Potential role of p53 deregulation in modulating immune responses in human malignancies: A paradigm to develop immunotherapy.

The crucial role played by the oncogenic expression of TP53, stemming from mutation or amyloid formation, in various human malignancies has been extensively studied over the past two decades. Interestingly, the potential role of TP53 as a crucial player in modulating immune responses has provided new insight into the field of cancer biology. The loss of p53's transcriptional functions and/or the acquisition of tumorigenic properties can efficiently modulate the recruitment and functions of myeloid and lymphoid cells, ultimately leading to the evasion of immune responses in human tumors. Consequently, the oncogenic nature of the tumor suppressor p53 can dynamically alter the function of immune cells, providing support for tumor progression and metastasis. This review comprehensively explores the dual role of p53 as both the guardian of the genome and an oncogenic driver, especially in the context of regulation of autophagy, apoptosis, the tumor microenvironment, immune cells, innate immunity, and adaptive immune responses. Additionally, the focus of this review centers on how p53 status in the immune response can be harnessed for the development of tailored therapeutic strategies and their potential application in immunotherapy against human malignancies.

Multifaceted role of mTOR (mammalian target of rapamycin) signaling pathway in human health and disease.

The mammalian target of rapamycin (mTOR) is a protein kinase that controls cellular metabolism, catabolism, immune responses, autophagy, survival, proliferation, and migration, to maintain cellular homeostasis. The mTOR signaling cascade consists of two distinct multi-subunit complexes named mTOR complex 1/2 (mTORC1/2). mTOR catalyzes the phosphorylation of several critical proteins like AKT, protein kinase C, insulin growth factor receptor (IGF-1R), 4E binding protein 1 (4E-BP1), ribosomal protein S6 kinase (S6K), transcription factor EB (TFEB), sterol-responsive element-binding proteins (SREBPs), Lipin-1, and Unc-51-like autophagy-activating kinases. mTOR signaling plays a central role in regulating translation, lipid synthesis, nucleotide synthesis, biogenesis of lysosomes, nutrient sensing, and growth factor signaling. The emerging pieces of evidence have revealed that the constitutive activation of the mTOR pathway due to mutations/amplification/deletion in either mTOR and its complexes (mTORC1 and mTORC2) or upstream targets is responsible for aging, neurological diseases, and human malignancies. Here, we provide the detailed structure of mTOR, its complexes, and the comprehensive role of upstream regulators, as well as downstream effectors of mTOR signaling cascades in the metabolism, biogenesis of biomolecules, immune responses, and autophagy. Additionally, we summarize the potential of long noncoding RNAs (lncRNAs) as an important modulator of mTOR signaling. Importantly, we have highlighted the potential of mTOR signaling in aging, neurological disorders, human cancers, cancer stem cells, and drug resistance. Here, we discuss the developments for the therapeutic targeting of mTOR signaling with improved anticancer efficacy for the benefit of cancer patients in clinics.

p53 amyloid pathology is correlated with higher cancer grade irrespective of the mutant or wild-type form.

p53 (also known as TP53) mutation and amyloid formation are long associated with cancer pathogenesis; however, the direct demonstration of the link between p53 amyloid load and cancer progression is lacking. Using multi-disciplinary techniques and 59 tissues (53 oral and stomach cancer tumor tissue samples from Indian individuals with cancer and six non-cancer oral and stomach tissue samples), we showed that p53 amyloid load and cancer grades are highly correlated. Furthermore, next-generation sequencing (NGS) data suggest that not only mutant p53 (e.g. single-nucleotide variants, deletions, and insertions) but wild-type p53 also formed amyloids either in the nucleus (50%) and/or in the cytoplasm in most cancer tissues. Interestingly, in all these cancer tissues, p53 displays a loss of DNA-binding and transcriptional activities, suggesting that the level of amyloid load correlates with the degree of loss and an increase in cancer grades. The p53 amyloids also sequester higher amounts of the related p63 and p73 (also known as TP63 and TP73, respectively) protein in higher-grade tumor tissues. The data suggest p53 misfolding and/or aggregation, and subsequent amyloid formation, lead to loss of the tumor-suppressive function and the gain of oncogenic function, aggravation of which might determine the cancer grade.

Transcriptome wide functional analysis of HBx expressing human hepatocytes stimulated with endothelial cell cross-talk.

Identification of genes dysregulated during the hepatitis B virus (HBV)-host cell interaction adds to the understanding of underlying molecular mechanisms and aids in discovering effective therapies to improve prognosis in hepatitis B virus (HBV)-infected individuals. Through bioinformatics analyses of transcriptomics data, this study aimed to identify potential genes involved in the cross-talk of human hepatocytes expressing the HBV viral protein HBx with endothelial cells. Transient transfection of HBV viral gene X (HBx) was performed in THLE2 cells using pcDNA3 constructs. Through mRNA Sequencing (RNA Seq) analysis, differentially expressed genes (DEGs) were identified. THLE2 cells transfected with HBx (THLE2x) were further treated with conditioned medium from cultured human umbilical vein derived endothelial cells (HUVEC-CM). Gene Ontology (GO) enrichment analysis revealed that interferon and cytokine signaling pathways were primarily enriched for the downregulated DEGs in THLE2x cells treated with HUVEC-CM. One significant module was selected following protein-protein interaction (PPI) network generation, and thirteen hub genes were identified from the module. The prognostic values of the hub genes were evaluated using Kaplan-Meier (KM) plotter, and three genes (IRF7, IFIT1, and IFITM1) correlated with poor disease specific survival (DSS) in HCC patients with chronic hepatitis. A comparison of the DEGs identified in HUVEC-stimulated THLE2x cells with four publicly available HBV-related HCC microarray datasets revealed that PLAC8 was consistently downregulated in all four HCC datasets as well as in HUVEC-CM treated THLE2x cells. KM plots revealed that PLAC8 correlated with worse relapse free survival and progression free survival in HCC patients with hepatitis B virus infection. This study provided molecular insights which may help develop a deeper understanding of HBV-host stromal cell interaction and open avenues for future research.

Transition of amyloid/mutant p53 from tumor suppressor to an oncogene and therapeutic approaches to ameliorate metastasis and cancer stemness.

The tumor suppressor p53 when undergoes amyloid formation confers several gain-of-function (GOF) activities that affect molecular pathways crucial for tumorigenesis and progression like some of the p53 mutants. Even after successful cancer treatment, metastasis and recurrence can result in poor survival rates. The major cause of recurrence is mainly the remnant cancer cells with stem cell-like properties, which are resistant to any chemotherapy treatment. Several studies have demonstrated the role of p53 mutants in exacerbating cancer stemness properties and epithelial-mesenchymal transition in these remnant cancer cells. Analyzing the amyloid/mutant p53-mediated signaling pathways that trigger metastasis, relapse or chemoresistance may be helpful for the development of novel or improved individualized treatment plans. In this review, we discuss the changes in the metabolic pathways such as mevalonate pathway and different signaling pathways such as TGF-ß, PI3K/AKT/mTOR, NF-κB and Wnt due to p53 amyloid formation, or mutation. In addition to this, we have discussed the role of the regulatory microRNAs and lncRNAs linked with the mutant or amyloid p53 in human malignancies. Such changes promote tumor spread, potential recurrence, and stemness. Importantly, this review discusses the cancer therapies that target either mutant or amyloid p53, restore wild-type functions, and exploit the synthetic lethal interactions with mutant p53.

Co-aggregation and secondary nucleation in the life cycle of human prolactin/galanin functional amyloids.

Synergistic-aggregation and cross-seeding by two different proteins/peptides in the amyloid aggregation are well evident in various neurological disorders including Alzheimer's disease. Here, we show co-storage of human Prolactin (PRL), which is associated with lactation in mammals, and neuropeptide galanin (GAL) as functional amyloids in secretory granules (SGs) of the female rat. Using a wide variety of biophysical studies, we show that irrespective of the difference in sequence and structure, both hormones facilitate their synergic aggregation to amyloid fibrils. Although each hormone possesses homotypic seeding ability, a unidirectional cross-seeding of GAL aggregation by PRL seeds and the inability of cross seeding by mixed fibrils suggest tight regulation of functional amyloid formation by these hormones for their efficient storage in SGs. Further, the faster release of functional hormones from mixed fibrils compared to the corresponding individual amyloid, suggests a novel mechanism of heterologous amyloid formation in functional amyloids of SGs in the pituitary.

The formation of plaques of proteins called 'amyloids' in the brain is one of the hallmark characteristics of both Alzheimer's and Parkinson's disease, but amyloids can form in many tissues and organs, often disrupting normal activity. A lot of the research into amyloids has focused on their role in disease, but it turns out that amyloids can also appear in healthy tissues. For example, some protein hormones form amyloids that act as storage depots, helping cells to release the hormone when it is needed. Normally, amyloids are made mostly of a single type of protein or protein fragment associated with a particular disease like Alzheimer's. Often, this type of amyloid promotes plaque formation in other proteins, which aggravates other diseases (for example, the amyloids that form in Alzheimer's can lead to Parkinson's disease or type II diabetes getting worse).The plaques start growing from small amyloid fragments called seeds. In mixed amyloids ­ amyloids made of two types of proteins ­ seeds made of one protein can trigger the formation of amyloids of the other protein. This raises the question, is this true for hormones? The body often releases more than one hormone at a time from the same tissue; for example, the pituitary gland releases prolactin and galanin simultaneously. However, these hormones have completely different structures, so whether they can form a mixed amyloid is unclear. To answer this question, Chatterjee et al. first determined that, within the pituitary gland of female rats, prolactin and galanin could be found together in the same cells, forming mixed amyloids. To understand out how this happens, Chatterjee et al. tried seeding new amyloids using either prolactin or galanin. This revealed that only prolactin seeds were able to trigger the formation of galanin amyloids. Chatterjee et al. also found that the mixed amyloids could release the hormones faster than amyloids made from either protein alone. Together, these results suggest that the collaboration between these two proteins may help maintain hormone balance in the body. Problems with hormone storage and release lead to various human diseases, including prolactinoma. Understanding amyloid storage depots could reveal new ways to control hormone levels. Further research could also help to explain more about well-studied diseases linked to amyloids, like Alzheimer's.

Transporter engineering for the development of cyanobacteria as cell factories: A text analytics guided survey.

Cyanobacteria are attractive candidates for photoautotrophic production of platform chemicals due to their inherent ability to utilize carbon dioxide as the sole carbon source. Metabolic pathways can be engineered more readily in cyanobacteria compared to higher photosynthetic organisms. Although significant progress has been made in pathway engineering, intracellular accumulation of the product is a potential bottleneck in large-scale production. Likewise, substrate uptake is known to limit growth and product formation. These limitations can potentially be addressed by targeted and controlled expression of transporter proteins in the metabolically engineered strains. This review focuses on the transporters that have been explored in cyanobacteria. To highlight the progress on characterization and application of cyanobacterial transporters, we applied text analytics to extract relevant information from over 1000 publications. We have categorized the transporters based on their source, their function and the solute they transport. Further, the review provides insights into the potential of transporters in the metabolic engineering of cyanobacteria for improved product titer.

Adaptive laboratory evolution of the fast-growing cyanobacterium Synechococcus elongatus PCC 11801 for improved solvent tolerance.

Cyanobacteria hold promise as cell factories for the photoautotrophic conversion of carbon dioxide to useful chemicals. For the eventual commercial viability of such processes, cyanobacteria need to be engineered for (i) efficient channeling of carbon flux toward the product of interest and (ii) improved product tolerance, the latter being the focus of this study. We chose the recently reported, fast-growing, high light and CO2 tolerant cyanobacterium Synechococcus elongatus PCC 11801 for adaptive laboratory evolution. In two parallel experiments that lasted over 8400 h of culturing and 100 serial passages, S. elongatus PCC 11801 was evolved to tolerate 5 g/L n-butanol or 30 g/L 2,3-butanediol representing a 100% improvement in concentrations tolerated. The evolved strains retained alcohol tolerance even after being passaged several times without the alcohol stress suggesting that the changes were permanent. Whole genome sequencing of the n-butanol evolved strains revealed mutations in a number of stress responsive genes encoding translation initiation factors, RpoB and an ABC transporter. In 2,3-butanediol evolved strains, genes for ClpC, a different ABC transporter, glyceraldehyde-3-phosphate dehydrogenase and phosphoribulokinase were found to be mutated. Furthermore, the evolved strains showed significant improvement in tolerance toward several other alcohols. Notably, the n-butanol evolved strain could tolerate up to 32 g/L ethanol, thereby making it a promising host for photosynthetic production of biofuels via metabolic engineering.

Metabolic engineering of a fast-growing cyanobacterium Synechococcus elongatus PCC 11801 for photoautotrophic production of succinic acid.

BACKGROUND: Cyanobacteria, a group of photosynthetic prokaryotes, are being increasingly explored for direct conversion of carbon dioxide to useful chemicals. However, efforts to engineer these photoautotrophs have resulted in low product titers. This may be ascribed to the bottlenecks in metabolic pathways, which need to be identified for rational engineering. We engineered the recently reported, fast-growing and robust cyanobacterium, Synechococcus elongatus PCC 11801 to produce succinate, an important platform chemical. Previously, engineering of the model cyanobacterium S. elongatus PCC 7942 has resulted in succinate titer of 0.43 g l-1 in 8 days. RESULTS: Building on the previous report, expression of α-ketoglutarate decarboxylase, succinate semialdehyde dehydrogenase and phosphoenolpyruvate carboxylase yielded a succinate titer of 0.6 g l-1 in 5 days suggesting that PCC 11801 is better suited as host for production. Profiling of the engineered strains for 57 intermediate metabolites, a number of enzymes and qualitative analysis of key transcripts revealed potential flux control points. Based on this, we evaluated the effects of overexpression of sedoheptulose-1,7-bisphosphatase, citrate synthase and succinate transporters and knockout of succinate dehydrogenase and glycogen synthase A. The final construct with seven genes overexpressed and two genes knocked out resulted in photoautotrophic production of 0.93 g l-1 succinate in 5 days. CONCLUSION: While the fast-growing strain PCC 11801 yielded a much higher titer than the model strain, the efficient photoautotrophy of this novel isolate needs to be harnessed further for the production of desired chemicals. Engineered strains of S. elongatus PCC 11801 showed dramatic alterations in the levels of several metabolites suggesting far reaching effects of pathway engineering. Attempts to overexpress enzymes deemed to be flux controlling led to the emergence of other potential rate-limiting steps. Thus, this process of debottlenecking of the pathway needs to be repeated several times to obtain a significantly superior succinate titer.

A Novel Cyanobacterium Synechococcus elongatus PCC 11802 has Distinct Genomic and Metabolomic Characteristics Compared to its Neighbor PCC 11801.

Cyanobacteria, a group of photosynthetic prokaryotes, are attractive hosts for biotechnological applications. It is envisaged that future biorefineries will deploy engineered cyanobacteria for the conversion of carbon dioxide to useful chemicals via light-driven, endergonic reactions. Fast-growing, genetically amenable, and stress-tolerant cyanobacteria are desirable as chassis for such applications. The recently reported strains such as Synechococcus elongatus UTEX 2973 and PCC 11801 hold promise, but additional strains may be needed for the ongoing efforts of metabolic engineering. Here, we report a novel, fast-growing, and naturally transformable cyanobacterium, S. elongatus PCC 11802, that shares 97% genome identity with its closest neighbor S. elongatus PCC 11801. The new isolate has a doubling time of 2.8 h at 1% CO2, 1000 µmole photons.m-2.s-1 and grows faster under high CO2 and temperature compared to PCC 11801 thus making it an attractive host for outdoor cultivations and eventual applications in the biorefinery. Furthermore, S. elongatus PCC 11802 shows higher levels of key intermediate metabolites suggesting that this strain might be better suited for achieving high metabolic flux in engineered pathways. Importantly, metabolite profiles suggest that the key enzymes of the Calvin cycle are not repressed under elevated CO2 in the new isolate, unlike its closest neighbor.

Genome Features and Biochemical Characteristics of a Robust, Fast Growing and Naturally Transformable Cyanobacterium Synechococcus elongatus PCC 11801 Isolated from India.

Cyanobacteria provide an interesting platform for biotechnological applications due to their efficient photoautotrophic growth, amenability to genetic engineering and the ability to grow on non-arable land. An ideal industrial strain of cyanobacteria would need to be fast growing and tolerant to high levels of temperature, light, carbon dioxide, salt and be naturally transformable. In this study, we report Synechococcus elongatus PCC 11801, a strain isolated from India that fulfills these requirements. The physiological and biochemical characteristics of PCC 11801 under carbon and light-limiting conditions were investigated. PCC 11801 shows a doubling time of 2.3 h, that is the fastest growth for any cyanobacteria reported so far under ambient CO2 conditions. Genome sequence of PCC 11801 shows genome identity of ~83% with its closest neighbors Synechococcus elongatus PCC 7942 and Synechococcus elongatus UTEX 2973. The unique attributes of PCC 11801 genome are discussed in light of the physiological characteristics that are needed in an industrial strain. The genome of PCC 11801 shows several genes that do not have homologs in neighbor strains PCC 7942 and UTEX 2973, some of which may be responsible for adaptation to various abiotic stresses. The remarkably fast growth rate of PCC 11801 coupled with its robustness and ease of genetic transformation makes it an ideal candidate for the photosynthetic production of fuels and chemicals.

p53 amyloid formation leading to its loss of function: implications in cancer pathogenesis.

The transcriptional regulator p53 has an essential role in tumor suppression. Almost 50% of human cancers are associated with the loss of p53 functions, where p53 often accumulates in the nucleus as well as in cytoplasm. Although it has been previously suggested that amyloid formation could be a cause of p53 loss-of-function in subset of tumors, the characterization of these amyloids and its structure-function relationship is not yet established. In the current study, we provide several evidences for the presence of p53 amyloid formation (in human and animal cancer tissues); along with its isolation from human cancer tissues and the biophysical characterization of these tissue-derived fibrils. Using amyloid seed of p53 fragment (P8, p53(250-257)), we show that p53 amyloid formation in cells not only leads to its functional inactivation but also transforms it into an oncoprotein. The in vitro studies further show that cancer-associated mutation destabilizes the fold of p53 core domain and also accelerates the aggregation and amyloid formation by this protein. Furthermore, we also show evidence of prion-like cell-to-cell transmission of different p53 amyloid species including full-length p53, which is induced by internalized P8 fibrils. The present study suggests that p53 amyloid formation could be one of the possible cause of p53 loss of function and therefore, inhibiting p53 amyloidogenesis could restore p53 tumor suppressor functions.

Evidence of a Prion-Like Transmission of p53 Amyloid in Saccharomyces cerevisiae.

Loss of p53 function is largely responsible for the occurrence of cancer in humans. Aggregation of mutant p53 has been found in multiple cancer cell types, suggesting a role of aggregation in loss of p53 function and cancer development. The p53 protein has recently been hypothesized to possess a prion-like conformation, although experimental evidence is lacking. Here, we report that human p53 can be inactivated upon exposure to preformed fibrils containing an aggregation-prone sequence-specific peptide, PILTIITL, derived from p53, and the inactive state was found to be stable for many generations. Importantly, we provide evidence of a prion-like transmission of these p53 aggregates. This study has significant implications for understanding cancer progression due to p53 malfunctioning without any loss-of-function mutation or occurrence of transcriptional inactivation. Our data might unlock new possibilities for understanding the disease and will lead to rational design of p53 aggregation inhibitors for the development of drugs against cancer.

Enzymatic attributes of an l-isoaspartyl methyltransferase from Candida utilis and its role in cell survival.

BACKGROUNDS: Spontaneous deamidation and isoaspartate (IsoAsp) formation contributes to aging and reduced longevity in cells. A protein-l-isoaspartate (d-aspartate) O-methyltransferase (PCMT) is responsible for minimizing IsoAsp moieties in most organisms. METHODS: PCMT was purified in its native form from yeast Candida utilis. The role of the native PCMT in cell survival and protein repair was investigated by manipulating intracellular PCMT levels with Oxidized Adenosine (AdOx) and Lithium Chloride (LiCl). Proteomic Identification of possible cellular targets was carried out using 2-dimensional gel electrophoresis, followed by on-Blot methylation and mass spectrometric analysis. RESULTS: The 25.4 kDa native PCMT from C. utilis was found to have a Km of 3.5 µM for AdoMet and 33.36 µM for IsoAsp containing Delta Sleep Inducing Peptide (DSIP) at pH 7.0. Native PCMT comprises of 232 amino acids which is coded by a 698 bp long nucleotide sequence. Phylogenetic comparison revealed the PCMT to be related more closely with the prokaryotic homologs. Increase in PCMT levels in vivo correlated with increased cell survival under physiological stresses. PCMT expression was seen to be linked with increased intracellular reactive oxygen species (ROS) concentration. Proteomic identification of possible cellular substrates revealed that PCMT interacts with proteins mainly involved with cellular housekeeping. PCMT effected both functional and structural repair in aged proteins in vitro. GENERAL SIGNIFICANCE: Identification of PCMT in unicellular eukaryotes like C. utilis promises to make investigations into its control machinery easier owing to the familiarity and flexibility of the system.

Purification, characterization, sequencing and molecular cloning of a novel cysteine methyltransferase that regulates trehalose-6-phosphate synthase from Saccharomyces cerevisiae.

BACKGROUND: In Saccharomyces cerevisiae methylation at cysteine residue displayed enhanced activity of trehalose-6-phosphate synthase (TPS). METHODS: The cysteine methyltransferase (CMT) responsible for methylating TPS was purified and characterized. The amino acid sequence of the enzyme protein was determined by a combination of N-terminal sequencing and MALDI-TOF/TOF analysis. The nucleotide sequence of the CMT gene was determined, isolated from S. cerevisiae and expressed in E. coli. Targeted disruption of the CMT gene by PCR based homologous recombination in S. cerevisiae was followed by metabolite characterization in the mutant. RESULTS: The purified enzyme was observed to enhance the activity of TPS by a factor of 1.76. The 14kDa enzyme was found to be cysteine specific. The optimum temperature and pH of enzyme activity was calculated as 30°C and 7.0 respectively. The Km Vmax and Kcat against S-adenosyl-l-methionine (AdoMet) were 4.95µM, 3.2U/mg and 6.4s(-1) respectively. Competitive inhibitor S-Adenosyl-l-homocysteine achieved a Ki as 10.9µM against AdoMet. The protein sequence contained three putative AdoMet binding motifs. The purified recombinant CMT activity exhibited similar physicochemical characteristics with the native counterpart. The mutant, Mataα, cmt:: kan(r) exhibited almost 50% reduction in intracellular trehalose concentration. CONCLUSION: A novel cysteine methyltransferase is purified, which is responsible for enhanced levels of trehalose in S. cerevisiae. GENERAL SIGNIFICANCE: This is the first report about a cysteine methyltransferase which performs S methylation at cysteine residue regulating TPS activity by 50%, which resulted in an increase of the intercellular stress sugar, trehalose.

Enzymatic and regulatory attributes of trehalose-6-phosphate phosphatase from Candida utilis and its role during thermal stress.

Trehalose-6-phosphate phosphatase (TPP) catalyzes the final step in the biosynthesis of the anti-stress sugar trehalose. An 82 kDa TPP enzyme was isolated from Candida utilis with 61% yield and 43-fold purification. The protein sequence, determined by N-terminal sequencing and MALDI-TOF analysis, showed significant homology with known TPP sequences from related organisms. The full length gene sequence of TPP of C. utilis was identified using rapid amplification of cDNA ends-PCR reaction (RACE-PCR). The gene was cloned and expressed in Escherichia coli BL21. Recombinant TPP enzyme was isolated using affinity chromatography. CD spectroscopy and steady-state fluorescence revealed that the structural and conformational aspects were identical in both native and recombinant forms. The biochemical properties of the two forms were also similar. Km was determined to be ~0.8 mM. Optimum temperature and pH were found to be 30 °C and 8.5, respectively. Activity was dependent on the presence of divalent cations and inhibited by metal chelators. Methylation-mediated regulation of TPP enzyme and its effect on the overall survival of the organism under stress were investigated. The results indicated that enhancement of TPP activity by methylation at the Cysteine residues increased resistance of Candida cells against thermal stress. This work involves extensive investigations toward understanding the physico-chemical properties of the first TPP enzyme from any yeast strain. The mechanism by which methylation regulates its activity has also been studied. A correlation between regulation of trehalose synthesis and survivability of the organism under thermal stress was established.

Methylation dependent enhancement of trehalose production in Candida utilis.

Trehalose metabolism plays a central role in various stress responses in yeasts. Methylation dependant enhancement of trehalose synthesis has been reported from yeast Saccharomyces cerevisiae. In order to establish the role of methylation on trehalose metabolism in yeast, it was further investigated in Candida utilis. Universal methyl group donor, S-adenosyl-l-methionine (AdoMet) and its inhibitor, oxidized adenosine (Adox) were used to study the effect of methylation on trehalose metabolism in C. utilis. Treatment of early stationary phase cells of C. utilis with AdoMet and Adox exhibited increase in both intracellular metabolite levels and activities of the trehalose synthesizing enzymes, trehalose-6-phosphate synthase (TPS) and trehalose phosphate phosphatase (TPP). Among the intracellular metabolites studied, trehalose levels were enhanced in presence of AdoMet which correlated with the increasing levels of trehalose synthesizing enzymes. TPS was purified in presence of AdoMet and Adox, following an established protocol reported from this laboratory. Differences in the mobility of control TPS, methylated TPS, and methylation-inhibited TPS during acidic native gel electrophoresis confirmed the occurrence of induced methylation. MALDI-TOF analysis of trypsin-digested samples of the same further strengthened the presence of methylation in TPS. The data presented in this paper strongly indicate a positive role of methylation on trehalose synthesis which finally leads to enhanced trehalose production during the stationary growth phase of C. utilis.

Regulation of enzymatic activity by deamidation and their subsequent repair by protein L-isoaspartyl methyl transferase.

The present study explored both spontaneous and stress-induced deamidation in acid trehalase and endo-xylanase. An alteration in optimum pH by 1.5 units and optimum temperature by 20 °C accelerated the process of deamidation with a rise in isoaspartate formation and ammonia loss. Spontaneous deamidation during an enzyme-substrate reaction at physiological conditions resulted in accretion of isoaspartyl residues within the enzymes which gradually impaired their catalytic efficacy. Deamidation appeared to be more pronounced in endo-xylanase owing to its secondary structure conformation and high asparagine content. The active sites, Ala 549 in acid trehalase and His184 and Trp188 in endo-xylanase contributed to the loss of enzyme activity as they were flanking the deamidation-susceptible Asn residues. Protein L-isoaspartyl methyl transferase seemed to have a repairing capability, which enabled the heat-damaged enzymes to regain their partial activity as evident from there rise in K (cat)/K (m). Endo-xylanase could regain 38.1 % of its biological activity while a lesser 17.5 % reactivation was obtained in acid trehalase. A unique protein L-isoaspartyl methyl transferase recognition site, Asn 151 was also identified in acid trehalase. A mass increment of the tryptic peptides of repaired enzyme due to methylation catalyzed by protein L-isoaspartyl methyl transferase substantiated the repair hypothesis.

Purification and characterization of a trehalase-invertase enzyme with dual activity from Candida utilis.

Trehalose and sucrose, two important anti-stress non-reducing natural disaccharides, are catabolized by two enzymes, namely trehalase and invertase respectively. In this study, a 175 kDa enzyme protein active against both substrates was purified from wild type Candida utilis and characterized in detail. Substrate specificity assay and activity staining revealed the enzyme to be specific for both sucrose and trehalose. The ratio between trehalase and invertase activity was found to be constant at 1:3.5 throughout the entire study. Almost 40-fold purification and 30% yield for both activities were achieved at the final step of purification. The presence of common enzyme inhibitors, thermal and pH stress had analogous effects on its trehalase and invertase activity. Km values for two activities were similar while Vmax and Kcat also differed by a factor of 3.5. Competition plot for both substrates revealed the two activities to be occurring at the single active site. N-terminal sequencing and MALDI-TOF data analysis revealed higher similarity of the purified protein to previously known neutral trehalases. While earlier workers mentioned independent purification of neutral trehalase or invertase from different sources, the present study reports the purification of a single protein showing dual activity.