Synthetic transcription factors establish the function of nine amino acid transactivation domains of Komagataella phaffii Mxr1.

The zinc finger transcription factor Mxr1 (methanol expression regulator 1) of the methylotrophic yeast Komagataella phaffii (formerly Pichia pastoris) harbors a DNA-binding domain (DBD) consisting of two CH zinc fingers (Mxr1ZF) between amino acids 36-101 and a previously identified nine amino acid transactivation domain (9aaTAD) between residues 365-373 (TAD A, QELESSLNA). Beyond this, 21 putative 9aaTADs (designated TAD B-V) located between amino acids 401-1155 remain to be characterized. Here, we demonstrate that a compact synthetic transcription factor composed of Mxr1ZF and three tandem copies of TAD A can activate the transcription of Mxr1 target genes for ethanol and methanol metabolism with specificity and efficiency comparable to the full-length protein.

Unlocking Nature's Toolbox: glutamate-inducible recombinant protein production from the Komagatella phaffii PEPCK promoter.

Background: Komagataella phaffii (a.k.a.

Coordinate regulation of methanol utilization pathway genes of by transcription factors and chromatin modifiers.

The methylotrophic yeast () harbors a methanol utilization (MUT) pathway, enabling it to utilize methanol as the sole source of carbon. The nexus between transcription factors such as Mxr1p and Trm1p and chromatin-modifying enzymes in the regulation of genes of MUT pathway has not been well studied in . Using transcriptomics, we demonstrate that Gcn5, a histone acetyltransferase, and Gal83, one of the beta subunits of nuclear-localized SNF1 (sucrose non-fermenting 1) kinase complex are essential for the transcriptional regulation by the zinc finger transcription factors Mxr1p and Trm1p.

Histidine is essential for growth of Komagataella phaffii cultured in YPA medium.

Komagataella phaffii (a.k.a.

Posttranscriptional regulation of glutamate dehydrogenase 2 and phosphoenolpyruvate carboxykinase in Komagataella phaffii.

The yeast Komagataella phaffii (a.k.a.

Carbon starvation-induced synthesis of GDH2 and PEPCK is essential for the survival of Pichia pastoris.

The industrial yeast Pichia pastoris can utilize amino acids as the sole source of carbon. It possesses a post-transcriptional regulatory circuit that governs the synthesis of cytosolic glutamate dehydrogenase 2 (GDH2) and phosphoenolpyruvate carboxykinase (PEPCK), key enzymes of amino acid catabolism. Here, we demonstrate that the post-transcriptional regulatory circuit is activated during carbon starvation resulting in the translation of GDH2 and PEPCK mRNAs.

Characterization of the transactivation and nuclear localization functions of Pichia pastoris zinc finger transcription factor Mxr1p.

The zinc finger transcription factor Mxr1p regulates the transcription of genes involved in methanol, acetate, and amino acid metabolism of the industrial yeast Pichia pastoris (a.k.a.

Apolipoprotein L9 interacts with LC3/GABARAP and is a microtubule-associated protein with a widespread subcellular distribution.

Mouse Apolipoprotein L9 is a 34-kDa phosphatidylethanolamine (PE)-binding protein. The gene is present only in mouse and rat genomes; hence it is restricted to two species. To understand why, it is essential to uncover details about its functions in cellular processes.

The nuclear transcription factor Rtg1p functions as a cytosolic, post-transcriptional regulator in the methylotrophic yeast .

Rtg1p and Rtg3p are two basic helix-loop-helix, retrograde transcription factors in the budding yeast Both factors heterodimerize to activate the transcription of nuclear genes in response to mitochondrial dysfunction and glutamate auxotrophy, but are not well characterized in other yeasts. Here, we demonstrate that the Rtg1p/Rtg3p-mediated retrograde signaling pathway is absent in the methylotrophic yeast We observed that Rtg1p (PpRtg1p) heterodimerizes with Rtg3p and functions as a nuclear, retrograde transcription factor in , but not in We noted that Rtg3p lacks a functional leucine zipper and interacts with neither Rtg1p (ScRtg1p) nor PpRtg1p. In the absence of an interaction with Rtg3p, PpRtg1p has apparently acquired a novel function as a cytosolic regulator of multiple metabolic pathways, including biosynthesis of glutamate dehydrogenase 2 and phosphoenolpyruvate carboxykinase required for the utilization of glutamate as the sole carbon source.

Methionine synthase is localized to the nucleus in and and to the cytoplasm in .

Methionine synthase (MS) catalyzes methylation of homocysteine, the last step in the biosynthesis of methionine, which is essential for the regeneration of tetrahydrofolate and biosynthesis of -adenosylmethionine. Here, we report that MS is localized to the nucleus of and but is cytoplasmic in The strain carrying a deletion of the gene encoding MS () exhibits methionine as well as adenine auxotrophy indicating that MS is required for methionine as well as adenine biosynthesis. Nuclear localization of MS (PpMS) was abrogated by the deletion of 107 C-terminal amino acids or the R742A mutation.

Mouse Apolipoprotein L9 is a phosphatidylethanolamine-binding protein.

Mouse Apolipoprotein L9 (ApoL9) is an understudied cytoplasmic, interferon-inducible protein. The details of its intracellular localization and normal cellular functions are unclear. We report here that ApoL9 localizes to small puncta diffusely distributed in the cytoplasm, as well as to larger granules of varying size and number that are similar to aggresome-like induced structures (ALIS) and contain the autophagy receptor Sqstm1/p62, the autophagosome marker Lc3, and ubiquitin.

Methanol Expression Regulator 1 (Mxr1p) Is Essential for the Utilization of Amino Acids as the Sole Source of Carbon by the Methylotrophic Yeast, Pichia pastoris.

Unlike Saccharomyces cerevisiae, the methylotrophic yeast Pichia pastoris can assimilate amino acids as the sole source of carbon and nitrogen. It can grow in media containing yeast extract and peptone (YP), yeast nitrogen base (YNB) + glutamate (YNB + Glu), or YNB + aspartate (YNB + Asp). Methanol expression regulator 1 (Mxr1p), a zinc finger transcription factor, is essential for growth in these media.

Regulation of Acetate Metabolism and Acetyl Co-a Synthetase 1 (ACS1) Expression by Methanol Expression Regulator 1 (Mxr1p) in the Methylotrophic Yeast Pichia pastoris.

Methanol expression regulator 1 (Mxr1p) is a zinc finger protein that regulates the expression of genes encoding enzymes of the methanol utilization pathway in the methylotrophic yeast Pichia pastoris by binding to Mxr1p response elements (MXREs) present in their promoters. Here we demonstrate that Mxr1p is a key regulator of acetate metabolism as well. Mxr1p is cytosolic in cells cultured in minimal medium containing a yeast nitrogen base, ammonium sulfate, and acetate (YNBA) but localizes to the nucleus of cells cultured in YNBA supplemented with glutamate or casamino acids as well as nutrient-rich medium containing yeast extract, peptone, and acetate (YPA).

Curcumin as an Adjunct Drug for Infectious Diseases.

Curcumin, by virtue of its ability to function as an immunomodulator, has the potential to serve as an adjunct drug to treat infectious diseases and provide long-term protection. The current need is to establish clinical trials with curcumin as an adjunct drug against specific infectious diseases.

Simultaneously targeting inflammatory response and parasite sequestration in brain to treat Experimental Cerebral Malaria.

Malaria afflicts around 200 million people annually, with a mortality number close to 600,000. The mortality rate in Human Cerebral Malaria (HCM) is unacceptably high (15-20%), despite the availability of artemisinin-based therapy. An effective adjunct therapy is urgently needed.

Trm1p, a Zn(II)₂Cys₆-type transcription factor, is essential for the transcriptional activation of genes of methanol utilization pathway, in Pichia pastoris.

The zinc finger transcription factors Mxr1p and Rop are key regulators of methanol metabolism in the methylotrophic yeast, Pichia pastoris, while Trm1p and Trm2p regulate methanol metabolism in Candida boidinii. Here, we demonstrate that Trm1p is essential for the expression of genes of methanol utilization (mut) pathway in P. pastoris as well.

The zinc finger proteins Mxr1p and repressor of phosphoenolpyruvate carboxykinase (ROP) have the same DNA binding specificity but regulate methanol metabolism antagonistically in Pichia pastoris.

The methanol-inducible alcohol oxidase I (AOXI) promoter of the methylotrophic yeast, Pichia pastoris, is used widely for the production of recombinant proteins. AOXI transcription is regulated by the zinc finger protein Mxr1p (methanol expression regulator 1). ROP (repressor of phosphoenolpyruvate carboxykinase, PEPCK) is a methanol- and biotin starvation-inducible zinc finger protein that acts as a negative regulator of PEPCK in P.

Curcumin-arteether combination therapy of Plasmodium berghei-infected mice prevents recrudescence through immunomodulation.

Earlier studies in this laboratory have shown the potential of artemisinin-curcumin combination therapy in experimental malaria. In a parasite recrudescence model in mice infected with Plasmodium berghei (ANKA), a single dose of alpha,beta-arteether (ART) with three oral doses of curcumin prevented recrudescence, providing almost 95% protection. The parasites were completely cleared in blood with ART-alone (AE) or ART+curcumin (AC) treatments in the short-term, although the clearance was faster in the latter case involving increased ROS generation.

Catabolite repression of phosphoenolpyruvate carboxykinase by a zinc finger protein under biotin- and pyruvate carboxylase-deficient conditions in Pichia pastoris.

We have identified a methanol- and biotin-starvation-inducible zinc finger protein named ROP [repressor of phosphoenolpyruvate carboxykinase (PEPCK)] in the methylotrophic yeast Pichia pastoris. When P. pastoris strain GS115 (wild-type, WT) is cultured in biotin-deficient, glucose-ammonium (Bio(-)) medium, growth is suppressed due to the inhibition of anaplerotic synthesis of oxaloacetate, catalysed by the biotin-dependent enzyme pyruvate carboxylase (PC).

Protoporphyrinogen IX oxidase from Plasmodium falciparum is anaerobic and is localized to the mitochondrion.

Earlier studies in this laboratory had shown that the malarial parasite can synthesize heme de novo and inhibition of the pathway leads to death of the parasite. It has been proposed that the pathway for the biosynthesis of heme in Plasmodium falciparum is unique involving three different cellular compartments, namely mitochondrion, apicoplast and cytosol. Experimental evidences are now available for the functionality and localization of all the enzymes of this pathway, except protoporphyrinogen IX oxidase (PfPPO), the penultimate enzyme.

Recombinant mid gut antigen (Bm95) as a vaccine against Indian Rhiphicephalus haemaphysaloides in Bos indicus cattle.

In the present study, we report for the first time the efficacy of recombinant Bm95 mid gut antigen isolated from an Argentinean strain of Rhipicephalus microplus strain A in controlling the tick infestations in India. The synthetic gene for Bm95 optimized for expression in yeast was obtained and used to generate yeast transformants expressing Bm95 which was purified to apparent homogeneity. Liquid chromatography-mass spectrometry analysis of the purified protein confirmed its identity as Bm95.

Identification of protein interaction regions of VINC/NEAT1/Men epsilon RNA.

The virus inducible non-coding RNA (VINC) was detected initially in the brain of mice infected with Japanese encephalitis virus (JEV) and rabies virus. VINC is also known as NEAT1 or Men epsilon RNA. It is localized in the nuclear paraspeckles of several murine as well as human cell lines and is essential for paraspeckle formation.

Identification of Mxr1p-binding sites in the promoters of genes encoding dihydroxyacetone synthase and peroxin 8 of the methylotrophic yeast Pichia pastoris.

Expression of genes involved in methanol metabolism of Pichia pastoris is regulated by Mxr1p, a zinc finger transcription factor. In this study, we studied the target gene specificity of Mxr1p by examining its ability to bind to promoters of genes encoding dihydroxyacetone synthase (DHAS) and peroxin 8 (PEX8), since methanol-inducible expression of these genes is abrogated in mxr1-null mutant strains of P. pastoris.

Characterization of coproporphyrinogen III oxidase in Plasmodium falciparum cytosol.

A unique hybrid pathway has been proposed for de novo heme biosynthesis in Plasmodium falciparum involving three different compartments of the parasite, namely mitochondrion, apicoplast and cytosol. While parasite mitochondrion and apicoplast have been shown to harbor key enzymes of the pathway, there has been no experimental evidence for the involvement of parasite cytosol in heme biosynthesis. In this study, a recombinant P.