Identification of the potential dual inhibitor of protein tyrosine phosphatase sigma and leukocyte common antigen-related phosphatase by virtual screen, molecular dynamic simulations and post-analysis

A highly sensitive and continuous protein tyrosine phosphatase (PTPase) assay using 3,6-fluorescein diphosphate (FDP) is described. Leukocyte phosphatase CD45 (leukocyte common antigen), protein tyrosine phosphatase-lB, and leukocyte common antigen-related protein LAR preferentially hydrolyze FDP to fluorescein monophosphate (FMP) with Vmax and Km values comparable with those of phosphotyrosine peptide substrates. Further hydrolysis of FMP to fluorescein was less efficient because of increased Km values compared with those of FDP. FMP absorbs strongly at 445 nm and fluoresces intensely near 515 nm, both of which are insensitive to pH perturbations above pH 6. Its high catalytic efficiency, coupled with the highly sensitive dual detection in the visible wavelength region and wider pH operating range, make FDP the substrate of choice for PTPase inhibitor screening in HTS format and assay miniaturization.

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PTP-3(LAR PTPR) promotes intramolecular folding of SYD-2(liprin-α) to inactivate UNC-104(KIF1A) in neurons

10.1101/723288 ◽

2019 ◽

Author(s):

Muniesh Muthaiyan Shanmugam ◽

Syed Nooruzuha Barmaver ◽

Hsin-Yi Huang ◽

Oliver Ingvar Wagner

Keyword(s):

Synaptic Vesicle ◽

Protein Tyrosine Phosphatase ◽

Protein Transport ◽

Tyrosine Phosphatase ◽

Common Antigen ◽

Leukocyte Common Antigen ◽

C Elegans ◽

Open Conformation ◽

Transport Vesicles ◽

Conformation State

AbstractThis study aims to demonstrate how PTP-3 regulates SYD-2 to control UNC-104-mediated axonal transport. UNC-104 is the C. elegans homolog of kinesin-3 KIF-1A known for its fast shuttling of STVs (synaptic vesicle protein transport vesicles) in axons. SYD-2 is the homolog of liprin-α in C. elegans known to directly regulate UNC-104 as well as being a substrate of LAR PTPR (leukocyte common antigen-related (LAR) protein tyrosine phosphatase (PTP) transmembrane receptor) with PTP-3 as the closest homolog in C. elegans. CoIP assays revealed increased interaction between UNC-104 and SYD-2 in lysates from ptp-3 knockout worms. Intramolecular FRET analysis revealed that SYD-2 predominantly exists in an open conformation state in ptp-3 mutants. These assays also revealed that non-phosphorylatable SYD-2 (Y741F) exists predominately in folded conformations while phosphomimicking SYD-2 (Y741E) exists predominantly in open conformations. In ptp-3 mutants, SNB-1 cargo accumulates in soma while at the same time UNC-104 motors increasingly cluster along initial segments of axons. Interestingly, the unc-104 gene is downregulated in ptp-3 mutants that might explain the vesicle retention phenotype. More strikingly, the few visibly moving motors and STVs were overly active in neurons of these mutants. We propose a model in which the lack of PTP-3 promotes increased open conformations of SYD-2 that in turn facilitates UNC-104/SYD-2 interactions boosting motor and STVs moving speeds.

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