Development, validation and application of single molecule molecular inversion probe based novel integrated genetic screening method for 29 common lysosomal storage disorders in India.

BACKGROUND: Current clinical diagnosis pathway for lysosomal storage disorders (LSDs) involves sequential biochemical enzymatic tests followed by DNA sequencing, which is iterative, has low diagnostic yield and is costly due to overlapping clinical presentations. Here, we describe a novel low-cost and high-throughput sequencing assay using single-molecule molecular inversion probes (smMIPs) to screen for causative single nucleotide variants (SNVs) and copy number variants (CNVs) in genes associated with 29 common LSDs in India. RESULTS: 903 smMIPs were designed to target exon and exon-intron boundaries of targeted genes (n = 23; 53.7 kb of the human genome) and were equimolarly pooled to create a sequencing library. After extensive validation in a cohort of 50 patients, we screened 300 patients with either biochemical diagnosis (n = 187) or clinical suspicion (n = 113) of LSDs. A diagnostic yield of 83.4% was observed in patients with prior biochemical diagnosis of LSD. Furthermore, diagnostic yield of 73.9% (n = 54/73) was observed in patients with high clinical suspicion of LSD in contrast with 2.4% (n = 1/40) in patients with low clinical suspicion of LSD. In addition to detecting SNVs, the assay could detect single and multi-exon copy number variants with high confidence. Critically, Niemann-Pick disease type C and neuronal ceroid lipofuscinosis-6 diseases for which biochemical testing is unavailable, could be diagnosed using our assay. Lastly, we observed a non-inferior performance of the assay in DNA extracted from dried blood spots in comparison with whole blood. CONCLUSION: We developed a flexible and scalable assay to reliably detect genetic causes of 29 common LSDs in India. The assay consolidates the detection of multiple variant types in multiple sample types while having improved diagnostic yield at same or lower cost compared to current clinical paradigm.

Late infantile and adult-onset metachromatic leukodystrophy due to novel missense variants in the PSAP gene: Case report from India.

Metachromatic leukodystrophy (MLD) due to Sap-B deficiency is a rare autosomal recessive disorder caused due to biallelic variants in the PSAP gene. The PSAP gene encodes a precursor protein prosaposin, which is subsequently cleaved to form four active glycoproteins: Sap-A, Sap-B, Sap-C, and Sap-D. In case of deficiency of the sphingolipid activator protein Sap-B, there is a gradual accumulation of cerebroside-3-sulfate in the myelin of the nervous system resulting in progressive demyelination. Only 12 variants have been reported in the PSAP gene causing Sap-B deficiency to date. Here, we report two cases of MLD due to Sap-B deficiency (late-infantile and adult-onset form) harboring two novel missense variants c.688T > G and c.593G > A in the PSAP gene respectively. This study reports the third case of adult-onset MLD due to Sap-B deficiency in the world. The proband, a 3-year-old male child presented with complaints of hypotonia, lower limb tremors and global developmental delay. His MRI showed hyperintense signals in the bilateral cerebellar white matter. Overall, the findings were suggestive of metachromatic leukodystrophy. The second case was a 19-year-old male child with clinical features of regression of speech, gait ataxia and bilateral tremors referred to our clinic. MRI data suggested metachromatic leukodystrophy. Normal enzyme activity of arylsulfatase-A led to a suspicion of saposin B deficiency. For both cases, targeted sequencing was performed. This identified homozygous variant c.688T > G (p.Cys230Gly) and c.593G > A (p.Cys198Tyr) in exon 6 of the PSAP gene, respectively.

Design and characterization of a biosensor with lipase immobilized nanoparticles in polymer film for the detection of triglycerides.

High levels of triglycerides in blood can harden and block the arteries increasing the risk of heart disease and strokes. Triglycerides are important constituents of oils and fats used in various foods. The triglyceride content in commercial preparations of oils is estimated using conventional methods. In the present study, an electrochemical biosensor with lipase immobilized novel conductive polymer film has been developed for estimating triglyceride content in a variety of products. The portable biosensor can bring down the detection costs dramatically and can be used for varied purposes. It is based on cyclic voltammetry and has a three-electrode configuration system. Glassy carbon electrode is functionalized with nanoparticles embedded in polyethyleneimine and lipase is immobilized using glutaraldehyde. The strategy increases the electrochemical conductance manifold and overcomes the hindrance to lipase posed by membranes as it is oriented on the outside of the membrane. Thus, it increases the sensitivity and selectivity of detection. Results of scanning electron microscopy and FT-IR spectroscopy were used for characterizing the electrode surface. Linear range of the electrode for triglycerides is 100-500 mg/dL. The sensor was used successfully to determine triglyceride content in several real samples and the average recovery values lie from 95.47 % to 101.05 %.

Characterization of the chimeric protein cUBC1 engineered by substituting the linker of E2-25K into UBC1 enzyme of Saccharomyces cerevisiae.

Ubiquitination is an important posttranslational modification of proteins in eukaryotic cells, wherein ubiquitin molecules are conjugated to target proteins. Ubiquitination is catalyzed by the cascade of ubiquitin activating enzyme (E1), ubiquitin conjugating enzyme (E2), and ubiquitin ligase (E3). The number of E2s encoded in eukaryotes partly explains their contribution to the inherent specificity of the ubiquitin system. The ubiquitin conjugating enzyme UBC1 of Saccharomyces cerevisiae participates the degradation of short-lived and abnormal proteins. UBC1 consists of two well-defined domains separated by a long flexible linker. E2-25K, the human homolog of UBC1 is crucial to neurons and its failure leads to neurodegenerative disorders. The linker of UBC1 is of 22 amino acids, while that of E2-25K has 6 amino acids. To understand the importance of the linker, the chimeric protein, cUBC1 was constructed by substituting the linker of E2-25K in UBC1. cUBC1 shows minor changes in its secondary structure. cUBC1 expression in ubc1 deletion mutants showed no effect over growth, thermotolerance and resistance to antibiotic stress. However, survival under heat stress was enhanced with cUBC1. Western blot analysis of the enzymatic activity showed cUBC1 performed equally well as UBC1. Hence, cUBC1 demonstrates that the shorter linker increased the stability of UBC1.

Assessing suitability of commercial fibre reinforced plastic solar still for sustainable potable water production in rural India through detailed energy-exergy-economic analyses and environmental impacts.

Fibre reinforced plastic (FRP) basin solar still is manufactured and marketed commercially in developing/underdeveloped nations. Unfortunately, these commercial units have not been widely adopted by the public due to its low water productivity. In the present investigation, an effort has been made to assess the performance and enviro-economic aspects of the commercial unit and its low-cost improved versions under Coimbatore climatic conditions through real-time experiments and mathematical models, respectively. The commercial unit has distillate productivity of about 2.76 L/d under cumulative solar radiation intensity of 24.37 MJ/m2d and it increased up to 6.03 L/d with the addition of black dye and thermocol insulation. Yearly average distillate production rate, thermal efficiency, and exergy efficiency of the improved version is about 4.36 L/d, 53.62%, and 6.89%, respectively. The greenhouse payback time of the commercial unit and insulated commercial unit with black dye is about 2.39 and 1.81 Years, respectively. Global warming potential, acidification potential and photochemical oxidant formation potential of the commercial unit decreased from 0.27 to 0.12 kg of carbon di-oxide eq/L of distillate, 2.40 to 1.05 g of sulphur di-oxide eq/L of distillate and 0.73 to 0.32 g of ethylene eq/L of distillate, respectively with the incorporation of low-cost improvement techniques. The sustainability index of the improved version is around 5.94% higher than the sustainability index of the commercial unit. The distillate production cost of improved version (1.32 INR/L) is closer to reverse osmosis (RO) water supplied to Indian houses (1.00 INR/L). The incorporation of low-cost improved techniques to the commercial unit makes it an attractive option for sustainable potable water production. However, proper marketing and awareness strategies must be adopted by stakeholders to make this improved version acceptable among the public.

Probing the effects of double mutations on the versatile protein ubiquitin in Saccharomyces cerevisiae.

Ubiquitin is an indispensable protein of eukaryotic origin with an extraordinarily high degree of sequence conservation. It is used to tag proteins post-translationally and the process of ubiquitination regulates the activity of the modified proteins or drives them for degradation. Double mutations produce varied effects in proteins, depending on the structural relationship of the mutated residues, their role in the overall structure and functions of a protein. Six double mutants derived from the ubiquitin mutant UbEP42, namely S20F-A46S, S20F-L50P, S20F-I61T, A46S-L50P, A46S-I61T, and L50P-I61T, have been studied here to understand how they influence the ubiquitination related functions, by analysing their growth and viability, Cdc28 levels, K-48 linked polyubiquitination, UFD pathway, lysosomal degradation, endosomal sorting, survival under heat, and antibiotic stresses. The double mutation L50P-I61T is the most detrimental, followed by S20F-I61T and A46S-I61T. The double mutations studied here, in general, make cells more sensitive than the wild type to one or the other stress. However, the excessive negative effects of L50P and I61T are compensated under certain conditions by S20F and A46S mutations. The competitive inhibition produced by these substitutions could be used to manage certain ubiquitination associated diseases.

Structural and Functional Characterisation of the Domains of Ubiquitin-Activating Enzyme (E1) of Saccharomyces cerevisiae.

Ubiquitin-activating enzyme (E1) is the first enzyme of the ubiquitination pathway and is required to activate ubiquitin. E1 of Saccharomyces cerevisiae is a large multidomain monomeric protein. There are no studies available on the domains of yeast E1 as independent entities. Four domains of E1 namely, first catalytic cysteine half-domain (FCCH), four-helix bundle (4HB), second catalytic cysteine half-domain (SCCH) and ubiquitin fold domain (UFD) were characterised to understand their structural and functional independence vis-a-vis full length E1. Spectroscopic characterisation using circular dichroism and fluorescence suggested that these domains can act as independent folding units and attain native-like secondary structure. The structural features obtained with the peptides SCCH and FCCH of S. cerevisiae bear a high degree of structural similarity to the corresponding fragments of mouse from literature. Nearly 50% of the residues of the 4HB domain of the S. cerevisiae sample showed helical conformation. They displayed a high degree of conservation when compared with 4HB of mouse with respect to their identity and arrangement. The fragment UFD of yeast formed an α/ß domain as in the whole protein and exhibited 45% homology with that of mouse, showing a similar arrangement of α and ß elements in its secondary structure. Overexpression of the domains in vivo indicated that the SCCH domain and to some extent UFD apparently interfere with cellular functions such as survival under various stresses.

Construction and Characterization of UBC4 Mutants with Single Residues Swapped from UBC5.

Ubiquitination is tightly regulated to control degradation, localization and function of various proteins. Ubiquitination is catalysed by three enzymes, namely E1, E2 and E3. The specificity shown by E2s for E3s holds key to regulation of ubiquitination. Here we focussed on the E2 enzymes, UBC4 and UBC5 of Saccharomyces cerevisiae, which are almost identical differing only by 11 residues. They show functional complementation in protein degradation, especially during stress response. Existence of two almost identical proteins suggests specialized requirement of one of them under selective conditions. To understand the reasons for the residue differences between them, mutations were introduced in the UBC4 gene to generate single residue variants by swapping with codons from UBC5. Though the variants are found to be functionally active in Δubc4Δubc5 strain of yeast, they cause reduced growth under normal conditions, altered survival under heat and antibiotic stresses, when compared with UBC4. The variants indicated decrease in protein stability theoretically. Hence, the residues of UBC5 individually do not confer any structural advantage to UBC4. Interactive proteins of UBC4 are nearly three times more than those of UBC5. UBC5, therefore, is a functionally minimized version, evolved as another means of regulation to meet cell stage specific needs.

Acetylcholine esterase enzyme doped multiwalled carbon nanotubes for the detection of organophosphorus pesticide using cyclic voltammetry.

Use of immobilized acetylcholine esterase (AChE) for detecting organophosphorus pesticides in water sources and body fluids can bring down the detection costs dramatically. In the present study, AChE was directly doped on multiwalled carbon nanotube (MWCNT) surface modified with carboxylic groups through amide bond and used for organophosphorus pesticide detection. Amide bond formation between MWCNTs and the enzyme molecules avoid use of any intermediate membranes, cross-linkers or binding materials. This strategy overcomes the hindrance to electron transfer posed by membranes or cross-linkers and increases the sensitivity of detection. MWCNTs carrying carboxyl groups were deposited on glassy carbon electrode and were subsequently immobilized with AChE. The activity of AChE was monitored by cyclic voltammetry after immobilization. Scanning electron microscopy and atomic force microscopy were used to characterize the electrode surface. FT-IR spectra were taken to characterize enzyme-MWCNT complex. Under optimized parameters, the electrode showed linear range between 10 and 50 nM, which is promising for detection of trace amounts of the pesticide. The lower and higher detection limits of the sensor are 0.1 nM and 500 nM respectively. The stability and reusability of the electrode were determined. Finally, successful detection of organophosphorus compounds in real samples established it as reliable for sensor applications.

Recent advances in carbon nanotube based electrochemical biosensors.

There is an increasing need for rapid, low cost, reusable, reliable and sensitive detection systems for diagnosing infectious diseases, metabolic disorders, rapidly advancing cancers and detecting the presence of environmental pollutants. Most traditional methods are invasive, slow, expensive and laborious, requiring highly specialized instruments. Introduction of biosensors with nanomaterials as transducers of signals have helped in removing the disadvantages associated with traditional detectors. The properties of high mechanical strength, better electrical conductivity and ability to serve as efficient signal transducers make carbon nanotubes (CNTs) ideal material for biosensor applications among the gamut of nanomaterials. Further, CNTs with their high surface areas, easily functionalizable surfaces for receptor immobilization are gaining importance in the construction of biosensors. The expanding field of CNTs bridges the physical sciences with biology, as chemical methods are employed to develop novel tools and platforms for understanding biological systems, in disease diagnosis and treatment. This review presents recent advances in surface functionalization of CNTs necessary for immobilization of enzymes and antibodies for biosensor applications and the methodologies used for the detection of a number of chemical and biological species. The review ends with a speculation on future prospects for CNTs in biology and medicine.

Structural changes induced by L50P and I61T single mutations of ubiquitin affect cell cycle progression while impairing its regulatory and degradative functions in Saccharomyces cerevisiae.

Posttranslational conjugation of ubiquitin to proteins either regulates their function directly or concentration through ubiquitination dependent degradation. High degree of conservation of ubiquitin's sequence implies structural and functional importance of the conserved residues. Ubiquitin gene of Saccharomyces cerevisiae was evolved in vitro by us to study the significance of conserved residues. Present study investigates the structural changes in the protein resulting from the single mutations UbS20F, UbA46S, UbL50P, UbI61T and their functional consequences in the SUB60 strain of S. cerevisiae. Expression of UbL50P and UbI61T decreased Cdc28 protein kinase, enhanced Fus3 levels, caused dosage dependent lethality and at sublethal level produced drastic effects on stress tolerance, protein sorting, protein degradation by ubiquitin fusion degradation pathway and by lysosomes. UbS20F and UbA46S produced insignificant effects over the cells. All four mutations of ubiquitin were incorporated into polyubiquitin. However, polyubiquitination with K63 linkage decreased significantly in cells expressing UbL50P and UbI61T. Structural studies on UbL50P and UbI61T revealed distorted structure with greatly reduced α-helical and elevated ß-sheet contents, while UbS20F and UbA46S show mild structural alterations. Our results on functional efficacy of ubiquitin in relation to structural integrity may be useful for designing inhibitors to investigate and modulate eukaryotic cellular dynamics.

Degrons of yeast and mammalian ornithine decarboxylase enzymes make potent combination for regulated targeted protein degradation.

Elevation of polyamine levels in eukaryotes leads to rapid degradation of ornithine decarboxylase (ODC), the first enzyme of polyamine biosynthesis pathway. ODC in yeast (yODC) has two domains, the Nα/ß domain consisting of α/ß barrel domain (α/ß) preceded by an overhang of 50 residues at its N-terminus (N50) and ß sheet domain at its C-terminus. Two degradation determinant signals or degrons in yODC sequence, namely the N50 and the antizyme-binding element (AzBE) housed in the α/ß domain, are responsible for its degradation by proteasomes. Antizyme (Az) induced under polyamine excess binds to AzBE and delivers ODC to proteasome, while the N50 threads the protein into proteasome. It was previously reported by us that the peptide Nα/ß of yODC acts as an independent transplantable degron, whose action can be modulated with the help of antizyme by varying polyamine levels. Mammalian ODC (mODC), in spite of its 40% sequence homology with yODC, is devoid of N50 of yODC and instead sports a C-terminal tail of 37 residues (CmODC). CmODC acts as an independent transplantable degron with no equivalent in yODC. The present study investigates the merits of employing the two degrons Nα/ß and CmODC together for targeted protein degradation by expressing them in a chimeric fusion with green fluorescent protein (GFP). Our results establish that under the regulation of antizyme, the signals Nα/ß and CmODC acting together enhance degradation better than either degron in isolation. The combination of Nα/ß and CmODC can be employed to study the function of novel proteins through their rapid removal.

Characterization of a Kunitz-type serine protease inhibitor from Solanum tuberosum having lectin activity.

Plant lectins and protease inhibitors constitute a class of proteins which plays a crucial role in plant defense. In our continuing investigations on lectins from plants, we have isolated, purified and characterized a protein of about 20 kDa, named PotHg, showing hemagglutination activity from tubers of Indian potato, Solanum tuberosum. De novo sequencing and MS/MS analysis confirmed that the purified protein was a Kunitz-type serine protease inhibitor having two chains (15 kDa and 5 kDa). SDS and native PAGE analysis showed that the protein was glycosylated and was a heterodimer of about 15 and 5 kDa subunits. PotHg agglutinated rabbit erythrocytes with specific activity of 640 H.U./mg which was inhibited by complex sugars like fetuin. PotHg retained hemagglutination activity over a pH range 4-9 and up to 80°C. Mannose and galactose interacted with the PotHg with a dissociation constant (Kd) of 1.5×10(-3) M and 2.8×10(-3) M, respectively as determined through fluorescence studies. Fluorescence studies suggested the involvement of a tryptophan in sugar binding which was further confirmed through modification of tryptophan residues using N-bromosuccinimide. Circular dichroism (CD) studies showed that PotHg contains mostly ß sheets (∼45%) and loops which is in line with previously characterized protease inhibitors and modeling studies. There are previous reports of Kunitz-type protease inhibitors showing lectin like activity from Peltophorum dubium and Labramia bojeri. This is the first report of a Kunitz-type protease inhibitor showing lectin like activity from a major crop plant and this makes PotHg an interesting candidate for further investigation.

Q2N and E64G double mutation of ubiquitin confers a stress sensitive phenotype on Saccharomyces cerevisiae.

The eukaryotic protein, ubiquitin harbours a parallel ß-bulge in its structure which is formed by residues Glu64(1), Ser65(2) and Gln2(X). Despite their low % frequency of occurrence in parallel ß-bulges, the residues Gln2 and Glu64 have been totally conserved in ubiquitin. In a previous study, two single mutants UbQ2N and UbE64G were constructed by replacing the residues Gln2 and Glu64 with Asn and Gly, respectively to understand their importance. The choice of the residues for substitution was made on the basis of their high preference for existence in parallel ß-bulge, so that the structure of mutants remains unaltered and any functional differences observed would highlight the importance of Gln2 and Glu64 in ubiquitin biology. The results from this study established that yeast cells expressing either UbQ2N or UbE64G, displayed functional differences with respect to survival upon exposure to cycloheximide and degradation of substrates by ubiquitin fusion degradation (UFD) pathway. It describes construction of the double mutant UbQ2N-E64G and its characterization. Our results showed expression of UbQ2N-E64G in stress hypersensitive SUB60 cells led to significant decrease in growth rate and prolonged half-life of substrates of UFD pathway, besides failure of complementation under heat and antibiotic stresses, providing the reason for conservation of Gln2 and Glu64 in ubiquitin sequence.

Engineering degrons of yeast ornithine decarboxylase as vehicles for efficient targeted protein degradation.

BACKGROUND: Ornithine decarboxylase (ODC), which catalyzes the first step of polyamine biosynthesis, undergoes rapid targeted degradation (TPD) with the help of its two degron sequences, namely the N-terminal 50 residues (N50) and α/ß domain (α/ß) housing antizyme binding element (AzBE), in response to increased polyamine levels. Antizyme binds to AzBE of ODC and delivers it to proteasome for degradation. Entire ODC was used as a tag to demonstrate TPD of chimeric proteins. METHODS: Here we fashioned three peptide sequences from yeast ODC to test their capability to act as degrons, namely N50, α/ß and Nα/ß (a combination of N50 and α/ß), and monitored their degradation potentials in chimeric proteins. We have examined the correlation between degradation potentials and structural integrity of the peptides, to find mechanistic explanations. RESULTS: Nα/ß with two signals in tandem is a better degron, under the regulation of antizyme. N50 like N44 reported earlier could drive chimeric proteins to degradation, while α/ß could not act as an independent degron. Strong correlation was observed between functional efficacy of the peptides and their structural integrity. N50, which was believed to be unstructured, displayed propensity for helical conformation. Nα/ß exhibited optimal structure, while α/ß failed to adopt native like conformation. CONCLUSIONS AND GENERAL SIGNIFICANCE: Functional efficacy of the degron Nα/ß is a consequence of its structural integrity. Nα/ß and N50 could target chimeric proteins to degradation. However, α/ß failed in the quest. Nα/ß, regulated by antizyme, is better suited than N50 for TPD to understand the function of novel proteins.

Functional characterization of dosage-dependent lethal mutation of ubiquitin in Saccharomyces cerevisiae.

Ubiquitin is a eukaryotic protein with 96% sequence conservation from yeast to human. Ubiquitin plays a central role in protein homeostasis and regulation of protein function. We have reported on the generation of variants of ubiquitin by in vitro evolution in Saccharomyces cerevisiae to advance our understanding of the role of the invariant amino acid residues of ubiquitin in relation to its function. One of the mutants generated, namely UbEP42, was a dosage-dependent lethal form of the ubiquitin gene, causing lethality to UBI4-deficient cells but not to ubiquitin wild-type cells. In the present study we investigated the functional reasons for the observed lethality. Expression of UbEP42 in a UBI4-deleted stress-sensitive strain resulted in an increased generation time due to a delayed S phase caused by decreased levels of Cdc28 protein kinase. Cells expressing UbEP42 displayed heightened sensitivity towards heat stress and exposure to cycloheximide. Furthermore, its expression had a negative effect on the degradation of substrates of the ubiquitin fusion degradation pathway. However, UbEP42 is incorporated into polyubiquitin chains. Collectively, our results establish that the effects seen with the mutant ubiquitin protein UbEP42 are not due to malfunction at the stage of polyubiquitination.

The ends and means of artificially induced targeted protein degradation.

Studies on knockout mutants and conditional mutants are invaluable to biological research and have been used extensively to probe the intricacies of biological systems through loss of function associated with attenuation of a particular protein. Besides, RNAi technology has been developed in recent years to further aid the process of scientific inquiry. Even though, the methods, dealing with DNA and RNA have met with great success, are not without their shortcomings. In order to overcome the inadequacies of existing methods, a host of new techniques, aimed at knockdowns at the protein rather than the nucleic acid level, have been devised. Essentially, these methods can achieve rapid degradation of cellular pools of a target protein in response to an inducible signal coupled with dose-dependent modulation and exquisite temporal control, features which are absent from techniques involving manipulations at the DNA or RNA level. This review aims to provide a broad overview of a gamut of these methods, while highlighting the strengths and weaknesses of each one. Last two decades of advances presented here in the field of targeted protein degradation serve as a beacon to further research and are likely to find applications in the areas of medicine and allied fields of biology.

Synthesis, spectroscopic characterization and DNA nuclease activity of Cu(II) complexes derived from pyrazolone based NSO-donor Schiff base ligands.

Two neutral mononuclear Cu(II) complexes have been prepared in EtOH using Schiff bases derived from 4-toluoyl pyrazolone and thiosemicarbazide. Both the ligands have been characterized on the basis of elemental analysis, IR, (1)H NMR, (13)C NMR and mass spectral data. The molecular geometry of one of these ligands has been determined by single crystal X-ray study. It reveals that these ligands exist in amine-one tautomeric form in the solid state. Microanalytical data, Cu-estimation, molar conductivity, magnetic measurements, IR, UV-Visible, FAB-Mass, TG-DTA data and ESR spectral studies were used to confirm the structures of the complexes. Electronic absorption and IR spectra of the complexes suggest a square-planar geometry around the central metal ion. The interaction of complexes with pET30a plasmid DNA was investigated by spectroscopic measurements. Results suggest that the copper complexes bind to DNA via an intercalative mode and can quench the fluorescence intensity of EB bound to DNA. The interaction between the complexes and DNA has also been investigated by agarose gel electrophoresis, interestingly, we found that the copper(II) complexes can cleave circular plasmid DNA to nicked and linear forms.

Q2N and S65D substitutions of ubiquitin unravel functional significance of the invariant residues Gln2 and Ser65.

Ubiquitin is a small, globular protein, structure of which has been perfected and conserved through evolution to manage diverse functions in the macromolecular metabolism of eukaryotic cells. Several non-homologous proteins interact with ubiquitin through entirely different motifs. Though the roles of lysines in the multifaceted functions of ubiquitin are well documented, very little is known about the contribution of other residues. In the present study, the importance of two invariant residues, Gln2 and Ser65, have been examined by substituting them with Asn and Asp, respectively, generating single residue variants of ubiquitin UbQ2N and UbS65D. Gln2 and Ser65 form part of parallel G1 ß-bulge adjacent to Lys63, a residue involved in DNA repair, cell-cycle regulated protein synthesis and imparting resistance to protein synthesis inhibitors. The secondary structure of variants is similar to that of UbF45W, a structural homologue of wild-type ubiquitin (UbWt). However, there are certain functional differences observed in terms of resistance to cycloheximide, while there are no major differences pertaining to growth under normal conditions, adherence to N-end rule and survival under heat stress. Further, expression of UbQ2N impedes protein degradation by ubiquitin fusion degradation (UFD) pathway. Such differential responses with respect to functions of ubiquitin produced by mutations may be due to interference in the interactions of ubiquitin with selected partner proteins, hint at biomedical implications.

Construction and functional characterization of double and triple mutants of parallel beta-bulge of ubiquitin.

Ubiquitin, a small eukaryotic protein serving as a post-translational modification on many important proteins, plays central role in cellular homeostasis and cell cycle regulation. Ubiquitin features two beta-bulges, the second beta-bulge, located at the C-terminal region of the protein along with type II turn, holds 3 residues Glu64(1), Ser65(2) and Gln2(X). Percent frequency of occurrence of such a sequence in parallel beta-bulge is very low. However, the sequence and structure have been conserved in ubiquitin through out the evolution. Present study involves replacement of residues in unusual beta-bulge of ubiquitin by introducing mutations in combination through site directed mutagenesis, generating double and triple mutants and their functional characterization. Mutant ubiquitins cloned in yeast expression vector YEp96 tested for growth profile, viability assay and heat stress complementation study have revealed significant decrease in growth rate, loss of viability and non-complementation of heat sensitive phenotype with UbE64G-S65D and UbQ2N-E64G-S65D mutations. However, UbQ2N-S65D did not show any negative effects in the above assays. Present results show that, replacement of residues in beta-bulge of ubiquitin exerts severe effects on growth and viability in Saccharomyces cerevisiae due to functional failure of the mutant ubiquitins UbE64G-S65D and UbQ2N-E64G-S65D.