Temporal analysis of melanogenesis identifies fatty acid metabolism as key skin pigment regulator.

Therapeutic methods to modulate skin pigmentation has important implications for skin cancer prevention and for treating cutaneous hyperpigmentary conditions. Towards defining new potential targets, we followed temporal dynamics of melanogenesis using a cell-autonomous pigmentation model. Our study elucidates 3 dominant phases of synchronized metabolic and transcriptional reprogramming. The melanogenic trigger is associated with high MITF levels along with rapid uptake of glucose. The transition to pigmented state is accompanied by increased glucose channelisation to anabolic pathways that support melanosome biogenesis. SREBF1-mediated up-regulation of fatty acid synthesis results in a transient accumulation of lipid droplets and enhancement of fatty acids oxidation through mitochondrial respiration. While this heightened bioenergetic activity is important to sustain melanogenesis, it impairs mitochondria lately, shifting the metabolism towards glycolysis. This recovery phase is accompanied by activation of the NRF2 detoxication pathway. Finally, we show that inhibitors of lipid metabolism can resolve hyperpigmentary conditions in a guinea pig UV-tanning model. Our study reveals rewiring of the metabolic circuit during melanogenesis, and fatty acid metabolism as a potential therapeutic target in a variety of cutaneous diseases manifesting hyperpigmentary phenotype.

A Comprehensive Pilot Study to Elucidate the Distinct Gut Microbial Composition and Its Functional Significance in Cardio-Metabolic Disease.

Cardio-metabolic disease is a significant global health challenge with increasing prevalence. Recent research underscores the disruption of gut microbial balance as a key factor in disease susceptibility. We aimed to characterize the gut microbiota composition and function in cardio-metabolic disease and healthy controls. For this purpose, we collected stool samples of 18 subjects (12 diseased, 6 healthy) and we performed metagenomics analysis and functional prediction using QIIME2 and PICRUSt. Furthermore, we carried out assessments of microbe-gene interactions, gene ontology, and microbe-disease associations. Our findings revealed distinct microbial patterns in the diseased group, particularly evident in lower taxonomic levels with significant variations in 14 microbial features. The diseased cohort exhibited an enrichment of Lachnospiraceae family, correlating with obesity, insulin resistance, and metabolic disturbances. Conversely, reduced levels of Clostridium, Gemmiger, and Ruminococcus genera indicated a potential inflammatory state, linked to compromised butyrate production and gut permeability. Functional analyses highlighted dysregulated pathways in amino acid metabolism and energy equilibrium, with perturbations correlating with elevated branch-chain amino acid levels-a known contributor to insulin resistance and type 2 diabetes. These findings were consistent across biomarker assessments, microbe-gene associations, and gene ontology analyses, emphasizing the intricate interplay between gut microbial dysbiosis and cardio-metabolic disease progression. In conclusion, our study unveils significant shifts in gut microbial composition and function in cardio-metabolic disease, emphasizing the broader implications of microbial dysregulation. Addressing gut microbial balance emerges as a crucial therapeutic target in managing cardio-metabolic disease burden.

Urine Metabolomics Reveals Overlapping Metabolic Associations Between Preeclampsia and Gestational Diabetes.

Pregnancy is associated with numerous metabolic adaptations to meet the demands of the growing foetus. These adaptations could be perturbed during pregnancy due to preeclampsia (PE) and gestational diabetes (GDM). As these two obstetric aliments show some overlapping pathophysiology and similar biochemical dysregulation, the present study was undertaken to compare urine metabolome of PE and GDM with normal pregnancy (NT) in all trimesters of gestation using nuclear magnetic resonance spectroscopy-based metabolomics analysis to ascertain and compare metabolome in the study groups. We observed overlapping metabolic perturbations in PE and GDM. Though a study with a small sample size, this is the first report which confirms significantly differential metabolites in urine of both PE and GDM. Dimethylglycine and oxoglutaric acid were decreased while benzoic acid was increased in both the cases in all trimesters. Alanine, aspartate and glutamate metabolism, aminoacyl-tRNA biosynthesis, citrate and butanoate metabolism were the most perturbed pathways in both PE and GDM across pregnancy. These pathways have an association with energy metabolism, glucose homeostasis, insulin sensitivity and oxidative stress which play an important role in the development and progression of PE and GDM. In conclusion, our study showed that urine metabolome could reflect metabolic associations between PE and GDM and also in the identification of biomolecules that could be used as potential biomarker(s) for early detection of the metabolic diseases in pregnancy. Supplementary Information: The online version contains supplementary material available at 10.1007/s12291-022-01103-2.

Comparative analysis of the efficiency of seed protein profiles in assessing genetic variation and population structure among indigenous Manipur black rice cultivars.

BACKGROUND: The state of Manipur, North East India has distinct topology of hill and valley regions with vast agroclimatic variability, being considered as one of the centers of rice diversity. The indigenous Manipur black rice cultivars exhibit wide range of diversity in morphology, pericarp color, shape and size of grain, aroma, glutinous or non-glutinous features but remain less characterised. Many of these cultivars, such as those named Chakhao, are endowed with multiple health benefits due to high anthocyanins, and hold special importance for the local people. It is important to analyse the genetic diversity and population structure for this germplasm with unique allelic combinations to utilize in rice breeding programme. METHODS AND RESULTS: We characterized total soluble seed protein fractions to not only fingerprint the 45 indigenous black rice cultivars but assess their genetic relatedness. Cluster analyses generated mainly two groups, complemented by PCoA scatter plot ascertaining geographical distinction. The hill black rice were more diverse. The population structure analysis revealed seven subpopulations indicating high genetic variability. The 24 polymorphic bands were scored in the range of 127.8 to 10.3 kDa comprised of four protein fractions. Three polypeptide bands each were ascribed to known fractions of glutelins and prolamins, while one band each could be described for albumin and globulin fractions, besides other diagnostic bands. CONCLUSION: Some diverse cultivars were Amubi, Chedo Anal, Chipi Buh, Athebu, Poireton, BuPu Mui, Kotha Chahao II. These cultivars can be used in future black rice breeding programmes. This can further prevent genetic erosion and protect intellectual property rights.

Hinge like domain motion facilitates human RBMS1 protein binding to proto-oncogene c-myc promoter.

DNA binding proteins recognize DNA specifically or non-specifically using direct and indirect readout mechanisms like sliding, hopping, and diffusion. However, a common difficulty in explicitly elucidating any particular mechanism of site-specific DNA-protein recognition is the lack of knowledge regarding target sequences and inadequate account of non-specific interactions, in general. Here, we decipher the structural basis of target search performed by the key regulator of expression of c-myc proto-oncogene, the human RBMS1 protein. In this study, we have shown the structural reorganization of this multi-domain protein required for recognizing the specific c-myc promoter sequence. The results suggest that a synergy between structural re-organization and thermodynamics is necessary for the recognition of target sequences. The study presents another perspective of looking at the DNA-protein interactions.

Structure of an Unfolding Intermediate of an RRM Domain of ETR-3 Reveals Its Native-like Fold.

Prevalence of one or more partially folded intermediates during protein unfolding with different secondary and ternary conformations has been identified as an integral character of protein unfolding. These transition-state species need to be characterized structurally for elucidation of their folding pathways. We have determined the three-dimensional structure of an intermediate state with increased conformational space sampling under urea-denaturing condition. The protein unfolds completely at 10 M urea but retains residual secondary structural propensities with restricted motion. Here, we describe the native state, observable intermediate state, and unfolded state for ETR-3 RRM-3, which has canonical RRM fold. These observations can shed more light on unfolding events for RRM-containing proteins.

Interaction studies on bacterial stringent response protein RelA with uncharged tRNA provide evidence for its prerequisite complex for ribosome binding.

The bacterial stringent response is regulated by the synthesis of (p)ppGpp which is mediated by RelA in a complex with uncharged tRNA and ribosome. We intended to probe RelA-uncharged tRNA interactions off the ribosome to understand the sequential activation mechanism of RelA. Stringent response is a key regulatory pleiotropic mechanism which allows bacteria to survive in unfavorable conditions. Since the discovery of RelA, it has been believed that it is activated upon binding to ribosomes which already have uncharged tRNA on acceptor site (A-site). However, uncharged tRNA occupied in the A-site of the ribosome prior to RelA binding could not be observed; therefore, recently an alternate model for RelA activation has been proposed in which RelA first binds to uncharged tRNA and then RelA-uncharged tRNA complex is loaded on to the ribosome to synthesize (p)ppGpp. To explore the alternate hypothesis, we report here the in vitro binding of uncharged tRNA to RelA in the absence of ribosome using formaldehyde cross-linking, fluorescence spectroscopy, surface plasmon resonance, size-exclusion chromatography, and hydrogen-deuterium exchange mass spectrometry. Altogether, our results clearly indicate binding between RelA and uncharged tRNA without the involvement of ribosome. Moreover, we have analyzed their binding kinetics and mapping of tRNA-interacting regions of RelA structure. We have also co-purified TGS domain in complex with tRNA to further establish in vivo RelA-tRNA binding. We have observed that TGS domain recognizes all types of uncharged tRNA similar to EF-Tu and tRNA interactions. Altogether, our results demonstrate the complex formation between RelA and uncharged tRNA that may be loaded to the ribosome for (p)ppGpp synthesis.

Protein kinase G confers survival advantage to Mycobacterium tuberculosis during latency-like conditions.

Protein kinase G (PknG), a thioredoxin-fold-containing eukaryotic-like serine/threonine protein kinase, is a virulence factor in Mycobacterium tuberculosis, required for inhibition of phagolysosomal fusion. Here, we unraveled novel functional facets of PknG during latency-like conditions. We found that PknG mediates persistence under stressful conditions like hypoxia and abets drug tolerance. PknG mutant displayed minimal growth in nutrient-limited conditions, suggesting its role in modulating cellular metabolism. Intracellular metabolic profiling revealed that PknG is necessary for efficient metabolic adaptation during hypoxia. Notably, the PknG mutant exhibited a reductive shift in mycothiol redox potential and compromised stress response. Exposure to antibiotics and hypoxic environment resulted in higher oxidative shift in mycothiol redox potential of PknG mutant compared with the wild type. Persistence during latency-like conditions required kinase activity and thioredoxin motifs of PknG and is mediated through phosphorylation of a central metabolic regulator GarA. Finally, using a guinea pig model of infection, we assessed the in vivo role of PknG in manifestation of disease pathology and established a role for PknG in the formation of stable granuloma, hallmark structures of latent tuberculosis. Taken together, PknG-mediated GarA phosphorylation is important for maintenance of both mycobacterial physiology and redox poise, an axis that is dispensable for survival under normoxic conditions but is critical for non-replicating persistence of mycobacteria. In conclusion, we propose that PknG probably acts as a modulator of latency-associated signals.

Recombinant human islet amyloid polypeptide forms shorter fibrils and mediates ß-cell apoptosis via generation of oxidative stress.

Protein misfolding and aggregation play an important role in many human diseases including Alzheimer's, Parkinson's and type 2 diabetes mellitus (T2DM). The human islet amyloid polypeptide (hIAPP) forms amyloid plaques in the pancreas of T2DM subjects (>95%) that are involved in deteriorating islet function and in mediating ß-cell apoptosis. However, the detailed mechanism of action, structure and nature of toxic hIAPP species responsible for this effect remains elusive to date mainly due to the high cost associated with the chemical synthesis of pure peptide required for these studies. In the present work, we attempted to obtain structural and mechanistic insights into the hIAPP aggregation process using recombinant hIAPP (rhIAPP) isolated from Escherichia coli Results from biophysical and structural studies indicate that the rhIAPP self-assembled into highly pure, ß-sheet-rich amyloid fibrils with uniform morphology. rhIAPP-mediated apoptosis in INS-1E cells was associated with increased oxidative stress and changes in mitochondrial membrane potential. The transcript levels of apoptotic genes - Caspase-3 and Bax were found to be up-regulated, while the levels of the anti-apoptotic gene - Bcl2 were down-regulated in rhIAPP-treated cells. Additionally, the expression levels of genes involved in combating oxidative stress namely Catalase, SOD1 and GPx were down-regulated. rhIAPP exposure also affected glucose-stimulated insulin secretion from isolated pancreatic islets. The aggregation of rhIAPP also occurred significantly faster when compared with that of the chemically synthesized peptide. We also show that the rhIAPP fibrils were shorter and more cytotoxic. In summary, our study is one among the few to provide comprehensive evaluation of structural, biophysical and cytotoxic properties of rhIAPP.

N-acetylglucosamine (GlcNAc)-inducible gene GIG2 is a novel component of GlcNAc metabolism in Candida albicans.

Candida albicans is an opportunistic fungal pathogen that resides in the human body as a commensal and can turn pathogenic when the host is immunocompromised. Adaptation of C. albicans to host niche-specific conditions is important for the establishment of pathogenicity, where the ability of C. albicans to utilize multiple carbon sources provides additional flexibility. One alternative sugar is N-acetylglucosamine (GlcNAc), which is now established as an important carbon source for many pathogens and can also act as a signaling molecule. Although GlcNAc catabolism has been well studied in many pathogens, the importance of several enzymes involved in the formation of metabolic intermediates still remains elusive. In this context, microarray analysis was carried out to investigate the transcriptional responses induced by GlcNAc under different conditions. A novel gene that was highly upregulated immediately following the GlcNAc catabolic genes was identified and was named GIG2 (GlcNAc-induced gene 2). This gene is regulated in a manner distinct from that of the GlcNAc-induced genes described previously in that GlcNAc metabolism is essential for its induction. Furthermore, this gene is involved in the metabolism of N-acetylneuraminate (sialic acid), a molecule equally important for initial host-pathogen recognition. Mutant cells showed a considerable decrease in fungal burden in mouse kidneys and were hypersensitive to oxidative stress conditions. Since GIG2 is also present in many other fungal and enterobacterial genomes, targeted inhibition of its activity would offer insight into the treatment of candidiasis and other fungal or enterobacterial infections.

Modulation of Krüppel-like factors (KLFs) interaction with their binding partners in cancers through acetylation and phosphorylation.

Post-translational modifications (PTMs) of transcription factors regulate transcriptional activity and play a key role in essentially all biological processes and generate indispensable insight towards biological function including activity state, subcellular localization, protein solubility, protein folding, substrate trafficking, and protein-protein interactions. Amino acids modified chemically via PTMs, function as molecular switches and affect the protein function and characterization and increase the proteome complexity. Krüppel-like transcription factors (KLFs) control essential cellular processes including proliferation, differentiation, migration, programmed cell death and various cancer-relevant processes. We investigated the interactions of KLF group-2 members with their binding partners to assess the role of acetylation and phosphorylation in KLFs on their binding affinity. It was observed that acetylation and phosphorylation at different positions in KLFs have a variable effect on binding with specific partners. KLF2-EP300, KLF4-SP1, KLF6-ATF3, KLF6-JUN, and KLF7-JUN show stabilization upon acetylation or phosphorylation at variable positions. On the other hand, KLF4-CBP, KLF4-EP300, KLF5-CBP, KLF5-WWP1, KLF6-SP1, and KLF7-ATF3 show stabilization or destabilization due to acetylation or phosphorylation at variable positions in KLFs. This provides a molecular explanation of the experimentally observed dual role of KLF group-2 members as a suppressor or activator of cancers in a PTM-dependent manner.

Short-Chain Fatty Acid (SCFA) as a Connecting Link between Microbiota and Gut-Lung Axis-A Potential Therapeutic Intervention to Improve Lung Health.

The microbiome is an integral part of the human gut, and it plays a crucial role in the development of the immune system and homeostasis. Apart from the gut microbiome, the airway microbial community also forms a distinct and crucial part of the human microbiota. Furthermore, several studies indicate the existence of communication between the gut microbiome and their metabolites with the lung airways, called "gut-lung axis". Perturbations in gut microbiota composition, termed dysbiosis, can have acute and chronic effects on the pathophysiology of lung diseases. Microbes and their metabolites in lung stimulate various innate immune pathways, which modulate the expression of the inflammatory genes in pulmonary leukocytes. For instance, gut microbiota-derived metabolites such as short-chain fatty acids can suppress lung inflammation through the activation of G protein-coupled receptors (free fatty acid receptors) and can also inhibit histone deacetylase, which in turn influences the severity of acute and chronic respiratory diseases. Thus, modulation of the gut microbiome composition through probiotic/prebiotic usage and fecal microbiota transplantation can lead to alterations in lung homeostasis and immunity. The resulting manipulation of immune cells function through microbiota and their key metabolites paves the way for the development of novel therapeutic strategies in improving the lung health of individuals affected with various lung diseases including SARS-CoV-2. This review will shed light upon the mechanistic aspect of immune system programming through gut and lung microbiota and exploration of the relationship between gut-lung microbiome and also highlight the therapeutic potential of gut microbiota-derived metabolites in the management of respiratory diseases.

MediatorWeb: a protein-protein interaction network database for the RNA polymerase II Mediator complex.

The protein-protein interaction (PPI) network of the Mediator complex is very tightly regulated and depends on different developmental and environmental cues. Here, we present an interactive platform for comparative analysis of the Mediator subunits from humans, baker's yeast Saccharomyces cerevisiae, and model plant Arabidopsis thaliana in a user-friendly web-interface database called MediatorWeb. MediatorWeb provides an interface to visualize and analyze the PPI network of Mediator subunits. The database facilitates downloading the untargeted and unweighted network of Mediator complex, its submodules, and individual Mediator subunits to better visualize the importance of individual Mediator subunits or their submodules. Further, MediatorWeb offers network visualization of the Mediator complex and interacting proteins that are functionally annotated. This feature provides clues to understand functions of Mediator subunits in different processes. In an additional tab, MediatorWeb provides quick access to secondary and tertiary structures, as well as residue-level contact information for Mediator subunits in each of the three model organisms. Another useful feature of MediatorWeb is detection of interologs based on orthologous analyses, which can provide clues to understand the functions of Mediator complex in less explored kingdoms. Thus, MediatorWeb and its features can help the user to understand the role of Mediator complex and its subunits in the transcription regulation of gene expression.

CD4+ T cell-derived novel peptide Thp5 induces interleukin-4 production in CD4+ T cells to direct T helper 2 cell differentiation.

The differentiation of naïve CD4(+) T cells into T helper 2 (Th2) cells requires production of the cytokine IL-4 in the local microenvironment. It is evident that naïve/quiescently activated CD4(+) T cells produce the IL-4 that drives Th2 cell differentiation. Because early production of IL-4 in naïve T cells leads to preferential Th2 cell differentiation, this process needs to be tightly regulated so as to avoid catastrophic and misdirected Th2 cell differentiation. Here, we show that Thp5, a novel peptide with structural similarity to vasoactive intestinal peptide, regulates production of early IL-4 in newly activated CD4(+) T cells. Induction of IL-4 in CD4(+) T cells by Thp5 is independent of the transcription factor STAT6 but dependent on ERK1/2 signaling. Furthermore, cytokines (IL-12 and TGF-ß) that promote the differentiation of Th1 or Th17 cells inhibit Thp5 induction, thus suppressing Th2 cell differentiation. We further showed that Thp5 enhances Th2 responses and exacerbates allergic airway inflammation in mice. Taken together, our findings reveal that early activated CD4(+) T cells produce Thp5, which plays a critical role as a molecular switch in the differentiation of Th cells, biasing the response toward the Th2 cell phenotype.

Upregulation of galactose metabolic pathway by N-acetylglucosamine induced endogenous synthesis of galactose in Candida albicans.

N-Acetylglucosamine (GlcNAc) is an important signaling molecule that plays multiple roles in Candida albicans. Induction of galactose metabolic pathway by GlcNAc is an intriguing aspect of C. albicans biology. In order to investigate the role of galactose metabolic genes (GAL genes) in presence of GlcNAc, we created knockouts of galactokinase (GAL1) and UDP galactose epimerase (GAL10) genes. These mutants failed to grow on galactose and also showed lower growth rate in presence of GlcNAc. Interestingly, expression of GAL genes in presence of GlcNAc was higher in gal1Δ strain relative to that of wild type strain. Moreover, no GlcNAc induced upregulation of GAL genes was observed in the gal10Δ strain suggesting that UDP galactose epimerase is essential for GlcNAc induced activation of GAL genes. GlcNAc induced expression of GAL genes was also investigated in GlcNAc metabolic pathway triple mutant N216 (hxk1Δ nag1Δ dac1Δ). Interestingly, in this mutant the GAL genes are neither induced nor repressed and remain derepressed as found on a neutral carbon source such as glycerol, suggesting that catabolism of GlcNAc play an important role in the expression of GAL genes. GC/MS analysis of derivatized metabolites revealed a significant accumulation of galactose in the gal1Δ strain while no galactose was detected in gal10Δ and N216 strain. Solution-state NMR spectroscopy using N-acetyl-¹³C1-glucosamine confirmed the flow of ¹³C label from GlcNAc to galactose. Thus, internal galactose synthesized via UDP galactose pathway from GlcNAc metabolites acts as the inducer of GAL genes in presence of GlcNAc.

Purification, crystallization and preliminary crystallographic studies of C-terminal RNA recognition motif (RRM-3) of human ELAV-type RNA-binding protein 3 (ETR-3).

Human embryonically lethal abnormal vision (ELAV)-type RNA-binding protein 3 (ETR-3) has been implicated in many aspects of RNA-processing events including alternative splicing, stability, editing and translation. RNA recognition motif 3 (RRM-3) is an independent C-terminal RNA-binding domain of ETR-3 that preferentially binds to UG-rich repeats of the nuclear or cytoplasmic pre-mRNA, and along with the other domains mediates the inclusion of cardiac troponin T (c-TNT) exon 5 in embryonic muscle, which is otherwise excluded in the adult. In the present study, RRM-3 was cloned, overexpressed, purified and crystallized by the hanging-drop vapour-diffusion method. The crystals diffracted to 3 Šresolution at the home source and belonged to space group P213, with unit-cell parameters a=b=c=118.5 Å, α=ß=γ=90°. There were two molecules of RRM-3 in the asymmetric unit and the calculated Matthews coefficient (VM) was 6.35 Å3 Da(-1), with a solvent content of 80.62%. Initial phases were determined by molecular replacement.

Microbiome analysis of saliva from oral squamous cell carcinoma (OSCC) patients and tobacco abusers with potential biomarkers for oral cancer screening.

Oral squamous cell carcinoma (OSCC) is the most common type of oral cancer and accounts for about 95% of all head and neck cancers with high mortality, usually at a late stage. Dysbiosis in the oral microbiome can lead to chronic inflammatory responses and may predispose to the development and progression of OSCC. Tobacco abuse plays an essential role in oral microbiome dysregulation and OSCC pathogenesis. We used 16S rRNA gene amplicon next-generation sequencing to examine microbial signatures unique to saliva from OSCC patients, tobacco abusers (TA) and controls (n = 10 for each group) to elucidate oral microbiome changes associated with tobacco abuse and OSCC. Overall, the oral microbiome compositions of class Betaproteobacteria and Epsilonproteobacteria, order Neisseriales, Burkholderiales and Campylobacterales, family Burkholderiaceae and Campylobacteraceae and genera Campylobacter and Leptotrichia revealed significant differences among OSCC patients, TA and control. Our preliminary pilot study not only serves as a basis for future studies with large sample size but also gives an indication of microbiome-based potential non-invasive biomarkers for early screening and monitoring of oral carcinogenesis transition due to tobacco abuse.

Structural delineation of histone post-translation modifications in histone-nucleosome assembly protein complex.

Nucleosome assembly proteins (Nap) are histone chaperones with vital roles in chromatin assembly and disassembly. Decoding of histone post-translational modifications by histone chaperones is central in regulation of gene expression. We probed binding interfaces in Nap-histone complexes using histone peptide interaction arrays and chemical shift perturbation techniques. Phosphorylation at H3T32 and H3T118 enhanced Nap-peptide interactions while post-translational modifications like H3K122, H4K59 and H2AK36 diminished it. Nap recognition regions in histones H2A, H2B, H3 and H4 lie in core regions of the histone octamer, and form a surface exposed contiguous patch. Nap binding sites on H2A-H2B and H3-H4 fall on opposite surfaces of histone octamer, and the latter can therefore accommodate two dimeric Nap molecules simultaneously. Interfacial residues between Nap and histone octamer overlap with route of wrapped DNA on histone octamer, implying non-concurrent residency on the octamer by either Nap or DNA. Using NMR, we mapped Nap residues that undergo chemical shift perturbations in presence of histone peptides. Taken together, we provide an architectural model of a Nap-octamer complex which likely forms during chromatin reorganisation.

Cloning, purification, crystallization and preliminary X-ray diffraction studies of Escherichia coli PapD-like protein (EcpD).

Many Gram-negative bacteria are characterized by hair-like proteinaceous appendages on their surface known as fimbriae. In uropathogenic strains of Escherichia coli, fimbriae mediate attachment by binding to receptors on the host cell, often contributing to virulence and disease. E. coli PapD-like protein (EcpD) is a periplasmic chaperone that plays an important role in the proper folding and guiding of Yad fimbrial proteins to the outer membrane usher protein in a process known as pilus biogenesis. EcpD is essential for pilus biogenesis in uropathogenic E. coli and plays an important role in virulence. In the present study, EcpD was cloned, overexpressed, purified and crystallized by the hanging-drop vapour-diffusion method. The crystals diffracted to 1.67 Šresolution and belonged to the orthorhombic space group C222(1), with unit-cell parameters a = 100.3, b = 127.6, c = 45.9 Å. There was a single molecule in the asymmetric unit and the corresponding Matthews coefficient was calculated to be 3.02 Å(3) Da(-1), with 59% solvent content. Initial phases were determined by molecular replacement.

Cloning, purification, crystallization and preliminary X-ray crystallographic analysis of a cyclophilin A-like protein from Piriformospora indica.

Cyclophilins are widely distributed both in eukaryotes and prokaryotes and have a primary role as peptidyl-prolyl cis-trans isomerases (PPIases). This study focuses on the cloning, expression, purification and crystallization of a salinity-stress-induced cyclophilin A (CypA) homologue from the symbiotic fungus Piriformospora indica. Crystallization experiments in the presence of 56 mM sodium phosphate monobasic monohydrate, 1.34 M potassium phosphate dibasic pH 8.2 yielded crystals that were suitable for X-ray diffraction analysis. The crystals belonged to the orthorhombic space group C222(1), with unit-cell parameters a = 121.15, b = 144.12, c = 110.63 Å. The crystals diffracted to a resolution limit of 2.0 Å. Analysis of the diffraction data indicated the presence of three molecules of the protein per asymmetric unit (V(M) = 4.48 Å(3) Da(-1), 72.6% solvent content).