YtvA from Bacillus subtilis is a blue-light responsive, flavin-binding photoreceptor, built of a light-sensing LOV domain (aa 25-126) and an NTP (nucleoside triphosphate)-binding STAS domain (aa 147-261). The STAS domain is supposed to be the effector part of the protein or a secondary switch. Both domains are connected by a linker polypeptide. The active form of YtvA is generated upon light excitation, causing the formation of a covalent bond between a cysteine residue (Cys62) in the LOV domain and the position 4a of the flavin chromophore. This photoadduct formation within the LOV domain results in a conformational change of the NTP-binding cavity, evidencing intra-protein signal transmission. We have previously shown that Glu105, localized on the beta-scaffold of the LOV-core, is involved in this process. Here, we extend this work by the identification of further residues that upon mutation supress or strongly impair signal transmission by interfering with the communication between the two domains. These comprise L106 and D109 on the LOV domain; K130 and K134 on the linker region; D193, L194 and G196 within the DLSG GTP-binding motif (switch region) and N201 on the STAS domain. Furthermore in the mutated S195A and D193A proteins, GTP affinity is diminished. Other mutations investigated have little or no effect on signal transmission and GTP-binding affinity: R63K that was found to accelerate the thermal recovery of the parent state ca. ten-fold; K128A, Q129A and Y132A within the linker region, and S183A and S212A on the STAS domain. The results show a key role of the LOV domain beta-scaffold and of positively charged residues within the linker for intra-protein signal transmission. Furthermore they evidence the conformational switch function of a structurally conserved strand-loop-helix region (bearing the DLSG GTP-binding motif and N201) within the STAS domain that constitutes a novel GTP-binding fold.
LOV2 domains (1), members of the superfamily of PAS domains (2, 3), are abundant in all domains of life and were first identified in plant phototropins (4). These photoreceptors regulate stomatal opening, phototropism, etc. and contain two N-terminal LOV domains that confer light regulation on the C-terminal Ser/Thr kinase domain (4). They also occur in bacteria, in which YtvA from Bacillus subtilis has been best characterized (for a review, see e.g. Ref. 5). Its N-terminal LOV domain binds FMN and shows the typical LOV photochemistry (6, 7): covalent adduct formation between a cysteine and the FMN chromophore. A linker helix, denoted J␣ (7), connects the LOV domain to a STAS domain. The latter domain is present in many regulators of the general stress response of B. subtilis (8,9). Stress via the addition of salt or ethanol (for a review, see Ref.10) and blue light (11, 12) activates the general stress response via the environmental pathway, which integrates various signals via a large multiprotein complex, called the stressosome (13,14). YtvA, which mediates light activation of B (11, 12, 15), co-purifies with other STAS domain proteins in the stressosomes (16). When cells are stressed, STAS domains of several stressosome proteins (e.g. RsbS and RsbR) are phosphorylated by another intrinsic stressosome component, the serine/threonine kinase RsbT (9,14,17,18). Next, RsbT is released from the complex to trigger RsbU, a protein phosphatase, thus (indirectly) activating B (19). Phosphorylation of YtvA, however, has never been detected. Rather, it has been demonstrated in vitro that YtvA shows light-dependent GTP binding, presumably at its NTP-binding site in the STAS domain (20).Little is known about the mechanism of signal transmission in and by YtvA, except that in the C62A mutant, photochemistry in vitro (12) and light activation of B in vivo (12, 15) are abolished. More detailed information is available for LOV domains of phototropins. A conserved glutamine, which is in hydrogen-bonding contact with the isoalloxazine ring of FMN, rotates its side chain by 180 o upon covalent adduct formation (21). Replacement of this residue by leucine in the LOV2 domain of Phy3 from Adiantum results in a considerable reduction of the light-induced structural change (22). The corresponding mutation in phototropin 1 from Arabidopsis impairs autophosphorylation activity (23). The signal generated in the LOV2 domain is transmitted to the downstream kinase domain of phototropin 1 of Avena sativa through disruption of the interaction between its central -sheet and the C-terminal linker region, the J␣-helix (24).Here, we study the mechanism of activation of YtvA in vivo, i.e. light-induced activation of the B response, with site-directed mutagenesis. We focus on three regions of the protein, the flavinbinding pocket, the -sheet of the LOV domain, and the GTPbinding site, and on potential phosphorylation sites of the STAS domain. We demonstrate that light-activated GTP binding is crucial for functional YtvA. A computational approac...
BackgroundAccumulation of β-amyloid peptides is an important hallmark of Alzheimer’s disease (AD). Tremendous efforts have been directed to elucidate the mechanisms of β-amyloid peptides degradation and develop strategies to remove β-amyloid accumulation. In this study, we demonstrated that a subpopulation of oligodendroglial precursor cells, also called NG2 cells, were a new cell type that can clear β-amyloid peptides in the AD transgene mice and in NG2 cell line.ResultsNG2 cells were recruited and clustered around the amyloid plaque in the APPswe/PS1dE9 mice, which is Alzheimer’s disease mouse model. In vitro, NG2 cell line and primary NG2 cells engulfed β-amyloid peptides through the mechanisms of endocytosis in a time dependent manner. Endocytosis is divided into pinocytosis and phagocytosis. Aβ42 internalization by NG2 cells was mediated by actin-dependent macropinocytosis. The presence of β-amyloid peptides stimulated the autophagic pathway in NG2 cells. Once inside the cells, the β-amyloid peptides in NG2 cells were transported to lysosomes and degraded by autophagy.ConclusionsOur findings suggest that NG2 cells are a new cell type that can clear β-amyloid peptides through endocytosis and autophagy.
Thiamine deficiency (TD) causes mild impairment of oxidative metabolism and region-selective neuronal loss in the brain, which may be mediated by neuronal oxidative stress, endoplasmic reticulum stress, and neuroinflammation. TD-induced brain damage is used to model neurodegenerative disorders, and the mechanism for the neuronal death is still unclear. We hypothesized that autophagy might be activated in the TD brain and play a protective role in TD induced neuronal death. Our results demonstrated that TD induced the accumulation of autophagosomes in neurons of the thalamus measured by transmission electron microscopy, and the upregulation of autophagic markers: LC3-II, Atg5 and Beclin1 as measured with western blotting. TD also increased the expression of autophagic markers and induced LC3 puncta in SH-SY5Y neuroblastoma cells. TD-induced expression of autophagic markers was reversed once thiamine was re-administered. Both inhibition of autophagy by wortmannin and Beclin1 siRNA potentiated TD-induced death of SH-SY5Y cells. In contrast, activation of autophagy by rapamycin alleviated cell death induced by TD. Intraperitoneal injection of rapamycin stimulated neuronal autophagy and attenuated TD-induced neuronal death and microglia activation in the submedial thalamus nucleus (SmTN). TD inhibited the phosphorylation of p70S6 kinase, suggesting mTOR/p70S6 kinase pathway was involved the TD-induced autophagy. These results suggest that autophagy is neuroprotective in response to TD-induced neuronal death in the central nervous system. This opens a potential therapeutic avenue for neurodegenerative diseases caused by mild impairment of oxidative metabolism.
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