Expressional Localization and Functionally Identifying an RNA Editing Enzyme BmADARa of the Silkworm Bombyx mori

The most common type of RNA editing in metazoans is the deamination of adenosine into inosine (A-to-I) catalyzed by the adenosine deaminase acting on the RNA (ADAR) family of proteins. The deletion or dysfunction of ADAR enzymes in higher eukaryotes can affect the efficiency of substrate editing and cause neurological disorders. However, the information concerning A-to-I RNA editing and ADAR members in the silkworm, Bombyx mori (BmADAR), is limited. In this study, a first molecular comprehensive cloning and sequence analysis of BmADAR transcripts was presented. A complete open reading frame (ORF) (BmADARa) was obtained using RT-PCR and RACE and its expression pattern, subcellular localization and A-to-I RNA-editing function on the silkworm synaptotagmin I (BmSyt I) were investigated. Subcellular localization analysis observed that BmADARa was mainly localized in the nucleus. To further study the A-to-I RNA-editing function of BmADARa, BmSyt I-pIZ-EGFP was constructed and co-transfected with BmADARa-pIZ-EGFP into BmN cells. The result demonstrates that BmADARa can functionally edit the specific site of BmSyt I. Taken together, this study not only provides insight into the function of the first ADAR enzyme in B. mori, but also lays foundations for further exploration of the functional domain of BmADARa and its editing substrates and target sites.

Proteins of the vesicular cycle as a marker of neuroplasticity of dopaminergic neurons in the substantia nigra of the brain

Nigrostriatal dopaminergic neurons (DNs) involved in the regulation of motor function, are characterized by high plasticity. Indeed, at the death of up to 50% of DNs in Parkinsons disease, the survived neurons provide normal regulation. This study was aimed to determine whether proteins of the vesicular cycle, syntaxin Ia (Syn Ia), synaptotagmin I (Syt I), Rab5a and complexins I and II (Cmpx I and II), are involved in the mechanisms of neuroplasticity in the substantia nigra, which mainly contains cell bodies and processes of the DNs. In neurotoxic models of Parkinsons disease in mice, it was shown that at the degeneration of up to 50% of DNs, the content of Syt I, Syn Ia, Cmpх I and II involved in vesicle exocytosis does not change in the substantia nigra as whole, while this is compensatory increased in individual survived DNs. Thus, the data obtained in this study suggest that the impairment of motor behavior that occurs at the death of half of nigrostriatal DNs is not caused by the impairment of the production of vesicular cycle proteins in surviving DNs.

High affinity interactions of Pb2+ with Synaptotagmin I

ABSTRACTLead (Pb) is a potent neurotoxin that disrupts synaptic neurotransmission. We report that Synaptotagmin I (SytI), a key regulator of Ca2+-evoked neurotransmitter release, has two high-affinity Pb2+ binding sites that belong to its cytosolic C2A and C2B domains. The crystal structures of Pb2+-complexed C2 domains revealed that protein-bound Pb2+ ions have holodirected coordination geometries and all-oxygen coordination spheres. The on-rate constants of Pb2+ binding to the C2 domains of SytI are comparable to those of Ca2+ and are diffusion-limited. In contrast, the off-rate constants are at least two orders of magnitude smaller, indicating that Pb2+ can serve as both thermodynamic and kinetic trap for the C2 domains. We demonstrate, using NMR spectroscopy, that population of these sites by Pb2+ ions inhibits further Ca2+ binding despite the existing coordination vacancies. Our work offers a unique insight into the bioinorganic chemistry of Pb(II) and suggests a mechanism by which low concentrations of Pb2+ ions can interfere with the Ca2+-dependent function of SytI in the cell.

High affinity interactions of Pb2+ with synaptotagmin I

Pb2+ binds C2 domains with high affinity, desensitizes them to Ca2+, and supports their membrane interactions.

Pull-down combined with proteomic strategy reveals functional diversity of synaptotagmin I

Synaptotagmin I (Syt I) is most abundant in the brain and is involved in multiple cellular processes. Its two C2 domains, C2A and C2B, are the main functional regions. Our present study employed a pull-down combined with proteomic strategy to identify the C2 domain-interacting proteins to comprehensively understand the biological roles of the C2 domains and thus the functional diversity of Syt I. A total of 135 non-redundant proteins interacting with the C2 domains of Syt I were identified. Out of them, 32 and 64 proteins only bound to C2A or C2B domains, respectively, and 39 proteins bound to both of them. Compared with C2A, C2B could bind to many more proteins particularly those involved in synaptic transmission and metabolic regulation. Functional analysis indicated that Syt I may exert impacts by interacting with other proteins on multiple cellular processes, including vesicular membrane trafficking, synaptic transmission, metabolic regulation, catalysis, transmembrane transport and structure formation, etc. These results demonstrate that the functional diversity of Syt I is higher than previously expected, that its two domains may mediate the same and different cellular processes cooperatively or independently, and that C2B domain may play even more important roles than C2A in the functioning of Syt I. This work not only further deepened our understanding of the functional diversity of Syt I and the functional differences between its two C2 domains, but also provided important clues for the further related researches.