Assessing type I collagen expression and quality in cellular models of osteogenesis imperfecta.

Osteogenesis imperfecta (OI) is a group of genetic disorders of bone formation characterized by soft and shorter brittle bones in affected individuals. OI is generally considered a collagenopathy resulting from abnormal expression of type I collagen. As assay system to detect the cellular level and quality of type I collagen would help in rapid and correct detection of OI from the diagnostic perspectives. Here, we report an immunofluorescence assay for detection of type I collagen in fibroblast models of OI and represented them into two broad categories based on the expression level and aggregation characteristics of pro-α1(I). Cell phenotypic assays of pro-α1(I) in OI-related gene knocked down fibroblasts revealed aggregates of pro-α1(I) in conditions with knockdown of SERPINF1, CRTAP, P3H1, PPIB, SERPINH1, FKBP10, TMEM38B, MESD, and KDELR2, whereas pro-α1(I) expression was very low in fibroblasts which had knockdown of IFITM5, SP7, BMP1, WNT1, CREB3L1, MBTPS2, and CCDC134. The expression of pro-α1(I) showed abundant and non-aggregated distribution in the fibroblasts with knockdown of non-OI skeletal disorder-related genes (RAB33B and IFT52). The in vitro assay accurately detected abnormally expressed pro-α1(I) levels in cellular models of various types of OI. Thus, this procedure represents a promising point-of-detection assay for potential diagnosis and therapeutic decisions in OI.

Functional characterization of novel variants in SMPD1 in Indian patients with acid sphingomyelinase deficiency.

Pathogenic variations in SMPD1 lead to acid sphingomyelinase deficiency (ASMD), that is, Niemann-Pick disease (NPD) type A and B (NPA, NPB), which is a recessive lysosomal storage disease. The knowledge of variant spectrum in Indian patients is crucial for early and accurate NPD diagnosis and genetic counseling of families. In this study, we recruited 40 unrelated pediatric patients manifesting symptoms of ASMD and subnormal ASM enzyme activity. Variations in SMPD1 were studied using Sanger sequencing for all exons, followed by interpretation of variants based on American College of Medical Genetics and Genomics & Association for Molecular Pathology (ACMG/AMP) criteria. We identified 18 previously unreported variants and 21 known variants, including missense, nonsense, deletions, duplications, and splice site variations with disease-causing potential. Eight missense variants were functionally characterized using in silico molecular dynamic simulation and in vitro transient transfection in HEK293T cells, followed by ASM enzyme assay, immunoblot, and immunofluorescence studies. All the variants showed reduced ASM activity in transfected cells confirming their disease-causing potential. The study provides data for efficient prenatal diagnosis and genetic counseling of families with NPD type A and B.

An IRES-dependent translation of HYPK mRNA generates a truncated isoform of the protein that lacks the nuclear localization and functional ability.

Different mechanisms of translation initiation process exist to start the protein synthesis from various viral and eukaryotic mRNA. The cap-independent and tertiary structure directed translation initiation of mRNAs forms the basis of internal ribosome entry site (IRES) mediated translation initiation that helps in cellular protein production in different conditions. HYPK protein sequesters different aggregation-prone proteins to help in the cellular proteostasis. HYPK mRNA is differentially translated from an internal start/initiation codon to generate an amino terminal-truncated isoform (HSPC136) of HYPK protein. In this study, we report that an IRES-dependent translation initiation of HYPK mRNA results in the formation of the HSPC136/HYPK-ΔN isoform of HYPK protein. The IRES-driven translation product, HYPK-ΔN, lacks the N-terminal tri-arginine motif that acts as the nuclear localization signal (NLS) in the full-length HYPK protein. While the full-length HYPK protein translocates to the nucleus and prevents the aggregation of the mutant p53 (p53-R248Q) protein, the HYPK-ΔN lacks this activity. The NLS of HYPK is not evolutionarily conserved and its exclusive presence in the HYPK of higher eukaryotic animals imparts additional advantage to the HYPK protein in tackling the cytosolic as well as nuclear protein aggregates. The presence of the NLS in full-length HYPK also allows this protein to modulate the cell cycle. These results provide a mechanistic detail of HYPK mRNA's translation initiation control by an IRES that dictates the formation of HYPC136/HYPK-ΔN which lacks the nuclear localization and functional ability.

Disordered Nanostructure in Huntingtin Interacting Protein K Acts as a Stabilizing Switch To Prevent Protein Aggregation.

Protein misfolding due to mutation(s) and/or generation of unstable intermediate state(s) can be the cause of aberrant aggregations, leading to cellular degeneration. While molecular signatures like amyloidogenic regions cause aggregation, other features in proteins, like disorder and unique complexity regions, regulate and restrict such adhesive accumulation processes. Huntingtin interacting protein K (HYPK) is an aggregation-prone protein. Using various biophysical, microscopy, and computational techniques, we have deciphered how HYPK's N-terminal nanodisordered region plays a significant modulatory role in preventing its own aggregation and that of other proteins. HYPK's C-terminal hydrophobic regions lead to annular oligomerization and intermolecular charge interactions among the residues of low-complexity region (LCR) generate amorphous aggregates. The N-terminal disordered nanostructure loops toward the C-terminus, and a negative charge-rich patch in this region interacts with the LCR to shield LCR's positive charges. This interaction is required to prevent HYPK aggregation. Loss of this interaction causes partial unfolding of the structured C-terminus, resulting in HYPK's molten globule-like state and rapid annular oligomerization. The N-terminus also determines the specificity to mediate the differential bindings with aggregation-prone and wild type Huntingtin-exon1 proteins (Huntingtin97Q-exon1 and Huntingtin25Q-exon1). A sliding interaction of the specific N-terminal segment of HYPK along the extended polyglutamine region of Huntingtin-exon1 is responsible for HYPK's higher affinity for aggregation-prone Huntingtin than for its non-aggregating counterpart. Overall, our study provides evidence of the existence of disordered nanostructure in HYPK protein that mechanistically plays a decisive role in preventing both self and non-self protein aggregation.

Mce2R/Rv0586 of Mycobacterium tuberculosis is the functional homologue of FadRE. coli.

Lipid metabolism is critical to Mycobacterium tuberculosis survival and infection. Unlike Escherichia coli, which has a single FadR, the M. tuberculosis genome encodes five proteins of the FadR sub-family. While the role of E. coli FadR as a regulator of fatty acid metabolism is well known, the definitive functions of M. tuberculosis FadR proteins are still under investigation. An interesting question about the M. tuberculosis FadRs remains open: which one of these proteins is the functional homologue of E. coli FadR? To address this, we have applied two different approaches. The first one was the bioinformatics approach and the second one was the classical molecular genetic approach involving complementation studies. Surprisingly, the results of these two approaches did not agree. Among the five M. tuberculosis FadRs, Rv0494 shared the highest sequence similarity with FadRE. coli and Rv0586 was the second best match. However, only Rv0586, but not Rv0494, could complement E. coli ∆fadR, indicating that Rv0586 is the M. tuberculosis functional homologue of FadRE. coli. Further studies showed that both regulators, Rv0494 and Rv0586, show similar responsiveness to LCFA, and have conserved critical residues for DNA binding. However, analysis of the operator site indicated that the inter-palindromic distance required for DNA binding differs for the two regulators. The differences in the binding site selection helped in the success of Rv0586 binding to fadB upstream over Rv0494 and may have played a critical role in complementing E. coli ∆fadR. Further, for the first time, we report the lipid-responsive nature of Rv0586.

HosA, a MarR Family Transcriptional Regulator, Represses Nonoxidative Hydroxyarylic Acid Decarboxylase Operon and Is Modulated by 4-Hydroxybenzoic Acid.

Members of the Multiple antibiotic resistance Regulator (MarR) family of DNA binding proteins regulate transcription of a wide array of genes required for virulence and pathogenicity of bacteria. The present study reports the molecular characterization of HosA (Homologue of SlyA), a MarR protein, with respect to its target gene, DNA recognition motif, and nature of its ligand. Through a comparative genomics approach, we demonstrate that hosA is in synteny with nonoxidative hydroxyarylic acid decarboxylase (HAD) operon and is present exclusively within the mutS-rpoS polymorphic region in nine different genera of Enterobacteriaceae family. Using molecular biology and biochemical approach, we demonstrate that HosA binds to a palindromic sequence downstream to the transcription start site of divergently transcribed nonoxidative HAD operon and represses its expression. Furthermore, in silico analysis showed that the recognition motif for HosA is highly conserved in the upstream region of divergently transcribed operon in different genera of Enterobacteriaceae family. A systematic chemical search for the physiological ligand revealed that 4-hydroxybenzoic acid (4-HBA) interacts with HosA and derepresses HosA mediated repression of the nonoxidative HAD operon. Based on our study, we propose a model for molecular mechanism underlying the regulation of nonoxidative HAD operon by HosA in Enterobacteriaceae family.

Rv0494 is a starvation-inducible, auto-regulatory FadR-like regulator from Mycobacterium tuberculosis.

Fatty acid metabolism plays an important role in the survival and pathogenesis of Mycobacterium tuberculosis. Lipids are assumed to be the major source of energy during dormancy. Here, we report the characterization of a starvation-inducible, lipid-responsive transcriptional regulator, Rv0494, divergently transcribed from the Rv0493c probable operon. The striking difference in the transcriptional regulatory apparatus between mycobacteria and other well-studied organisms, such as Escherichia coli, is the organization of mycobacterial promoters. Mycobacterial promoters have diverse architectures and most of these promoters function inefficiently in E. coli. In this study, we characterized the promoter elements of Rv0494 along with the sigma factors required for transcription initiation. Rv0494 promoter activity increased under nutrient starvation conditions and was transcribed via two promoters: the promoter proximal to the translational start site was active under standard growth conditions, whilst both promoters contributed to the increased activity seen during starvation, with the major contribution from the distal promoter. Furthermore, Rv0494 translation initiated at a codon located 9 bp downstream of the annotated start codon. Rv0494 bound to its upstream sequence to auto-regulate its own expression; this binding was responsive to long-chain fatty acyl-CoA molecules. We further report Rv0494-mediated transcriptional regulation of the Rv2326c gene - a probable transmembrane ATP-binding transporter encoding gene.

Iron-regulated protein HupB of Mycobacterium tuberculosis positively regulates siderophore biosynthesis and is essential for growth in macrophages.

Mycobacterium tuberculosis expresses the 28-kDa protein HupB (Rv2986c) and the Fe(3+)-specific high-affinity siderophores mycobactin and carboxymycobactin upon iron limitation. The objective of this study was to understand the functional role of HupB in iron acquisition. A hupB mutant strain of M. tuberculosis, subjected to growth in low-iron medium (0.02 µg Fe ml(-1)), showed a marked reduction of both siderophores with low transcript levels of the mbt genes encoding the MB biosynthetic machinery. Complementation of the mutant strain with hupB restored siderophore production to levels comparable to that of the wild type. We demonstrated the binding of HupB to the mbtB promoter by both electrophoretic mobility shift assays and DNA footprinting. The latter revealed the HupB binding site to be a 10-bp AT-rich region. While negative regulation of the mbt machinery by IdeR is known, this is the first report of positive regulation of the mbt operon by HupB. Interestingly, the mutant strain failed to survive inside macrophages, suggesting that HupB plays an important role in vivo.

Identification and characterization of nuclear localization signals in the circumsporozoite protein of Plasmodium falciparum.

Secretory proteins of Plasmodium exhibit differential spatial and functional activity within the host cell nucleus. However, the nuclear localization signals (NLSs) for these proteins remain largely uncharacterized. In this study, we have identified and characterized two NLSs in the circumsporozoite protein of Plasmodium falciparum (Pf-CSP). Both NLSs in the Pf-CSP contain clusters of lysine and arginine residues essential for specific interactions with the conserved tryptophan and asparagine residues of importin-α, facilitating nuclear translocation of Pf-CSP. While the two NLSs of Pf-CSP function independently and are both crucial for nuclear localization, a single NLS of Pf-CSP leads to weak nuclear localization. These findings shed light on the mechanism of nuclear penetrability of secretory proteins of Plasmodium proteins.

Mutant MESD links cellular stress to type I collagen aggregation in osteogenesis imperfecta type XX.

Aberrant forms of endoplasmic reticulum (ER)-resident chaperones are implicated in loss of protein quality control in rare diseases. Here we report a novel mutation (p.Asp233Asn) in the ER retention signal of MESD by whole exome sequencing of an individual diagnosed with osteogenesis imperfecta (OI) type XX. While MESDD233N has similar stability and chaperone activity as wild-type MESD, its mislocalization to cytoplasm leads to imbalance of ER proteostasis, resulting in improper folding and aggregation of proteins, including LRP5 and type I collagen. Aggregated LRP5 loses its plasma membrane localization to disrupt the expression of WNT-responsive genes, such as BMP2, BMP4, in proband fibroblasts. We show that MESD is a direct chaperone of pro-α1(I) [COL1A1], and absence of MESDD233N in ER results in cytosolic type I collagen aggregates that remain mostly not secreted. While cytosolic type I collagen aggregates block the intercellular nanotubes, decreased extracellular type I collagen also results in loss of interaction of ITGB1 with type I collagen and weaker attachment of fibroblasts to matrix. Although proband fibroblasts show increased autophagy to degrade the aggregated type I collagen, an overall cellular stress overwhelms the proband fibroblasts. In summary, we present an essential chaperone function of MESD for LRP5 and type I collagen and demonstrating how the D233N mutation in MESD correlates with impaired WNT signaling and proteostasis in OI.

Deep intronic mutation in CRTAP results in unstable isoforms of the protein to induce type I collagen aggregation in a lethal type of osteogenesis imperfecta type VII.

Genetic mutations are involved in Mendelian disorders. Unbuffered intronic mutations in gene variants can generate aberrant splice sites in mutant transcripts, resulting in mutant isoforms of proteins with modulated expression, stability, and function in diseased cells. Here, we identify a deep intronic variant, c.794_1403A>G, in CRTAP by genome sequencing of a male fetus with osteogenesis imperfecta (OI) type VII. The mutation introduces cryptic splice sites in intron-3 of CRTAP, resulting in two mature mutant transcripts with cryptic exons. While transcript-1 translates to a truncated isoform (277 amino acids) with thirteen C-terminal non-wild-type amino acids, transcript-2 translates to a wild-type protein sequence, except that this isoform contains an in-frame fusion of non-wild-type twenty-five amino acids in a tetratricopeptide repeat sequence. Both mutant isoforms of CRTAP are unstable due to the presence of a unique 'GWxxI' degron, which finally leads to loss of proline hydroxylation and aggregation of type I collagen. Although type I collagen aggregates undergo autophagy, the overall proteotoxicity resulted in death of the proband cells by senescence. In summary, we present a genetic disease pathomechanism by linking a novel deep intronic mutation in CRTAP to unstable mutant isoforms of the protein in lethal OI type VII.

The parasite specific substitution matrices improve the annotation of apicomplexan proteins.

BACKGROUND: A number of apicomplexan genomes have been sequenced successfully in recent years and this would help in understanding the biology of apicomplexan parasites. The members of the phylum Apicomplexa are important protozoan parasites (Plasmodium, Toxoplasma and Cryptosporidium etc) that cause some of the deadly diseases in humans and animals. In our earlier studies, we have shown that the standard BLOSUM matrices are not suitable for compositionally biased apicomplexan proteins. So we developed a novel series (SMAT and PfFSmat60) of substitution matrices which performed better in comparison to standard BLOSUM matrices and developed ApicoAlign, a sequence search and alignment tool for apicomplexan proteins. In this study, we demonstrate the higher specificity of these matrices and make an attempt to improve the annotation of apicomplexan kinases and proteases. RESULTS: The ROC curves proved that SMAT80 performs best for apicomplexan proteins followed by compositionally adjusted BLOSUM62 (PSI-BLAST searches), BLOSUM90 and BLOSUM62 matrices in terms of detecting true positives. The poor E-values and/or bit scores given by SMAT80 matrix for the experimentally identified coccidia-specific oocyst wall proteins against hematozoan (non-coccidian) parasites further supported the higher specificity of the same. SMAT80 uniquely detected (missed by BLOSUM) orthologs for 1374 apicomplexan hypothetical proteins against SwissProt database and predicted 70 kinases and 17 proteases. Further analysis confirmed the conservation of functional residues of kinase domain in one of the SMAT80 detected kinases. Similarly, one of the SMAT80 detected proteases was predicted to be a rhomboid protease. CONCLUSIONS: The parasite specific substitution matrices have higher specificity for apicomplexan proteins and are helpful in detecting the orthologs missed by BLOSUM matrices and thereby improve the annotation of apicomplexan proteins which are hypothetical or with unknown function.

HYPK coordinates degradation of polyneddylated proteins by autophagy.

Selective degradation of protein aggregates by macroautophagy/autophagy is an essential homeostatic process of safeguarding cells from the effects of proteotoxicity. Among the ubiquitin-like proteins, NEDD8 conjugation to misfolded proteins is prominent in stress-induced protein aggregates, albeit the function of neddylation in autophagy is unclear. Here, we report that polyneddylation functions as a post-translational modification for autophagic degradation of proteotoxic-stress induced protein aggregates. We also show that HYPK functions as an autophagy receptor in the polyneddylation-dependent aggrephagy. The scaffolding function of HYPK is facilitated by its C-terminal ubiquitin-associated domain and N-terminal tyrosine-type LC3-interacting region which bind to NEDD8 and LC3 respectively. Both NEDD8 and HYPK are positive modulators of basal and proteotoxicity-induced autophagy, leading to protection of cells from protein aggregates, such as aggregates of mutant HTT exon 1. Thus, we propose an indispensable and additive role of neddylation and HYPK in clearance of protein aggregates by autophagy, resulting in cytoprotective effect during proteotoxic stress.Abbreviations: ATG5, autophagy related 5; ATG12, autophagy related 12; ATG14, autophagy related 14; BECN1, beclin 1; CBL, casitas B-lineage lymphoma; CBLB, Cbl proto-oncogene B; GABARAP, GABA type A receptor-associated protein; GABARAPL1, GABA type A receptor associated protein like 1; GABARAPL2, GABA type A receptor associated protein like 2; GFP, green fluorescent protein; HTT, huntingtin; HTT97Q exon 1, huntingtin 97-glutamine exon 1; HUWE1, HECT, UBA and WWE domain containing E3 ubiquitin protein ligase 1; HYPK, huntingtin interacting protein K; IgG, immunoglobulin G; IMR-32, Institute for Medical Research-32; KD, knockdown; Kd, dissociation constant; LAMP1, lysosomal associated membrane protein 1; LIR, LC3 interacting region; MAP1LC3/LC3, microtubule associated protein 1 light chain 3; MAP1LC3A/LC3A, microtubule associated protein 1 light chain 3 alpha; MAP1LC3B/LC3B, microtubule associated protein 1 light chain 3 beta; MARK1, microtubule affinity regulating kinase 1; MARK2, microtubule affinity regulating kinase 2; MARK3, microtubule affinity regulating kinase 3; MARK4, microtubule affinity regulating kinase 4; MCF7, Michigan Cancer Foundation-7; MTOR, mechanistic target of rapamycin kinase; NAE1, NEDD8 activating enzyme E1 subunit 1; NBR1, NBR1 autophagy cargo receptor; NEDD8, NEDD8 ubiquitin like modifier; Ni-NTA, nickel-nitrilotriacetic acid; NUB1, negative regulator of ubiquitin like proteins 1; PIK3C3, phosphatidylinositol 3-kinase catalytic subunit type 3; PolyQ, poly-glutamine; PSMD8, proteasome 26S subunit, non-ATPase 8; RAD23A, RAD23 homolog A, nucleotide excision repair protein; RAD23B, RAD23 homolog B, nucleotide excision repair protein; RFP, red fluorescent protein; RPS27A, ribosomal protein S27a; RSC1A1, regulator of solute carriers 1; SNCA, synuclein alpha; SIK1, salt inducible kinase 1; siRNA, small interfering ribonucleic acid; SOD1, superoxide dismutase 1; SPR, surface plasmon resonance; SQSTM1, sequestosome 1; SUMO1, small ubiquitin like modifier 1; TAX1BP1, Tax1 binding protein 1; TDRD3, tudor domain containing 3; TNRC6C, trinucleotide repeat containing adaptor 6C; TOLLIP, toll interacting protein; TUBA, tubulin alpha; TUBB, tubulin beta class I; UBA, ubiquitin-associated; UBA1, ubiquitin like modifier activating enzyme 1; UBA5, ubiquitin like modifier activating enzyme 5; UBAC1, UBA domain containing 1; UBAC2, UBA domain containing 2; UBAP1, ubiquitin associated protein 1; UBAP2, ubiquitin associated protein 2; UBASH3B, ubiquitin associated and SH3 domain containing B; UBD/FAT10, ubiquitin D; UBE2K, ubiquitin conjugating enzyme E2 K; UBLs, ubiquitin-like proteins; UBL7, ubiquitin like 7; UBQLN1, ubiquilin 1; UBQLN2, ubiquilin 2; UBQLN3, ubiquilin 3; UBQLN4, ubiquilin 4; UBXN1, UBX domain protein 1; ULK1, unc-51 like autophagy activating kinase 1; URM1, ubiquitin related modifier 1; USP5, ubiquitin specific peptidase 5; USP13, ubiquitin specific peptidase 13; VPS13D, vacuolar protein sorting 13 homolog D.

The E262K mutation in Lamin A links nuclear proteostasis imbalance to laminopathy-associated premature aging.

Deleterious, mostly de novo, mutations in the lamin A (LMNA) gene cause spatio-functional nuclear abnormalities that result in several laminopathy-associated progeroid conditions. In this study, exome sequencing in a sixteen-year-old male with manifestations of premature aging led to the identification of a mutation, c.784G>A, in LMNA, resulting in a missense protein variant, p.Glu262Lys (E262K), that aggregates in nucleoplasm. While bioinformatic analyses reveal the instability and pathogenicity of LMNAE262K , local unfolding of the mutation-harboring helical region drives the structural collapse of LMNAE262K into aggregates. The E262K mutation also disrupts SUMOylation of lysine residues by preventing UBE2I binding to LMNAE262K , thereby reducing LMNAE262K degradation, aggregated LMNAE262K sequesters nuclear chaperones, proteasomal proteins, and DNA repair proteins. Consequently, aggregates of LMNAE262K disrupt nuclear proteostasis and DNA repair response. Thus, we report a structure-function association of mutant LMNAE262K with toxicity, which is consistent with the concept that loss of nuclear proteostasis causes early aging in laminopathies.

ApicoAlign: an alignment and sequence search tool for apicomplexan proteins.

BACKGROUND: Over the recent years, a number of genomes have been successfully sequenced and this was followed by genome annotation projects to help understand the biological capabilities of newly sequenced genomes. To improve the annotation of Plasmodium falciparum proteins, we earlier developed parasite specific matrices (PfSSM) and demonstrated their (Smat80 and PfFSmat60) better performance over standard matrices (BLOSUM and PAM). Here we extend that study to nine apicomplexan species other than P. falciparum and develop a web application ApicoAlign for improving the annotation of apicomplexan proteins. RESULTS: The SMAT80 and PfFSmat60 matrices perform better for apicomplexan proteins compared to BLOSUM in detecting the orthologs and improving the alignment of these proteins with their potential orthologs respectively. Database searches against non-redundant (nr) database have shown that SMAT80 gives superior performance compared to BLOSUM series in terms of E-values, bit scores, percent identity, alignment length and mismatches for most of the apicomplexan proteins studied here. Using these matrices, we were able to find orthologs for rhomboid proteases of P. berghei, P. falciparum & P. vivax and large subunit of U2 snRNP auxiliary factor of Cryptosporidium parvum in Arabidopsis thaliana. We also show improved pairwise alignments of proteins from Apicomplexa viz. Cryptosporidium parvum and P. falciparum with their orthologs from other species using the PfFSmat60 matrix. CONCLUSIONS: The SMAT80 and PfFSmat60 substitution matrices perform better for apicomplexan proteins compared to BLOSUM series. Since they can be helpful in improving the annotation of apicomplexan genomes and their functional characterization, we have developed a web server ApicoAlign for finding orthologs and aligning apicomplexan proteins.

Cellular targets of mefloquine.

Mefloquine is a quinoline-based compound widely used as an antimalarial drug, particularly in chemoprophylaxis. Although decades of research have identified various aspects of mefloquine's anti-Plasmodium properties, toxic effects offset its robust use in humans. Mefloquine exerts harmful effects in several types of human cells by targeting many of the cellular lipids, proteins, and complexes, thereby blocking a number of downstream signaling cascades. In general, mefloquine modulates several cellular phenomena, such as alteration of membrane potential, induction of oxidative stress, imbalance of ion homeostasis, disruption of metabolism, failure of organelle function, etc., leading to cell cycle arrest and programmed cell death. This review aims to summarize the information on functional and mechanistic findings related to the cytotoxic effects of mefloquine.

Differential Stabilities of Mefloquine-Bound Human and Plasmodium falciparum Acyl-CoA-Binding Proteins.

Toxic effects of pharmacological drugs restrict their robust application against human diseases. Although used as a drug in the combinatorial therapy to treat malaria, the use of mefloquine is not highly recommended because of its adverse effects in humans. Mefloquine inhibits the binding of acyl-CoAs to acyl-CoA-binding proteins of Plasmodium falciparum (PfACBPs) and human (hACBP). In this study, we have used molecular dynamics simulation and other computational approaches to investigate the differences of stabilities of mefloquine-PfACBP749 and mefloquine-hACBP complexes. The stability of mefloquine in the binding cavity of PfACBP749 is less than its stability in the binding pocket of hACBP. Although the essential tyrosine residues (tyrosine-30 and tyrosine-33 of PfACBP749 and tyrosine-29 and tyrosine-32 of hACBP) mediate the initial binding of mefloquine to the proteins by π-stacking interactions, additional temporally longer interactions between mefloquine and aspartate-22 and methionine-25 of hACBP result in stronger binding of mefloquine to hACBP. The higher fluctuation of mefloquine-binding residues of PfACBP749 contributes to the instability of mefloquine in the binding cavity of the protein. On the contrary, in the mefloquine-bound state, the stability of hACBP protein is less than the stability of PfACBP749. The helix-to-coil transition of the N-terminal hydrophobic region of hACBP has a destabilizing effect upon the protein's structure. This causes the induction of aggregation properties in the hACBP in the mefloquine-bound state. Taken together, we describe the mechanistic features that affect the differential dynamic stabilities of mefloquine-bound PfACBP749 and hACBP proteins.

How Healthy Is State Mental Health System in Madhya Pradesh, India? An Assessment of Today to Plan for a Better Tomorrow.

BACKGROUND: Mental illness affects over one-third of the Indian population, and only a little is known about the exact situation of health systems in Madhya Pradesh, India. Therefore, the present research work provides an assessment of state mental health systems in Madhya Pradesh. METHODS: The present cross-sectional study was conducted as a part of National Mental Health Survey 2015-16 in 48 districts of Madhya Pradesh, to provide an overview of the status of mental health systems. Secondary data was also retrieved from the state office so as to present the situational analysis in a more comprehensive and inferential way. The proforma for the study was developed based on the experience gained from studies conducted earlier with World Health Organization's Assessment Instrument for Mental Health Systems (WHO-AIMS) and with WHO's Mental Health Atlas as the base for thematic analysis. RESULTS: Out of 51 districts, 13.7% of the districts of the state have been covered under District Mental Health Program (DMHP) in 2015-16. Around 11.8% of district/general hospitals were involved in providing mental health services. The availability of psychiatrist was 0.05 per Lakh population. Around 0.2% of the total health budget was allocated by the state for the last financial year for mental health. The overall average score of Madhya Pradesh in the assessment of qualitative indicators was 31 out of 100 in the year 2015-16. CONCLUSION: There is huge scope and an urgent need to increase mental healthcare facilities (with upgradation of existing one) along the availability of mental healthcare staff.

Genome bias influences amino acid choices: analysis of amino acid substitution and re-compilation of substitution matrices exclusive to an AT-biased genome.

The genomic era has seen a remarkable increase in the number of genomes being sequenced and annotated. Nonetheless, annotation remains a serious challenge for compositionally biased genomes. For the preliminary annotation, popular nucleotide and protein comparison methods such as BLAST are widely employed. These methods make use of matrices to score alignments such as the amino acid substitution matrices. Since a nucleotide bias leads to an overall bias in the amino acid composition of proteins, it is possible that a genome with nucleotide bias may have introduced atypical amino acid substitutions in its proteome. Consequently, standard matrices fail to perform well in sequence analysis of these genomes. To address this issue, we examined the amino acid substitution in the AT-rich genome of Plasmodium falciparum, chosen as a reference and reconstituted a substitution matrix in the genome's context. The matrix was used to generate protein sequence alignments for the parasite proteins that improved across the functional regions. We attribute this to the consistency that may have been achieved amid the target and background frequencies calculated exclusively in our study. This study has important implications on annotation of proteins that are of experimental interest but give poor sequence alignments with standard conventional matrices.

The metastable states of proteins.

The intriguing process of protein folding comprises discrete steps that stabilize the protein molecules in different conformations. The metastable state of protein is represented by specific conformational characteristics, which place the protein in a local free energy minimum state of the energy landscape. The native-to-metastable structural transitions are governed by transient or long-lived thermodynamic and kinetic fluctuations of the intrinsic interactions of the protein molecules. Depiction of the structural and functional properties of metastable proteins is not only required to understand the complexity of folding patterns but also to comprehend the mechanisms of anomalous aggregation of different proteins. In this article, we review the properties of metastable proteins in context of their stability and capability of undergoing atypical aggregation in physiological conditions.