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An important question in cell biology is how cytoskeletal proteins evolved and drove the development of novel structures and functions. Here we address the origin of SPIRE actin nucleators. Mammalian SPIREs work with RAB GTPases, formin (FMN)-subgroup actin assembly proteins and class-5 myosin (MYO5) motors to transport organelles along actin filaments towards the cell membrane. However, the origin and extent of functional conservation of SPIRE among species is unknown. Our sequence searches show that SPIRE exist throughout holozoans (animals and their closest single-celled relatives), but not other eukaryotes. SPIRE from unicellular holozoans (choanoflagellate), interacts with RAB, FMN and MYO5 proteins, nucleates actin filaments and complements mammalian SPIRE function in organelle transport. Meanwhile SPIRE and MYO5 proteins colocalise to organelles in Salpingoeca rosetta choanoflagellates. Based on these observations we propose that SPIRE originated in unicellular ancestors of animals providing an actin-myosin driven exocytic transport mechanism that may have contributed to the evolution of complex multicellular animals. Genome analysis and molecular cell biology studies indicate that SPIRE actin nucleators originated in unicellular ancestors of animals, providing an actin-myosin driven exocytic transport mechanism that may have contributed to animal evolution. [ABSTRACT FROM AUTHOR]

The derivatives of 1,2,4- triazole have attracted great attention among medicinal chemists due to their wide range of biological activity, good pharmacodynamic and pharmacokinetic profiles, and low toxicity, that necessitates the development of various synthesis methods and a comprehensive study of their reaction mechanisms. A detailed investigation of possible pathways for formation of new spiro-condensed [1,2,4]triazolo[1,5-c]quinazolines, that combine two structural domains with different biological properties, was performed by computational study at the SMD/B3lyp/6-31+G(d) theory level. The mechanism of interaction between [2-(3-hetaryl-1,2,4-triazol-5-yl)phenyl]amine and cyclohexanone in methanol involves three main processes: formation of carbinolamine by addition of an amine to double bond C=O, elimination of a water molecule, and intramolecular cyclization leading to formation of spiro compounds. Results show increase in reactivity of reactants during acid-catalyzed reaction compared to uncatalyzed one. The nature of the heterocyclic substituent on the triazole ring has little effect on the reaction energy, while the mechanism is unchanged. [ABSTRACT FROM AUTHOR]

Mechanisms for formation and hydrolysis of biologically active 2-substituted [1,2,4]triazolo[1,5-c]quinazolines were modeled at the SMD/B3LYP/6–31 + G(d) theory level. Obtained results revealed that internal heterocyclization of hetarylcarboxylic acid (3H-quinazolin-4-ylidene)-hydrazides involves a proton transfer from nitrogen atom of quinazoline system to oxygen atom of carbonyl group, cyclization with formation of [1,2,4]triazolo[4,3-c]quinazoline system, elimination of molecule of water. Dimroth rearrangement using the ANRORC mechanism of 2-hetaryl[1,2,4]triazolo[4,3-c]quinazolines leads to 2-hetaryl[1,2,4]triazolo[1,5-c]quinazolines. The rearrangement is catalyzed by molecule of water and consists of an addition of molecule of water to quinazoline cycle that facilitates its sequential opening, rotation of 1,2,4-triazole, cycle closure and elimination of molecule of water. Acid-catalyzed hydrolysis of 2-hetaryl[1,2,4]triazolo[1,5-c]quinazolines involves protonation of nitrogen atom of quinazoline cycle, an addition of molecule of water to protonated [1,2,4]triazolo[1,5-c]quinazoline system, opening of quinazoline cycle, an addition of second molecule of water, elimination of formic acid, and deprotonation. Substituents influence insignificantly on activation barriers for formation and hydrolysis of 2-hetaryl[1,2,4]triazolo[1,5-c]quinazolines. [ABSTRACT FROM AUTHOR]

1,2,4-Triazole is a popular scaffold in drug design. According to chemical nature, the triazole ring tends to prototropic tautomerism. Tautomeric phenomena are important for studying the chemical reactivity and interaction of drugs based on triazole with biomolecules in the human body. Theoretical modeling was used to assign structures of newly synthesized 2-(3-hetaryl-1,2,4-triazol-5-yl)anilines. The procedure included quantum-chemical SMD/M06-2X/6–311++G(d,p) calculation of the relative stability for possible tautomers, simulation of UV/vis spectra for the most stable forms, and comparison of the resulting curves with the experimental spectral data taking into account the Boltzmann weighting. The influence of the substituents in triazole ring on tautomeric equilibrium was elucidated. NBO charge distribution, dipole moment, molecular electrostatic potential, and HOMO/LUMO gap for the most stable tautomers were analyzed. [ABSTRACT FROM AUTHOR]

The actin and microtubule cytoskeletons play important roles in Golgi structure and function, but how they are connected remain poorly known. In this study, we investigated whether RAB6 GTPase, a Golgi-associated RAB involved in the regulation of several transport steps at the Golgi level, and two of its effectors, Myosin IIA and KIF20A participate in the coupling between actin and microtubule cytoskeleton. We have previously shown that RAB6–Myosin IIA interaction is critical for the fission of RAB6-positive transport carriers from Golgi/TGN membranes. Here we show that KIF20A is also involved in the fission process and serves to anchor RAB6 on Golgi/TGN membranes near microtubule nucleating sites. We provide evidence that the fission events occur at a limited number of hotspots sites. Our results suggest that coupling between actin and microtubule cytoskeletons driven by Myosin II and KIF20A ensures the spatial coordination between RAB6-positive vesicles fission from Golgi/ TGN membranes and their exit along microtubules. [ABSTRACT FROM AUTHOR]

Familial hyperkalemic hypertension (FHHt) is a Mendelian form of arterial hypertension that is partially explained by mutations in WNK1 and WNK4 that lead to increased activity of the Na+-Cl? cotransporter (NCC) in the distal nephron. Using combined linkage analysis and whole-exome sequencing in two families, we identified KLHL3 as a third gene responsible for FHHt. Direct sequencing of 43 other affected individuals revealed 11 additional missense mutations that were associated with heterogeneous phenotypes and diverse modes of inheritance. Polymorphisms at KLHL3 were not associated with blood pressure. The KLHL3 protein belongs to the BTB-BACK-kelch family of actin-binding proteins that recruit substrates for Cullin3-based ubiquitin ligase complexes. KLHL3 is coexpressed with NCC and downregulates NCC expression at the cell surface. Our study establishes a role for KLHL3 as a new member of the complex signaling pathway regulating ion homeostasis in the distal nephron and indirectly blood pressure. [ABSTRACT FROM AUTHOR]

JIP3 and JIP4 (JNK-interacting proteins 3 and 4) are adaptors for cargo recruitment by dynein/dynactin and kinesin1 motors. Both are dimers that are stabilised by two sections of leucine zipper coiled coils. The N-terminal Leucine Zipper I (LZI) belongs to a section that binds dynein-DLIC and kinesin1-KHC, whilst the medial Leucine Zipper II (LZII) binds dynactin-p150glued and kinesin1-KLC. Structural data is available for the LZII, but the LZI section is still uncharacterized. Here we characterize the N-terminal part of JIP3/4 which consists of an RH1 (RILP homology 1) domain followed by the LZI coiled coil using bioinformatical, biophysical and structural approaches. The RH1-LZI tandem of JIP3 associates as a high affinity homodimer exhibiting elongated alpha-helical fold. 3D homology modelling of the RH1-LZI tandem reveals that the kinesin1-KHC binding site mainly overlaps with the RH1 domain. A sequence comparison search indicates that only one other protein family has RH1 domains similar to those of JIP3/4, the RILP (Rab-interacting lysosomal protein) family which consists of adaptor proteins linking Rab GTPases to cytoskeletal motors. RILPL2 is recruited through its RH1 domain by the myosin 5a motor. Here, we showed that the RH1 domain of JIP3 also interacts with myosin 5 A in vitro, highlighting JIP3/4 as possible myosin 5a adaptors. Finally, we propose that JIP3/4 and RILP family members define a unique RH1/RH2-architecture adaptor superfamily linking cytoskeletal motors and Rab GTPases. [ABSTRACT FROM AUTHOR]