Neuron-specific gene NSG1 binds to and positively regulates sortilin ectodomain shedding via a metalloproteinase-dependent mechanism.

Increasing evidence suggests that aberrant regulation of sortilin ectodomain shedding can contribute to amyloid-ß pathology and frontotemporal dementia, although the mechanism by which this occurs has not been elucidated. Here, we probed for novel binding partners of sortilin using multiple and complementary approaches and identified two proteins of the neuron-specific gene (NSG) family, NSG1 and NSG2, that physically interact and colocalize with sortilin. We show both NSG1 and NSG2 induce subcellular redistribution of sortilin to NSG1- and NSG2-enriched compartments. However, using cell surface biotinylation, we found only NSG1 reduced sortilin cell surface expression, which caused significant reductions in uptake of progranulin, a molecular determinant for frontotemporal dementia. In contrast, we demonstrate NSG2 has no effect on sortilin cell surface abundance or progranulin uptake, suggesting specificity for NSG1 in the regulation of sortilin cell surface expression. Using metalloproteinase inhibitors and A disintegrin and metalloproteinase 10 KO cells, we further show that NSG1-dependent reduction of cell surface sortilin occurred via proteolytic processing by A disintegrin and metalloproteinase 10 with a concomitant increase in shedding of sortilin ectodomain to the extracellular space. This represents a novel regulatory mechanism for sortilin ectodomain shedding that is regulated in a neuron-specific manner. Furthermore, this finding has implications for the development of strategies for brain-specific regulation of sortilin and possibly sortilin-driven pathologies.

Proteome analysis of the circadian clock protein PERIOD2.

Circadian rhythms are a series of endogenous autonomous 24-h oscillations generated by the circadian clock. At the molecular level, the circadian clock is based on a transcription-translation feedback loop, in which BMAL1 and CLOCK transcription factors of the positive arm activate the expression of CRYPTOCHROME (CRY) and PERIOD (PER) genes of the negative arm as well as the circadian clock-regulated genes. There are three PER proteins, of which PER2 shows the strongest oscillation at both stability and cellular localization level. Protein-protein interactions (PPIs) or interactome of the circadian clock proteins have been investigated using classical methods such as two-dimensional gel electrophoresis, immunoprecipitation-coupled mass spectrometry, and yeast-two hybrid assay where the dynamic and weak interactions are difficult to catch. To identify the interactome of PER2 we have adopted proximity-dependent labeling with biotin and mass spectrometry-based identification of labeled proteins (BioID). In addition to known interactions with such as CRY1 and CRY2, we have identified several new PPIs for PER2 and confirmed some of them using co-immunoprecipitation technique. This study characterizes the PER2 protein interactions in depth, and it also implies that using a fast BioID method with miniTurbo or TurboID coupled to other major circadian clock proteins might uncover other interactors in the clock that have yet to be discovered.

Identification and Characterization of a New Class of (6-4) Photolyase from Vibrio cholerae.

Light is crucial for many biological activities of most organisms, including vision, resetting of circadian rhythm, photosynthesis, and DNA repair. The cryptochrome/photolyase family (CPF) represents an ancient group of UV-A/blue light sensitive proteins that perform different functions such as DNA repair, circadian photoreception, and transcriptional regulation. The CPF is widely distributed throughout all organisms, including marine prokaryotes. The bacterium Vibrio cholerae was previously shown to have a CPD photolyase that repairs UV-induced thymine dimers and two CRY-DASHs that repair UV-induced single-stranded DNA damage. Here, we characterize a hypothetical gene Vca0809 encoding a new member of CPF in this organism. The spectroscopic analysis of the purified protein indicated that this enzyme possessed a catalytic cofactor, FAD, and photoantenna chromophore 6,7-dimethyl 8-ribityl-lumazin. With a slot blot-based DNA repair assay, we showed that it possessed (6-4) photolyase activity. Further phylogenetic and computational analyses enabled us to classify this gene as a member of the family of iron-sulfur bacterial cryptochromes and photolyases (FeS-BCP). Therefore, we named this gene Vc(6-4) FeS-BCP.

Interactions of drosophila cryptochrome.

In this study, we investigate the intricate regulatory mechanisms underlying the circadian clock in Drosophila, focusing on the light-induced conformational changes in the cryptochrome (DmCry). Upon light exposure, DmCry undergoes conformational changes that prompt its binding to Timeless and Jetlag proteins, initiating a cascade crucial for the starting of a new circadian cycle. DmCry is subsequently degraded, contributing to the desensitization of the resetting mechanism. The transient and short-lived nature of DmCry protein-protein interactions (PPIs), leading to DmCry degradation within an hour of light exposure, presents a challenge for comprehensive exploration. To address this, we employed proximity-dependent biotinylation techniques, combining engineered BioID (TurboID) and APEX (APEX2) enzymes with mass spectrometry. This approach enabled the identification of the in vitro DmCry interactome in Drosophila S2 cells, uncovering several novel PPIs associated with DmCry. Validation of these interactions through a novel co-immunoprecipitation technique enhances the reliability of our findings. Importantly, our study suggests the potential of this method to reveal additional circadian clock- or magnetic field-dependent PPIs involving DmCry. This exploration of the DmCry interactome not only advances our understanding of circadian clock regulation but also establishes a versatile framework for future investigations into light- and time-dependent protein interactions in Drosophila.

Opposite Carcinogenic Effects of Circadian Clock Gene BMAL1.

The circadian clock confers daily rhythmicity on many biochemical and physiological functions and its disruption is associated with increased risks of developing obesity, diabetes, heart disease and cancer. Although, there are studies on the role of Bmal1 in carcinogenesis using germline, conditional or tissue-specific knockouts, it is still not well understood how BMAL1 gene affects cancer-related biological events at the molecular level. We, therefore, took an in vitro approach to understand the contribution of BMAL1 in this molecular mechanism using human breast epithelial cell lines by knocking out BMAL1 gene with CRISPR technology. We preferred epithelial cells over fibroblasts as the most of cancers originate from epithelial cells. After obtaining BMAL1 knockouts by targeting the gene at two different sites from non-tumorigenic MCF10A and invasive tumorigenic MDA-MB-231 cells, we analysed apoptosis and invasion properties of the cell lines as representative events in tumor development. BMAL1 disruption sensitized both cell lines to a bulky-DNA adduct forming agent (cisplatin) and a double-strand break-inducing agent (doxorubicin), while it enhanced the invasive properties of MDA-MB-231 cells. These results show that the disruption of clock genes may have opposing carcinogenic effects.