Background: Serine proteases are one of the largest groups of proteolytic enzymes found across all kingdoms of life and are associated with several essential physiological pathways. The availability of Arabidopsis thaliana and rice (Oryza sativa) genome sequences has permitted the identification and comparison of the repertoire of serine protease-like proteins in the two plant species.

UNC-104/KIF1A is a Kinesin-3 motor that transports synaptic vesicles from the cell body towards the synapse by binding to PI(4,5)P2 through its PH domain. The fate of the motor upon reaching the synapse is not known. We found that wild-type UNC-104 is degraded at synaptic regions through the ubiquitin pathway and is not retrogradely transported back to the cell body. As a possible means to regulate the motor, we tested the effect of cargo binding on UNC-104 levels. The unc-104(e1265) allele carries a point mutation (D1497N) in the PI(4,5)P2 binding pocket of the PH domain, resulting in greatly reduced preferential binding to PI(4,5)P2 in vitro and presence of very few motors on pre-synaptic vesicles in vivo. unc-104(e1265) animals have poor locomotion irrespective of in vivo PI(4,5)P2 levels due to reduced anterograde transport. Moreover, they show highly reduced levels of UNC-104 in vivo. To confirm that loss of cargo binding specificity reduces motor levels, we isolated two intragenic suppressors with compensatory mutations within the PH domain. These show partial restoration of in vitro preferential PI(4,5)P2 binding and presence of more motors on pre-synaptic vesicles in vivo. These animals show improved locomotion dependent on in vivo PI(4,5)P2 levels, increased anterograde transport, and partial restoration of UNC-104 protein levels in vivo. For further proof, we mutated a conserved residue in one suppressor background. The PH domain in this triple mutant lacked in vitro PI(4,5)P2 binding specificity, and the animals again showed locomotory defects and reduced motor levels. All allelic variants show increased UNC-104 levels upon blocking the ubiquitin pathway. These data show that inability to bind cargo can target motors for degradation. In view of the observed degradation of the motor in synaptic regions, this further suggests that UNC-104 may get degraded at synapses upon release of cargo.

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Kinesin-2 is an anterograde motor involved in intraflagellar transport and certain other intracellular transport processes. It consists of two different motor subunits and an accessory protein KAP (kinesin accessory protein). The motor subunits were shown to bind each other through the coiled-coil stalk domains, while KAP was proposed to bind the tail domains of the motor subunits. Although several genetic studies established that KAP plays an important role in kinesin-2 functions, its exact role remains unclear. Here, we report the results of a systematic analysis of the KAP binding sites by using recombinant Drosophila kinesin-2 subunits as well as the endogenous proteins. These show that at least one of the coiled-coil stalks is sufficient to bind the N-terminal region of DmKAP. The soluble complex involving the recombinant kinesin-2 fragments is reconstituted in vitro at high salt concentrations, suggesting that the interaction is primarily nonionic. Furthermore, independent distant homology modeling indicated that DmKAP may bind along the coiled-coil stalks through a combination of predominantly hydrophobic interactions and hydrogen bonds. These observations led us to propose that KAP would stabilize the motor subunit heterodimer and help assemble a greater kinesin-2 complex in vivo.

Background: Serine proteases are one of the most abundant groups of proteolytic enzymes found in all the kingdoms of life. While studies have established significant roles for many prokaryotic serine proteases in several physiological processes, such as those associated with metabolism, cell signalling, defense response and development, functional associations for a large number of prokaryotic serine proteases are relatively unknown. Current analysis is aimed at understanding the distribution and probable biological functions of the select serine proteases encoded in representative prokaryotic organisms.

Tyrosine phosphatases play an important role in cellular signalling and networking that is antagonistic to the kinases. Near completion of the human genome- sequencing project permits us to review the distribution of this family and study its involvement in different pathways. Ninety-six homologues of the classical and dual- specific tyrosine phosphatases (DuSPs) were identified in the human genome using sensitive sequence search techniques. Uncommon domain architectures were encountered, including an example where a kinase and a phosphatase domain are found to co-exist in a single polypeptide. The evolutionary rate is higher for the DuSP compared with the classical tyrosine phosphatases. Orthologues of the 96 putative human tyrosine phosphatases were identified in four model organisms to study the conservation of the family members. Three nuclear localized tyrosine phosphatases retain an orthologous relationship with all model systems considered but still differ in their domain architectures. The diversity in the multi-domain members of the superfamily occurs mainly through domain recruitment, especially in receptor tyrosine phosphatases. The curation of human tyrosine phosphatases provides a convenient framework for characterizing and analysing the functional and structural properties of this diverse family of proteins.

The serotonin receptors, also known as 5-hydroxytryptamine (5-HT) receptors, are a group of G protein-coupled receptors (GPCRs) and ligand-gated ion channels found in the central and peripheral nervous systems. GPCRs have a characteristic feature of activating different signalling pathways upon ligand binding and these ligands display several efficacy levels to differentially activate the receptor. GPCRs are primary drug targets due to their central role in several signal transduction pathways. Drug design for GPCRs is also most challenging due to their inherent promiscuity in ligand recognition, which gives rise to several side effects of existing drugs. Here, we have performed the ligand interaction study using the two prominent states of GPCR, namely the active and inactive state of the 5-HT2A receptor. Active state of 5-HT2A receptor model enhances the understanding of conformational difference which influences the ligand-binding site. A 5-HT2A receptor active state model was constructed by homology modelling using active state β2-adrenergic receptor (β2-AR). In addition, virtual screening and docking studies with both active and inactive state models reveal potential small molecule hits which could be considered as agonist-like and antagonist-like molecules. The results from the all-atom molecular dynamics simulations further confirmed that agonists and antagonists interact in different modes with the receptor.

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