Striatin is an intracellular protein characterized by four protein-protein interaction domains, a caveolinbinding motif, a coiled-coil structure, a calmodulinbinding domain, and a WD repeat domain, suggesting that it is a signaling or a scaffold protein. Down-regulation of striatin, which is expressed in a few subsets of neurons, impairs the growth of dendrites as well as rat locomotor activity (Bartoli, M., Ternaux, J. P., Forni, C., Portalier, P., Salin, P., Amalric, M., and Monneron, A. (1999) J. Neurobiol. 40, 234 -243). Zinedin, a "novel" protein described here, and SG2NA share with striatin identical protein-protein interaction domains and the same overall domain structure. A phylogenetic analysis supports the hypothesis that they constitute a multigenic family deriving from an ancestral gene. DNA probes and antibodies raised against specific domains of each protein showed that zinedin is mainly expressed in the central nervous system, whereas SG2NA, of more widespread occurrence, is mainly expressed in the brain and muscle. All three proteins are both cytosolic and membrane-bound. All three bind calmodulin in the presence of Ca 2؉ . In rat brain, SG2NA and striatin are generally not found in the same neurons. Both localize to the soma and dendrites, suggesting that they share a similar type of addressing and closely related functions.Striatin is an intracellular protein mostly present in neurons of mammalian basal ganglia and cranial and spinal motor nuclei (1, 2). Electron microscopy showed that it is present in the somato-dendritic compartment of neurons, especially in dendritic spines (1). Brain fractionation shows that striatin is both cytosolic and associated with membranes. This multimodular protein possesses, from the N to the C terminus, four domains mediating protein-protein interactions: a caveolinbinding domain (aa 1 55-63) (3), a putative coiled-coil structure (aa 70 -116), a Ca 2ϩ -calmodulin (CaM)-binding domain (aa 149 -166) (4),and a WD repeat domain (aa 419 -780). The WD repeat family is composed of homologous, structurally related, but functionally diverse proteins able to organize multiple simultaneous or consecutive protein-protein interactions (5). The richness of striatin in domains mediating protein-protein interactions suggests that striatin is both a signaling protein and a multimodular platform protein. A study aimed at elucidating the function of striatin revealed two sets of data demonstrating its central role both in embryonic neurons and in adult brain (6). On the one hand, we showed that the expression of striatin is essential for the maintenance and growth of dendrites in rat embryonic motoneurons in culture. On the other hand, we showed that striatin is involved in the control of motor function in adult rats. Albeit a quantitatively minor protein, striatin thus appears to play major cellular and physiological roles.We have previously reported that the sequence of SG2NA, a 713 aa, supposedly nuclear protein discovered by Muro et al. (7), is 80% similar to and 66% identical...
Green fluorescent genetically encoded calcium indicators (GECIs) are the most popular tool for visualization of calcium dynamics in vivo. However, most of them are based on the EGFP protein and have similar molecular brightnesses. The NTnC indicator, which is composed of the mNeonGreen fluorescent protein with the insertion of troponin C, has higher brightness as compared to EGFP-based GECIs, but shows a limited inverted response with an ΔF/F of 1. By insertion of a calmodulin/M13-peptide pair into the mNeonGreen protein, we developed a green GECI called NCaMP7. In vitro, NCaMP7 showed positive response with an ΔF/F of 27 and high affinity (Kd of 125 nM) to calcium ions. NCaMP7 demonstrated a 1.7-fold higher brightness and similar calcium-association/dissociation dynamics compared to the standard GCaMP6s GECI in vitro. According to fluorescence recovery after photobleaching (FRAP) experiments, the NCaMP7 design partially prevented interactions of NCaMP7 with the intracellular environment. The NCaMP7 crystal structure was obtained at 1.75 Å resolution to uncover the molecular basis of its calcium ions sensitivity. The NCaMP7 indicator retained a high and fast response when expressed in cultured HeLa and neuronal cells. Finally, we successfully utilized the NCaMP7 indicator for in vivo visualization of grating-evoked and place-dependent neuronal activity in the visual cortex and the hippocampus of mice using a two-photon microscope and an NVista miniscope, respectively.
Striatin, SG2NA and zinedin, the three mammalian members of the striatin family are multimodular WD-repeat, calmodulin and calveolin-binding proteins. These scaffolding proteins, involved in both signaling and trafficking, are highly expressed in neurons. Using ultrastructural immunolabeling, we showed that, in Purkinje cells and hippocampal neurons, SG2NA is confined to the somatodendritic compartment with the highest density in dendritic spines. In cultured hippocampal neurons, SG2NA is also highly concentrated in dendritic spines. By expressing truncated forms of HA-tagged SG2NAb, we demonstrated that the coiled-coil domain plays an essential role in the targeting of SG2NA within spines. Furthermore, co-immunoprecipitation experiments indicate that this coiled-coil domain is also crucial for the homo-and hetero-oligomerization of these proteins. Thus, oligomerization of the striatin family proteins is probably an obligatory step for their routing to the dendritic spines, and hetero-oligomerization explains why all these proteins are often co-expressed in the neurons of the rat brain and spinal cord. In the central nervous system, most excitatory terminals contact dendritic actin-rich protrusions called dendritic spines. The formation and maintenance of spines, including the postsynaptic components, requires precise targeting and coordinated activation of structural and signaling molecules (1-3). The mechanisms by which scaffolding proteins are sorted into pre-or postsynaptic compartments remain largely unclear. Multi-protein complexes, vesicles and organelles found at axon terminals and dendritic spines are transported by motor proteins along microtubules (4,5). Numerous proteins located in the postsynaptic density (PSD) undergo vesicular transport to dendritic spines in association with neurotransmitter receptors (6). Although the routing motifs of several trans-membrane proteins such as ionotropic glutamate receptors have been identified (7,8), much less is known about the targeting of soluble cytoplasmic proteins to dendritic spines, especially for proteins that do not belong to the PSD. To investigate this problem, we analyzed the routing mechanisms of cytoplasmic, non-PSD-associated, dendritic spine proteins: the striatin family.In mammals, this family consists of three scaffolding proteins composed of striatin, SG2NA and zinedin. They are mainly expressed in the cytoplasm of neurons of both the central and the peripheral nervous system (9-11). Ultrastructural studies show that striatin is strictly localized to the somatodendritic compartment of neurons and highly concentrated within the spines of the striatal GABAergic neurons (12). Information concerning the physiological role of the striatin family is beginning to emerge. First, we have shown that striatin expression is crucial for both dendritic growth in cultured rat embryonic motoneurons and for the control of motor function in adult rats (13). Second, striatin and SG2NA have been proposed to be regulatory subunits of protein phosphatase 2A (14). I...
Octaheme nitrite reductase from the haloalkaliphilic bacterium Thioalkalivibrio paradoxus was isolated and characterized. A comparative structural and functional analysis of two homologous octaheme nitrite reductases from closely related Thioalkalivibrio species was performed. It was shown that both enzymes have similar catalytic properties, owing to high structural similarity. Both enzymes are characterized by specific structural features distinguishing them from pentaheme cytochrome c nitrite reductases, such as the Tyr-Cys bond in the active site, the hexameric structure resulting in the formation of a void space inside the hexamer, and the product channel that opens into the void interior space of the hexamer. It is suggested that these specific structural features are responsible for the higher nitrite reductase activity, the greater preference for nitrite than for sulfite as a substrate, and the wider pH range of the catalytic activity of octaheme nitrite reductases than of pentaheme homologs.
BackgroundThe structure and function of bacterial nucleoid are controlled by histone-like proteins of HU/IHF family, omnipresent in bacteria and also founding archaea and some eukaryotes.HU protein binds dsDNA without sequence specificity and avidly binds DNA structures with propensity to be inclined such as forks, three/four-way junctions, nicks, overhangs and DNA bulges. Sequence comparison of thousands of known histone-like proteins from diverse bacteria phyla reveals relation between HU/IHF sequence, DNA–binding properties and other protein features.Methodology and principal findingsPerformed alignment and clusterization of the protein sequences show that HU/IHF family proteins can be unambiguously divided into three groups, HU proteins, IHF_A and IHF_B proteins. HU proteins, IHF_A and IHF_B proteins are further partitioned into several clades for IHF and HU; such a subdivision is in good agreement with bacterial taxonomy. We also analyzed a hundred of 3D fold comparative models built for HU sequences from all revealed HU clades. It appears that HU fold remains similar in spite of the HU sequence variations. We studied DNA–binding properties of HU from N. gonorrhoeae, which sequence is similar to one of E.coli HU, and HU from M. gallisepticum and S. melliferum which sequences are distant from E.coli protein. We found that in respect to dsDNA binding, only S. melliferum HU essentially differs from E.coli HU. In respect to binding of distorted DNA structures, S. melliferum HU and E.coli HU have similar properties but essentially different from M. gallisepticum HU and N. gonorrhea HU. We found that in respect to dsDNA binding, only S. melliferum HU binds DNA in non-cooperative manner and both mycoplasma HU bend dsDNA stronger than E.coli and N. gonorrhoeae. In respect to binding to distorted DNA structures, each HU protein has its individual profile of affinities to various DNA-structures with the increased specificity to DNA junction.Conclusions and significanceHU/IHF family proteins sequence alignment and classification are updated. Comparative modeling demonstrates that HU protein 3D folding’s even more conservative than HU sequence. For the first time, DNA binding characteristics of HU from N. gonorrhoeae, M. gallisepticum and S. melliferum are studied. Here we provide detailed analysis of the similarity and variability of DNA-recognizing and bending of four HU proteins from closely and distantly related HU clades.
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