Search

With varying clinical symptoms, most neurodegenerative diseases are associated with abnormal loss of neurons. They share the same common pathogenic mechanisms involving misfolding and aggregation, and these visible aggregates of proteins are deposited in the central nervous system. Amyloid formation is thought to arise from partial unfolding of misfolded proteins leading to the exposure of hydrophobic surfaces, which interact with other similar structures and give rise to form dimers, oligomers, protofibrils, and eventually mature fibril aggregates. Accumulating evidence indicates that amyloid oligomers, not amyloid fibrils, are the most toxic species that causes Alzheimer's disease (AD) and Parkinson's disease (PD). AD has recently been recognized as the ‘twenty-first century plague’, with an incident rate of 1% at 60 years of age, which then doubles every fifth year. Currently, 5.3 million people in the US are afflicted with this disease, and the number of cases is expected to rise to 13.5 million by 2050. PD, a disorder of the brain, is the second most common form of dementia, characterized by difficulty in walking and movement. Keeping the above views in mind, in this review we have focused on the roles of amyloid in neurodegenerative diseases including AD and PD, the involvement of amyloid in mitochondrial dysfunction leading to neurodegeneration, are also considered in the review.

Nanobodies (Nbs), the single-domain antigen-binding fragments of dromedary heavy-chain antibodies (HCAb), are excellent candidates as therapeutic and diagnostic tools in synucleinopathies because of their small size, solubility and stability. Here, we constructed an immune nanobody library specific to the monomeric form of alpha-synuclein (α-syn). Phage display screening of the library allowed the identification of a nanobody, Nbα-syn01, specific for α-syn. Unlike previously developed nanobodies, Nbα-syn01 recognized the N-terminal region which is critical for in vitro and in vivo aggregation and contains many point mutations involved in early PD cases. The affinity of the monovalent Nbα-syn01 and the engineered bivalent format BivNbα-syn01 measured by isothermal titration calorimetry revealed unexpected results where Nbα-syn01 and its bivalent format recognized preferentially α-syn fibrils compared to the monomeric form. Nbα-syn01 and BivNbα-syn01 were also able to inhibit α-syn-seeded aggregation in vitro and reduced α-syn-seeded aggregation and toxicity in cells showing their potential to reduce α-syn pathology. Moreover, both nanobody formats were able to recognize Lewy-body pathology in human post-mortem brain tissue from PD and DLB cases. Additionally, we present evidence through structural docking that Nbα-syn01 binds the N-terminal region of the α-syn aggregated form. Overall, these results highlight the potential of Nbα-syn01 and BivNbα-syn01 in developing into a diagnostic or a therapeutic tool for PD and related disorders.

The prevalence of homocystinuria in Qatar is 1:1800, mainly due to a founder missense mutation p.R336C. • The cystathionine beta-synthase (CBS) R336C mutant was bacterially expressed, purified and its molecular properties were compared to CBS wild type (WT) recombinant protein. • Our data revealed that p.R336C mutation results in a dramatic reduction (∼86%) of CBS enzymatic activity. • Circular Dichroism experiments suggested that the p.R336C mutation does not significantly alter the secondary structure of the CBS protein. • CD spectra also revealed distinct differences in the thermal unfolding mechanisms of CBS WT and R336C mutant protein species. • Chemical denaturation experiments indicated that the WT CBS protein is thermodynamically more stable than the R336C mutant, suggesting a destabilizing effect of the p.R336C mutation. • This study provides mechanistic insight into the pathogenicity of the p.R336C mutation that leads to a severe homocystinuria phenotype. This publication was made possible by GSRA grant, GSRA6-1-0413-19013, from the Qatar National Research Fund (a member of Qatar Foundation). In addition, NA would like to thank fund from The Medical Research Center at Hamad Medical Corporation for IRGC-06-SI-19-167 grant. The statements made herein are solely the responsibility of the authors. Open Access funding provided by the Qatar National Library.

Fibrin-based systems offer promises in drug and gene delivery as well as tissue engineering. We established earlier a fibrin-based plasma beads (PB) system as an efficient carrier of drugs and antigens. In the present work, attempts were made to further improve its therapeutic efficacy exploiting innovative ideas, including the use of plasma alginate composite matrices, proteolytic inhibitors, cross linkers, and dual entrapment in various liposomal formulations. In vitro efficacy of the different formulations was examined. Pharmacokinetics of the formulations encapsulating Amphotericin B (AmpB), an antifungal compound, were investigated in Swiss albino mice. While administration of the free AmpB led to its rapid elimination (144 h) and its gradual accumulation in the vital organs, also compared to the liposomal formulations alone. Interestingly, the slow release of AmpB from PB was unusual compared to other small molecules in our earlier findings, suggesting strong interaction with plasma proteins. Molecular interaction studies of bovine serum albumin constituting approximately 60% of plasma with AmpB using isothermal titration calorimetry and in silico docking verify these interactions, explaining the slow release of AmpB entrapped in PB alone. The above findings suggest that PB/liposome-PB could be used as safe and effective delivery systems to combat fungal infections in humans.

Higher eukaryotic development is a complex and tightly regulated process, whereby transcription factors (TFs) play a key role in controlling the gene regulatory networks. Dysregulation of these regulatory networks has also been associated with carcinogenesis. Transcription factors are key enablers of cancer stemness, which support the maintenance and function of cancer stem cells that are believed to act as seeds for cancer initiation, progression and metastasis, and treatment resistance. One key area of research is to understand how these factors interact and collaborate to define cellular fate during embryogenesis as well as during tumor development. This review focuses on understanding the role of TFs in cell development and cancer. The molecular mechanisms of cell fate decision are of key importance in efforts towards developing better protocols for directed differentiation of cells in research and medicine. We also discuss the dysregulation of TFs and their role in cancer progression and metastasis, exploring TF networks as direct or indirect targets for therapeutic intervention, as well as specific TFs’ potential as biomarkers for predicting and monitoring treatment responses.

MotivationX-ray crystallography has facilitated the majority of protein structures determined to date. Sequence-based predictors that can accurately estimate protein crystallization propensities would be highly beneficial to overcome the high expenditure, large attrition rate, and to reduce the trial-and-error settings required for crystallization.ResultsIn this study, we present a novel model, BCrystal, which uses an optimized gradient boosting machine (XGBoost) on sequence, structural and physio-chemical features extracted from the proteins of interest. BCrystal also provides explanations, highlighting the most important features for the predicted crystallization propensity of an individual protein using the SHAP algorithm. On three independent test sets, BCrystal outperforms state-of-the-art sequence-based methods by more than 12.5% in accuracy, 18% in recall and 0.253 in Matthew’s correlation coefficient, with an average accuracy of 93.7%, recall of 96.63% and Matthew’s correlation coefficient of 0.868. For relative solvent accessibility of exposed residues, we observed higher values to associate positively with protein crystallizability and the number of disordered regions, fraction of coils and tripeptide stretches that contain multiple histidines associate negatively with crystallizability. The higher accuracy of BCrystal enables it to accurately screen for sequence variants with enhanced crystallizability.Availability and implementationOur BCrystal webserver is at https://machinelearning-protein.qcri.org/ and source code is available at https://github.com/raghvendra5688/BCrystal.Supplementary informationSupplementary data are available at Bioinformatics online.

Liposomes have established themselves as great pharmaceutical carriers over the past three decades. These phospholipid vesicular systems have undergone great technical advances including remote drug loading, targeted delivery, and combinatorial drug therapy. Ionic gradient liposomes (IGL) necessitates active loading of the drug in preformed vesicles exhibiting a transmembrane pH or ion gradient, with a low intra liposome pH (∼ 4-5), and a high outside pH (∼7-8). It allows high drug encapsulation and prolonged release, particularly for amphipathic weak acids and weak bases. Most local anesthetics (Bupivacaine, Ropivacaine, Tetracaine, and others) have a pka in the range of 7-9, which makes them ideal candidates for their entrapment in IGL. The same is true for most anthracyclines which have great anti-tumor properties (Doxorubicin, Daunorubicin, Idarubicin, and others). Many FDA approved liposomal drugs utilise ion gradient for their encapsulation. Considering their immense utility, we summarize here in this review, the recent contributions made by various research groups utilizing IGL, to accentuate the development of these carriers in drug delivery. This would possibly be helpful in carrying new investigations and further contributions in the optimization and advancements of new drugs for better therapeutics.

Protein misfolding and aggregation can be induced by a wide variety of factors, such as dominant disease-associated mutations, changes in the environmental conditions (pH, temperature, ionic strength, protein concentration, exposure to transition metal ions, exposure to toxins, posttranslational modifications including glycation, phosphorylation, and sulfation). Misfolded intermediates interact with similar intermediates and progressively form dimers, oligomers, protofibrils, and fibrils. In amyloidoses, fibrillar aggregates are deposited in the tissues either as intracellular inclusion or extracellular plaques (amyloid). When such proteinaceous deposit occurs in the neuronal cells, it initiates degeneration of neurons and consequently resulting in the manifestation of various neurodegenerative diseases. Several different types of molecules have been designed and tested both in vitro and in vivo to evaluate their anti-amyloidogenic efficacies. For instance, the native structure of a protein associated with amyloidosis could be stabilized by ligands, antibodies could be used to remove plaques, oligomer-specific antibody A11 could be used to remove oligomers, or prefibrillar aggregates could be removed by affibodies. Keeping the above views in mind, in this review we have discussed protein misfolding and aggregation, mechanisms of protein aggregation, factors responsible for aggregations, and strategies for aggregation inhibition.

Motivation A global effort is underway to identify compounds for the treatment of COVID-19. Since de novo compound design is an extremely long, time-consuming and expensive process, efforts are underway to discover existing compounds that can be repurposed for COVID-19 and new viral diseases. We propose a machine learning representation framework that uses deep learning induced vector embeddings of compounds and viral proteins as features to predict compound-viral protein activity. The prediction model in-turn uses a consensus framework to rank approved compounds against viral proteins of interest. Results Our consensus framework achieves a high mean Pearson correlation of 0.916, mean R2 of 0.840 and a low mean squared error of 0.313 for the task of compound-viral protein activity prediction on an independent test set. As a use case, we identify a ranked list of 47 compounds common to three main proteins of SARS-COV-2 virus (PL-PRO, 3CL-PRO and Spike protein) as potential targets including 21 antivirals, 15 anticancer, 5 antibiotics and 6 other investigational human compounds. We perform additional molecular docking simulations to demonstrate that majority of these compounds have low binding energies and thus high binding affinity with the potential to be effective against the SARS-COV-2 virus. Availability and implementation All the source code and data is available at: https://github.com/raghvendra5688/Drug-Repurposing and https://dx.doi.org/10.17632/8rrwnbcgmx.3. We also implemented a web-server at: https://machinelearning-protein.qcri.org/index.html. Supplementary information Supplementary data are available at Bioinformatics online.

Amyloid fibrillation is the root cause of several neuro as well as non-neurological disorders. Understanding the molecular basis of amyloid aggregate formation is crucial for deciphering various neurodegenerative diseases. In our study, we have examined the lysozyme fibrillation process using nano-infrared spectroscopy (nanoIR). NanoIR enabled us to investigate both structural and chemical characteristics of lysozyme fibrillar species concurrently. The spectroscopic results indicate that lysozyme transformed into a fibrillar structure having mainly parallel β-sheets, with almost no antiparallel β-sheets. Features such as protein stiffness have a good correlation with obtained secondary structural information showing the state of the protein within the fibrillation state. The structural and chemical details were compared with transmission electron microscopy (TEM) and circular dichroism (CD). We have utilized nanoIR and measured infrared spectra to characterize lysozyme amyloid fibril structures in terms of morphology, molecular structure, secondary structure content, stability, and size of the cross-β core. We have shown that the use of nanoIR can complement other biophysical studies to analyze the aggregation process and is particularly useful for studying proteins involved in aggregation to help in designing molecules against amyloid aggregation. Specifically, the nanoIR spectra afford higher resolution information and a characteristic fingerprint for determining states of aggregation.Communicated by Ramaswamy H. Sarma.

Background PHF21A has been associated with intellectual disability and craniofacial anomalies based on its deletion in the Potocki-Shaffer syndrome region at 11p11.2 and its disruption in three patients with balanced translocations. In addition, three patients with de novo truncating mutations in PHF21A were reported recently. Here, we analyze genomic data from seven unrelated individuals with mutations in PHF21A and provide detailed clinical descriptions, further expanding the phenotype associated with PHF21A haploinsufficiency. Methods Diagnostic trio whole exome sequencing, Sanger sequencing, use of GeneMatcher, targeted gene panel sequencing, and MiSeq sequencing techniques were used to identify and confirm variants. RT-qPCR was used to measure the normal expression pattern of PHF21A in multiple human tissues including 13 different brain tissues. Protein-DNA modeling was performed to substantiate the pathogenicity of the missense mutation. Results We have identified seven heterozygous coding mutations, among which six are de novo (not maternal in one). Mutations include four frameshifts, one nonsense mutation in two patients, and one heterozygous missense mutation in the AT Hook domain, predicted to be deleterious and likely to cause loss of PHF21A function. We also found a new C-terminal domain composed of an intrinsically disordered region. This domain is truncated in six patients and thus likely to play an important role in the function of PHF21A, suggesting that haploinsufficiency is the likely underlying mechanism in the phenotype of seven patients. Our results extend the phenotypic spectrum of PHF21A mutations by adding autism spectrum disorder, epilepsy, hypotonia, and neurobehavioral problems. Furthermore, PHF21A is highly expressed in the human fetal brain, which is consistent with the neurodevelopmental phenotype. Conclusion Deleterious nonsense, frameshift, and missense mutations disrupting the AT Hook domain and/or an intrinsically disordered region in PHF21A were found to be associated with autism spectrum disorder, epilepsy, hypotonia, neurobehavioral problems, tapering fingers, clinodactyly, and syndactyly, in addition to intellectual disability and craniofacial anomalies. This suggests that PHF21A is involved in autism spectrum disorder and intellectual disability, and its haploinsufficiency causes a diverse neurological phenotype.

Alzheimer's disease (AD) is one of the most common age-related diseases that occurs because of the deposition of amyloid fibrils in a form of extracellular plaques containing β-amyloid peptide (Aβ) and tangles are found as intracellular deposit in the brain made up of twisted strands of aggregated microtubule binding protein. Scores of small molecule inhibitors have been designed for the treatment of AD. However some of these drugs cannot pass through the brain-blood-barrier (BBB). To overcome this problem, various nanoparticles (NPs) or nanomedicines (NMs) have been synthesized. These nanoparticles exploit the existing physiological mechanisms of passing through the BBB, including receptor- and adsorptive-mediated transcytosis that facilitate the transcellular transport of nanoparticle from the blood to the brain. During the last decades, varieties of nanoparticles that differ in the composition have been developed, and they have the potential application in the diagnostics and therapy of AD. The most common NP formulations that have major impact in the diagnosis and therapy of AD include polymeric NPs (PPs), gold NPs, gadolinium NPs, selenium NPs, protein-based NPs, polysaccharide-based NPs, etc. The goal of this review is to provide discussion of the application of different types of NP formulations in the diagnosis and therapy of AD.

Background: Aggregation of α-synuclein (α-syn) has been implicated in multiple neurodegenerative disorders including Parkinson’s disease (PD), dementia with Lewy bodies (DLB) and multiple system atrophy (MSA), collectively grouped as synucleinopathies. Recently, recombinant antibody fragments (Fab, scFvs and diabodies) against α-syn have emerged as an alternative to the traditional full-length antibody in immunotherapeutic approaches owing to their advantages including smaller size and higher stability, specificity and affinity. However, most of the recombinant antibody fragments tend to be expressed as inclusion bodies (IBs) making its purification extremely challenging. Results: In the current study, a single-chain variable fragment (scFv-F) antibody, targeting the pathogenic α-syn fibrils, was engineered and expressed in E.coli. Majority of the expressed scFv-F accumulated in insoluble aggregates as IBs. A variety of mild and harsh solubilizing conditions were tested to solubilize IBs containing scFv-F to obtain the active protein. To preserve secondary structure and bioactivity, a mild solubilizing protocol involving 100 mM Tris, pH 12.5 with 2 M urea was chosen to dissolve IBs. Slow on-column refolding method was employed to subsequently remove urea and obtain active scFv-F. A three-dimensional (3D) model was built using homology modeling and subjected to molecular docking with the known α-syn structure. Structural alignment was performed to delineate the potential binding pocket. Conclusion: The scFv-F thus purified demonstrated high specificity towards α-syn fibrils compared to monomers. Molecular modeling studies suggest that scFv-F shares the same structural topology with other known scFvs. We present evidence through structural docking and alignment that scFv-F binds to α-syn C-terminal region. In conclusion, mild solubilization followed by slow on-column refolding can be utilized as a generalized and efficient method for hard to purify disease relevant insoluble proteins and/or antibody molecules from IBs.

Hsp70: J-domain protein (JDP) machines, along with the cellular protein degradation systems play a central role in regulating cellular proteostasis. An equally robust surveillance system operates at the plasma membrane too that affects proper sorting, stability as well as the turnover of membrane proteins. Although plausible, a definitive role of the Hsp70: JDP machine in regulating the stability of plasma membrane proteins is not well understood in Saccharomyces cerevisiae. Here we show that a moderate over-expression of Caj1, one of the thirteen JDPs residing in the nucleo-cytosolic compartment of S. cerevisiae reduced the cold sensitivity of tryptophan auxotrophic yeast cells by stabilizing tryptophan permeases, Tat1 and Tat2 in a J-domain dependent manner. Concomitantly, higher Caj1 levels also caused slow growth and increased plasma membrane damage at elevated temperatures possibly due to the stabilization of thermolabile plasma membrane proteins. Finally, we show that although majorly cytosolic, Caj1 also co-localizes with the membrane dye FM4-64 at the cellular periphery suggesting that Caj1 might interact with the plasma membrane. Based on the results presented in this study, we implicate the Hsp70: Caj1 chaperone machine in regulating the stability or turnover of plasma membrane proteins in budding yeast.

Phosphate-activated glutaminases catalyze the deamidation of glutamine to glutamate and play key roles in several physiological and pathological processes. In humans, GLS encodes two multidomain splicing isoforms: KGA and GAC. In both isoforms, the canonical glutaminase domain is flanked by an N-terminal region that is folded into an EF-hand-like four-helix bundle. However, the splicing event replaces a well-structured three-repeat ankyrin domain in KGA with a shorter, unordered C-terminal stretch in GAC. The multidomain architecture, which contains putative protein-protein binding motifs, has led to speculation that glutaminases are involved in cellular processes other than glutamine metabolism; in fact, some proteins have been identified as binding partners of KGA and the isoforms of its paralogue gene, GLS2. Here, a yeast two-hybrid assay identified nuclear receptor peroxisome proliferator-activated receptor γ (PPARγ) as a new binding partner of the glutaminase. We show that KGA and GAC directly bind PPARγ with a low-micromolar dissociation constant; the interaction involves the N-terminal and catalytic domains of glutaminases as well as the ligand-binding domain of the nuclear receptor. The interaction occurs within the nucleus, and by sequestering PPARγ from its responsive element DR1, the glutaminases decreased nuclear receptor activity as assessed by a luciferase reporter assay. Altogether, our findings reveal an unexpected glutaminase-binding partner and, for the first time, directly link mitochondrial glutaminases to an unanticipated role in gene regulation.

The transcription factor Sox2 plays an important role in various phases of embryonic development, including cell fate and differentiation. These key regulatory functions are facilitated by binding to specific DNA sequences in combination with partner proteins to exert their effects. Recently, overexpression and gene amplification of Sox2 has been associated with tumor aggression and metastasis in various cancer types, including breast, prostate, lung, ovarian and colon cancer. All the different roles for Sox2 involve complicated regulatory networks consisting of protein-protein and protein-nucleic acid interactions. Their involvement in the EMT modulation is possibly enabled by Wnt/ β-catenin and other signaling pathways. There are number of in vivo models which show Sox2 association with increased cancer aggressiveness, resistance to chemo-radiation therapy and decreased survival rate suggesting Sox2 as a therapeutic target. This review will focus on the different roles for Sox2 in metastasis and tumorigenesis. We will also review the mechanism of action underlying the cooperative Sox2- DNA/partner factors binding where Sox2 can be potentially explored for a therapeutic opportunity to treat cancers.

The active transport of glycolytic pyruvate across the inner mitochondrial membrane is thought to involve two mitochondrial pyruvate carrier subunits, MPC1 and MPC2, assembled as a 150 kDa heterotypic oligomer. Here, the recombinant production of human MPC through a co-expression strategy is first described; however, substantial complex formation was not observed, and predominantly individual subunits were purified. In contrast to MPC1, which co-purifies with a host chaperone, we demonstrated that MPC2 homo-oligomers promote efficient pyruvate transport into proteoliposomes. The derived functional requirements and kinetic features of MPC2 resemble those previously demonstrated for MPC in the literature. Distinctly, chemical inhibition of transport is observed only for a thiazolidinedione derivative. The autonomous transport role for MPC2 is validated in cells when the ectopic expression of human MPC2 in yeast lacking endogenous MPC stimulated growth and increased oxygen consumption. Multiple oligomeric species of MPC2 across mitochondrial isolates, purified protein and artificial lipid bilayers suggest functional high-order complexes. Significant changes in the secondary structure content of MPC2, as probed by synchrotron radiation circular dichroism, further supports the interaction between the protein and ligands. Our results provide the initial framework for the independent role of MPC2 in homeostasis and diseases related to dysregulated pyruvate metabolism.

The mitochondrial phosphate-activated glutaminase C (GAC) is produced by the alternative splicing of the GLS gene. Compared to the other GLS isoform, the kidney-type glutaminase (KGA), GAC is more enzymatically efficient and of particular importance for cancer cell growth. Although its catalytic mechanism is well understood, little is known about how post-translational modifications can impact GAC function. Here, we identified by mass spectrometry a phosphorylated serine at the GLS N-terminal domain (at position 95) and investigated its role on regulating GAC activity. The ectopic expression of the phosphomimetic mutant (GAC.S95D) in breast cancer cells, compared to wild-type GAC (GAC.WT), led to decreased glutaminase activity, glutamine uptake, glutamate release and intracellular glutamate levels, without changing GAC sub-cellular localization. Interestingly, cells expressing the GAC.S95D mutant, compared to GAC.WT, presented decreased migration and vimentin level, an epithelial-to-mesenchymal transition marker. These results reveal that GAC is post-translationally regulated by phosphorylation, which affects cellular glutamine metabolism and glutaminase-related cell phenotype.

Classically, ankyrin repeat (ANK) proteins are built from tandems of two or more repeats and form curved solenoid structures that are associated with protein-protein interactions. These are short, widespread structural motif of around 33 amino acids repeats in tandem, having a canonical helix-loop-helix fold, found individually or in combination with other domains. The multiplicity of structural pattern enables it to form assemblies of diverse sizes, required for their abilities to confer multiple binding and structural roles of proteins. Three-dimensional structures of these repeats determined to date reveal a degree of structural variability that translates into the considerable functional versatility of this protein superfamily. Recent work on the ANK has proposed novel structural information, especially protein-lipid, protein-sugar and protein-protein interaction. Self-assembly of these repeats was also shown to prevent the associated protein in forming filaments. In this review, we summarize the latest findings and how the new structural information has increased our understanding of the structural determinants of ANK proteins. We discussed latest findings on how these proteins participate in various interactions to diversify the ANK roles in numerous biological processes, and explored the emerging and evolving field of designer ankyrins and its framework for protein engineering emphasizing on biotechnological applications.

Protein homeostasis (proteostasis) is achieved by the interplay among various components and pathways inside a cell. Dysfunction in proteostasis leads to protein misfolding and aggregation which is ubiquitously associated with many neurodegenerative disorders, although the exact role of these aggregate in the pathogenesis remains unknown. Many neurodegenerative diseases, including Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, and others are characterized by the conversion of specific protein aggregates into protein inclusions and/or plaques in degenerating brains. Apart from the conventional disease specific proteins, such as amyloid-β, α - synuclein, huntingtin protein, and prions that are known to aggregate, a number of other proteins play a vital role in aggravating the disease condition. In this review, we discuss the disease etiology, mechanism, the role of various pathways, molecular machinery including molecular chaperones, protein degradation pathways, and the active formation of inclusions in various neurodegenerative diseases. We also highlight the approaches, strategies, and methods that have been used for the treatment of these complex diseases over the years and the efforts that have potential in the near future.

On the basis of tissue-specific enzyme activity and inhibition by catalytic products, Hans Krebs first demonstrated the existence of multiple glutaminases in mammals. Currently, two human genes are known to encode at least four glutaminase isoforms. However, the phylogeny of these medically relevant enzymes remains unclear, prompting us to investigate their origin and evolution. Using prokaryotic and eukaryotic glutaminase sequences, we built a phylogenetic tree whose topology suggested that the multidomain architecture was inherited from bacterial ancestors, probably simultaneously with the hosting of the proto-mitochondrion endosymbiont. We propose an evolutionary model wherein the appearance of the most active enzyme isoform, glutaminase C (GAC), which is expressed in many cancers, was a late retrotransposition event that occurred in fishes from the Chondrichthyes class. The ankyrin (ANK) repeats in the glutaminases were acquired early in their evolution. To obtain information on ANK folding, we solved two high-resolution structures of the ANK repeat-containing C-termini of both kidney-type glutaminase (KGA) and GLS2 isoforms (glutaminase B and liver-type glutaminase). We found that the glutaminase ANK repeats form unique intramolecular contacts through two highly conserved motifs; curiously, this arrangement occludes a region usually involved in ANK-mediated protein-protein interactions. We also solved the crystal structure of full-length KGA and present a small-angle X-ray scattering model for full-length GLS2. These structures explain these proteins' compromised ability to assemble into catalytically active supra-tetrameric filaments, as previously shown for GAC. Collectively, these results provide information about glutaminases that may aid in the design of isoform-specific glutaminase inhibitors.

The conformation of bovine serum fetuin (BSF) was examined over the pH 7.0-12.9 regions by circular dichroism, intrinsic fluorescence and ANS binding. We observed that at higher pH, BSF exists in alkaline unfolded state. Our results provided evidence that correlates simultaneous formation of secondary structure followed by accumulation of hydrophobic clusters.

Maleylacetate reductase (EC 1.3.1.32) is an important enzyme that is involved in the degradation pathway of aromatic compounds and catalyzes the reduction of maleylacetate to 3-oxoadipate. The gene pnpD encoding maleylacetate reductase in Burkholderia sp. strain SJ98 was cloned, expressed in Escherichia coli and purified by affinity chromatography. The enzyme was crystallized in both native and SeMet-derivative forms by the sitting-drop vapour-diffusion method using PEG 3350 as a precipitant at 293 K. The crystals belonged to space group P2(1)2(1)2, with unit-cell parameters a = 72.91, b = 85.94, c = 53.07 A. X-ray diffraction data for the native and SeMet-derivative crystal were collected to 2.7 and 2.9 A resolution, respectively.

The riboflavin biosynthesis pathway has been shown to be essential in many pathogens and is absent in humans. Therefore, enzymes involved in riboflavin synthesis are considered as potential antibacterial drug targets. The enzyme 3,4-dihydroxy-2-butanone-4-phosphate synthase (DHBPS) catalyzes one of the two committed steps in the riboflavin pathway and converts d-ribulose 5-phosphate (Ru5P) to l-3,4-dihydroxy-2-butanone 4-phosphate and formate. Moreover, DHBPS is shown to be indispensable for Mycobacterium, Salmonella, and Helicobacter species. Despite the essentiality of this enzyme in bacteria, no inhibitor has been identified hitherto. Here, we describe kinetic and crystal structure characterization of DHBPS from Vibrio cholerae (vDHBPS) with a competitive inhibitor 4-phospho-d-erythronohydroxamic acid (4PEH) at 1.86-A resolution. In addition, we also report the structural characterization of vDHBPS in its apo form and in complex with its substrate and substrate plus metal ions at 1.96-, 1.59-, and 2.04-A resolution, respectively. Comparison of these crystal structures suggests that 4PEH inhibits the catalytic activity of DHBPS as it is unable to form a proposed intermediate that is crucial for DHBPS activity. Furthermore, vDHBPS structures complexed with substrate and metal ions reveal that, unlike Candida albicans, binding of substrate to vDHBPS induces a conformational change from an open to closed conformation. Interestingly, the position of second metal ion, which is different from that of Methanococcus jannaschii, strongly supports an active role in the catalytic mechanism. Thus, the kinetic and structural characterization of vDHBPS reveals the molecular mechanism of inhibition shown by 4PEH and that it can be explored further for designing novel antibiotics.