Sajid Rashid

Endosomal Adaptor Proteins APPL1 and APPL2 Are Novel Activators of β-Catenin/TCF-mediated Transcription

Canonical Wnt signaling regulates many aspects of cellular physiology and tissue homeostasis during development and in adult organisms. In molecular terms, stimulation by Wnt ligands leads to the stabilization of β-catenin, its translocation to the nucleus, and stimulation of TCF (T-cell factor)-dependent transcription of target genes. This process is controlled at various stages by a number of regulatory proteins, including transcriptional activators and repressors. Here we demonstrate that the endosomal proteins APPL1 and APPL2 are novel activators of β-catenin/TCF-mediated transcription. APPL proteins are multifunctional adaptors and effectors of the small GTPase Rab5, which localize to a subpopulation of early endosomes but are also capable of nucleocytoplasmic shuttling. Overexpression of APPL1 or APPL2 protein stimulates the activity of β-catenin/TCF-dependent reporter construct, whereas silencing of APPL1 reduces it. Both APPL proteins interact directly with Reptin, a transcriptional repressor binding to β-catenin and HDAC1 (histone deacetylase 1), and this interaction was mapped to the pleckstrin homology domain of APPL1. Moreover, APPL proteins are present in an endogenous complex containing Reptin, β-catenin, HDAC1, and HDAC2. Overexpression of either APPL protein relieves Reptin-dependent transcriptional repression and correlates with the reduced amounts of HDACs and β-catenin associated with Reptin as well as with the lower levels of Reptin and HDAC1 on the promoters of β-catenin target genes. We propose that APPL proteins exert their stimulatory effects on β-catenin/TCF-dependent transcription by decreasing the activity of a Reptin-containing repressive complex.

Disruption of the murine dynein light chain gene Tcte3-3 results in asthenozoospermia

To elucidate the role of the mouse gene Tcte3 (Tctex2), which encodes a putative light chain of the outer dynein arm of cilia and sperm flagella, we have inactivated this gene in mice using targeted disruption. Breeding of heterozygous males and females resulted in normal litter size; however, we were not able to detect homozygous Tcte3-deficent mice using standard genotype techniques. In fact, our results indicate the presence of at least three highly similar copies of the Tcte3 gene (Tcte3-1, Tcte3-2, and Tcte3-3) in the murine genome. Therefore, quantitative real-time PCR was established to differentiate between mice having one or two targeted Tcte3-3 alleles. By this approach, Tcte3-3(-/-) animals were identified, which were viable and revealed no obvious malformation. Interestingly, some homozygous Tcte3-3-deficient male mice bred with wild-type female produced no offspring while other Tcte3-3-deficient males revealed decreased sperm motility but were fertile. In infertile Tcte3-3(-/-) males, spermatogenesis was affected and sperm motility was reduced, too, resulting in decreased ability of Tcte3-3-deficient spermatozoa to move from the uterus into the oviduct. Impaired flagellar motility is not correlated with any gross defects in the axonemal structure, since outer dynein arms are detectable in sperm of Tcte3-3(-/-) males. However, in infertile males, deficient Tcte3-3 function is correlated with increased apoptosis during male germ cell development, resulting in a reduction of sperm number. Moreover, multiple malformations in developing haploid germ cells are present. Our results support a role of Tcte3-3 in generation of sperm motility as well as in male germ cell differentiation.

In Silico Analysis of Missense Mutations in LPAR6 Reveals Abnormal Phospholipid Signaling Pathway Leading to Hypotrichosis

Autosomal recessive hypotrichosis is a rare genetic irreversible hair loss disorder characterized by sparse scalp hair, sparse to absent eyebrows and eyelashes, and sparse axillary and body hair. The study, presented here, established genetic linkage in four families showing similar phenotypes to lysophosphatidic acid receptor 6 (LPAR6) gene on chromosome 13q14.11-q21.32. Subsequently, sequence analysis of the gene revealed two previously reported missense mutations including p.D63V in affected members of one and p.I188F in three other families. Molecular modeling and docking analysis was performed to investigate binding of a ligand oleoyl-L-alpha-lysophosphatidic acid (LPA) to modeled protein structures of normal and mutated (D63V, G146R, I188F, N248Y, S3T, L277P) LPAR6 receptors. The mutant receptors showed a complete shift in orientation of LPA at the binding site. In addition, hydropathy analysis revealed a significant change in the membrane spanning topology of LPAR6 helical segments. The present study further substantiated involvement of LPAR6-LPA signaling in the pathogenesis of hypotrichosis/woolly hair and provided additional insight into the molecular mechanism of hair development.

Identification of potential Plk1 targets in a cell-cycle specific proteome through structural dynamics of kinase and Polo box-mediated interactions.

Polo like kinase 1 (Plk1) is a key player in orchestrating the wide variety of cell-cycle events ranging from centrosome maturation, mitotic entry, checkpoint recovery, transcriptional control, spindle assembly, mitotic progression, cytokinesis and DNA damage checkpoints recovery. Due to its versatile nature, Plk1 is considered an imperative regulator to tightly control the diverse aspects of the cell cycle network. Interactions among Plk1 polo box domain (PBD) and its putative binding proteins are crucial for the activation of Plk1 kinase domain (KD). To date, only a few substrate candidates have been characterized through the inclusion of both polo box and kinase domain-mediated interactions. Thus it became compelling to explore precise and specific Plk1 substrates through reassessment and extension of the structure-function paradigm. To narrow this apparently wide gap in knowledge, here we employed a thorough sequence search of Plk1 phosphorylation signature containing proteins and explored their structure-based features like conceptual PBD-binding capabilities and subsequent recruitment of KD directed phosphorylation to dissect novel targets of Plk1. Collectively, we identified 4,521 phosphodependent proteins sharing similarity to the consensus phosphorylation and PBD recognition motifs. Subsequent application of filters including similarity index, Gene Ontology enrichment and protein localization resulted in stringent pre-filtering of irrelevant candidates and isolated unique targets with well-defined roles in cell-cycle machinery and carcinogenesis. These candidates were further refined structurally using molecular docking and dynamic simulation assays. Overall, our screening approach enables the identification of several undefined cell-cycle associated functions of Plk1 by uncovering novel phosphorylation targets.

Genetic analysis of four Pakistani families with achromatopsia and a novel S4 motif mutation of CNGA3.

BackgroundTo identify the causative variants of achromatopsia (ACHM) in four Pakistani families presenting autosomal recessive ACHM.MethodsFour families (50, 55, 70 and 74) exhibiting features of achromatopsia were subjected to homozygosity mapping with STS markers flanking known ACHM loci. Mutation screening was done for two of the families linked to CNGA3 and CNGB3 by direct sequencing of the coding regions and exon-intron boundaries of genes to find the pathogenic variant.ResultsHomozygosity mapping showed co-segregation of CNGA3 in family 50 and CNGB3 in family 74. Sequencing of coding regions of CNGA3 in family 50 revealed a novel missense mutation, c.827A>G, in exon 7, which results in p.N276S substitution. N276S is located in the S4 motif of the CNGA3 protein and is conserved in all channel proteins. Bioinformatics analysis showed that the N276S substitution altered the channel conformation by shifting the helix. No pathogenic variation was identified in any affected members of family 74 in the coding sequence of CNGB3. The other two families, 55 and 70, were not linked to any known ACHM loci, indicating further heterogeneity of the ACHM phenotype.ConclusionsWe describe a novel S4 motif mutation of CNGA3 in a Pakistani family.

Novel homozygous mutations in the WNT10B gene underlying autosomal recessive split hand/foot malformation in three consanguineous families.

Split-hand/split-foot malformation (SHFM), representing variable degree of median clefts of hands and feet, is a genetically heterogeneous group of limb malformations with seven loci mapped on different human chromosomes. However, only 3 genes (TP63, WNT10B, DLX5) for the seven loci have been identified. The study, presented here, described three consanguineous Pakistani families segregating SHFM in autosomal recessive manner. Linkage in the families was searched by genotyping microsatellite markers and mutation screening of candidate gene was performed by Sanger DNA sequencing. Clinical features of affected members of these families exhibited SHFM phenotype with involvement of hands and feet. Genotyping using microsatellite markers mapped the families to WNT10B gene at SHFM6 on chromosome 12q13.11-q13. Subsequently, sequence analysis of WNT10B gene revealed a novel 4-bp deletion mutation (c.1165_1168delAAGT) in one family and 7-bp duplication (c.300_306dupAGGGCGG) in two other families. Structure-based analysis showed a significant conformational shift in the active binding site of mutated WNT10B (p.Lys388Glufs*36), influencing binding with Fzd8. The mutations identified in the WNT10B gene extend the body of evidence implicating it in the pathogenesis of SHFM.

Exploration of Novel Inhibitors for Class I Histone Deacetylase Isoforms by QSAR Modeling and Molecular Dynamics Simulation Assays.

Histone deacetylases (HDAC) are metal-dependent enzymes and considered as important targets for cell functioning. Particularly, higher expression of class I HDACs is common in the onset of multiple malignancies which results in deregulation of many target genes involved in cell growth, differentiation and survival. Although substantial attempts have been made to control the irregular functioning of HDACs by employing various inhibitors with high sensitivity towards transformed cells, limited success has been achieved in epigenetic cancer therapy. Here in this study, we used ligand-based pharmacophore and 2-dimensional quantitative structure activity relationship (QSAR) modeling approaches for targeting class I HDAC isoforms. Pharmacophore models were generated by taking into account the known IC50 values and experimental energy scores with extensive validations. The QSAR model having an external R2 value of 0.93 was employed for virtual screening of compound libraries. 10 potential lead compounds (C1-C10) were short-listed having strong binding affinities for HDACs, out of which 2 compounds (C8 and C9) were able to interact with all members of class I HDACs. The potential binding modes of HDAC2 and HDAC8 to C8 were explored through molecular dynamics simulations. Overall, bioactivity and ligand efficiency (binding energy/non-hydrogen atoms) profiles suggested that proposed hits may be more effective inhibitors for cancer therapy.

Molecular docking studies of flavonoids for their inhibition pattern against β-catenin and pharmacophore model generation from experimentally known flavonoids to fabricate more potent inhibitors for Wnt signaling pathway.

Background: Canonical Wnt signaling plays a key role in tumor cell proliferation, which correlates with the accumulation of β-catenin in cell due to inactivation of glycogen synthetase kinase-3 β. However, uncontrolled expression of β-catenin leads to fibromatosis, sarcoma and mesenchymal tumor formation. Recently, a number of polyphenolic compounds of naturally occurring flavonoid family have been screened for the inhibition of Wnt signaling. Objective: Elucidation of the binding mode of inhibitors to β-catenin, reporting more potent inhibitors for the disease-causing protein and designing a pharmacophore model based on naturally occurring compounds, flavonoids. Materials and Methods: In this study, a comparative molecular docking analysis was performed to elucidate the binding mode of experimentally reported and unknown inhibitors. Based on the knowledge of geometry, binding affinity and drug score, we described a subset of novel inhibitors. Results: The binding energy of known inhibitors (isorhamnetin, fisetin, genistein and silibinin) was observed in a range of −5.68 to −4.98 kcal/mol, while novel inhibitors (catechin, luteolin, coumestrol and β-naphthoflavone) exhibited −6.50 to −5.22 kcal/mol. We observed good placement and strong interactions of selected compounds inside the binding pocket of β-catenin. Moreover, flavonoid family members and T cell factors 4 (TCF4) compete for β-catenin binding by sharing common binding residues. Conclusion: This study will largely help in understanding the molecular basis of β-catenin/TCF4 inhibition through flavonoids by exploring their structural details. Finally, the novel inhibitors proposed in this study need further attention to uncover cancer treatment and with the generated pharmacophore model, more and potent β-catenin inhibitors can be easily screened.

Pharmacoinformatics elucidation of potential drug targets against migraine to target ion channel protein KCNK18.

Migraine, a complex debilitating neurological disorder is strongly associated with potassium channel subfamily K member 18 (KCNK18). Research has emphasized that high levels of KCNK18 may be responsible for improper functioning of neurotransmitters, resulting in neurological disorders like migraine. In the present study, a hybrid approach of molecular docking and virtual screening were followed by pharmacophore identification and structure modeling. Screening was performed using a two-dimensional similarity search against recommended migraine drugs, keeping in view the physicochemical properties of drugs. LigandScout tool was used for exploring pharmacophore properties and designing novel molecules. Here, we report the screening of four novel compounds that have showed maximum binding affinity against KCNK18, obtained through the ZINC database, and Drug and Drug-Like libraries. Docking studies revealed that Asp-46, Ile-324, Ile-44, Gly-118, Leu-338, Val-113, and Phe-41 are critical residues for receptor–ligand interaction. A virtual screening approach coupled with docking energies and druglikeness rules illustrated that ergotamine and PB-414901692 are potential inhibitor compounds for targeting KCNK18. We propose that selected compounds may be more potent than the previously listed drug analogs based on the binding energy values. Further analysis of these inhibitors through site-directed mutagenesis could be helpful for exploring the details of ligand-binding pockets. Overall, the findings of this study may be helpful for designing novel therapeutic targets to cure migraine.

Comparative Molecular Docking Analysis of Cytoplasmic Dynein Light Chain DYNLL1 with Pilin to Explore the Molecular Mechanism of Pathogenesis Caused by Pseudomonas aeruginosa PAO

Cytoplasmic dynein light chain 1 (DYNLL1) is a component of large protein complex, which is implicated in cargo transport processes, and is known to interact with many cellular and viral proteins through its short consensus motif (K/R)XTQT. Still, it remains to be explored that bacterial proteins also exhibit similar recognition sequences to make them vulnerable to host defense mechanism. We employed multiple docking protocols including AUTODOCK, PatchDock, ZDOCK, DOCK/PIERR and CLUSPRO to explore the DYNLL1 and Pilin interaction followed by molecular dynamics simulation assays. Subsequent structural comparison of the predicted binding site for DYNLL1-Pilin complex against the experimentally verified DYNLL1 binding partners was performed to cross check the residual contributions and to determine the binding mode. On the basis of in silico analysis, here we describe a novel interaction of DYNLL1 and receptor binding domain of Pilin (the main protein constituent of bacterial type IV Pili) of gram negative bacteria Pseudomonas aeruginosa (PAO), which is the third most common nosocomial pathogen associated with the life-threatening infections. Evidently, our results underscore that Pilin specific motif (KSTQD) exhibits a close structural similarity to that of Vaccinia virus polymerase, P protein Rabies and P protein Mokola viruses. We speculate that binding of DYNLL1 to Pilin may trigger an uncontrolled inflammatory response of the host immune system during P. aeruginosa chronic infections thereby opening a new pioneering area to investigate the role of DYNLL1 in gram negative bacterial infections other than viral infections. Moreover, by manifesting a strict correspondence between sequence and function, our study anticipates a novel drug target site to control the complications caused by P. aeruginosa infections.