight of 39.981kDa and an isoelectric point of 5.75. Secondary structure analysis revealed that Pk-cbcp had 28.97% α-helices, 14.48% β-turns, 19.50% extended strands and 37.05% random coils. Semi-quantitative PCR analysis revealed 157% higher expression of Pk-cbcp during senescence compared to that of pre-senescence. Further, application of phytohormones abscisic acid, jasmonic acid and cytokinin influenced the temporal expression status of Pk-cbcp. Abscisic acid and jasmonic acid increased the expression level whereas cytokinin reduced the expression. The findings suggest the role of Pk-cbcp in leaf senescence in Picrorhiza which may be differentially mediated through phytohormones

Anthocyanin-Rich Vegetables for Human Consumption—Focus on Potato, Sweetpotato and Tomato

Malnutrition, unhealthy diets, and lifestyle changes have become major risk factors for non-communicable diseases while adversely impacting economic growth and sustainable development. Anthocyanins, a group of flavonoids that are rich in fruits and vegetables, contribute positively to human health. This review focuses on genetic variation harnessed through crossbreeding and biotechnology-led approaches for developing anthocyanins-rich fruit and vegetable crops. Significant progress has been made in identifying genes involved in anthocyanin biosynthesis in various crops. Thus, the use of genetics has led to the development and release of anthocyanin-rich potato and sweet potato cultivars in Europe and the USA. The purple potato ’Kufri Neelkanth’ has been released for cultivation in northern India. In Europe, the anthocyanin-rich tomato cultivar ‘Sun Black’ developed via the introgression of Aft and atv genes has been released. The development of anthocyanin-rich food crops without any significant yield penalty has been due to the use of genetic engineering involving specific transcription factors or gene editing. Anthocyanin-rich food ingredients have the potential of being more nutritious than those devoid of anthocyanins. The inclusion of anthocyanins as a target characteristic in breeding programs can ensure the development of cultivars to meet the nutritional needs for human consumption in the developing world

Exploring possibilities of enhancing water use efficiency in potato: A review

Climate change threatens the global agriculture sustainability. Among different kinds of abiotic stresses, water stress is the most devastating component which curtails potato crop productivity. Our recent knowledge is limited concerning water stress tolerance and water use efficiency in potato. Many efforts are being made by the scientific community to reduce water use and to produce “more crop per drop”. This review elaborates quantitative and qualitative aspects of multiple stress mechanisms and their regulating system related to present scenario of water use efficiency (WUE) requirements. WUE can only be improved by using multidisciplinary promising research approaches like molecular breeding, high throughput genotyping, multi-gene transfer and bioinformatics applications to unleash the information needed to exploitation of required traits in potato

Developing germplasm and promoting consumption of anthocyanin-rich grains for health benefits

Malnutrition, unhealthy diets, and lifestyle changes are the major risk factors for overweight and obesity-linked chronic diseases in humans adversely impact achieving sustainable development goals. Colored grains are a source of anthocyanins, a group of flavonoids, that contribute positively to human health. This review focuses on genetic variation harnessed through breeding and biotechnology tools for developing anthocyanin-rich grain crops. Agronomic practices, genotype × environment interactions, different stresses, seed development and seed maturity are factors that impact the content and composition of anthocyanins. Significant progress has been made in characterizing genes associated with anthocyanin biosynthesis in cereal and other crops. Breeding has led to the development and release of grain anthocyanin-rich crop cultivars in Europe, America and in some countries in Asia. Notably, genetic engineering utilizing specific transcription factors and gene editing has led to the development of anthocyanin-rich genetic variants without any significant yield penalty. A variety of food products derived from colored grains or flours are now available in grocery stores and supermarkets worldwide. The public perception about anthocyanin-rich food is positive, but availability, affordability, and willingness to pay a higher price than before limit consumption. Together with other seed nutrition traits in breeding programs the inclusion of anthocyanins can ensure the development of cultivars that meet nutrition needs of humans, especially in the developing world

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Allosterically Regulated Phosphatase Activity from Peptide-PNA Conjugates Folded Through Hybridization

The importance of spatial organization in short peptide catalysts is well recognized. We synthesized and screened a library of peptides flanked by peptide nucleic acids (PNAs) such that the peptide would be constrained in a hairpin loop upon hybridization. A screen for phosphatase activity led to the discovery of a catalyst with >25-fold rate acceleration over the linear peptide. We demonstrated that the hybridization-enforced folding of the peptide is necessary for activity, and designed a catalyst that is allosterically controlled using a complementary PNA sequence

Hill slope instability of Nainital City, Kumaun Lesser Himalaya, Uttarakhand, India

Nainital City of Kumaun Lesser Himalaya is prone to mass wasting processes during monsoon season, which mischievously triggers the hill slope instability in this region. Slate, dolomitic limestone, silty sandstone and rhythmite of the Krol Formation are the main rock types. The present study focuses on the investigation of slope stability in the region in terms of potential seismicity and landslide. Geological and geotechnical mapping indicates that the major portion of the area is characterized by slope wash materials and buildings. The combination of 3–4 joint sets with one random joint is the main structure at outcrops. The major geological structures of this area are Nainital lake fault passing from the center of the lake, Main Boundary Thrust at SW, and Khuriya Fault passing from the SE direction of Nainital City. This work finds that different types of discontinuities (e.g. joints and faults), overburden due to unplanned civil structures, and neotectonic activity in the vicinity of this area affect the stability of the city. The slate forms the base of the city, dipping slightly towards the lake side along the NW direction, thus accelerating the instability of this area. Rock mass rating (RMR), slope mass rating, factor of safety (FOS) and graphical analysis of the discontinuity for slope kinematics indicate that the study area is a landslide-prone zone. This study can facilitate reducing the risk of human life, and contribute to the ongoing construction works in the area. Keywords: Hill slope instability, Himalaya region, Wedge failure, Rock mass rating (RMR), Factor of safety (FOS