Nucleobindin-derived nesfatin-1 and nesfatin-1-like peptide stimulate pro-opiomelanocortin synthesis in murine AtT-20 corticotrophs through the cAMP/PKA/CREB signaling pathway.

Nucleobindin (NUCB)-derived peptides, nesfatin-1 (NES-1) and nesfatin-1-like peptide (NLP) have several physiological roles in vertebrates. While NES-1 is implicated in stress, whether NUCB1/NLP and NUCB2/NES-1 have any effect on proopiomelanocortin (POMC) remains unknown. The main aim of this study was to determine if NES-1 and/or NLP affect POMC synthesis in mouse corticotrophs. Immunocytochemistry was employed to target NUCB colocalization with POMC in immortalized mouse tumoral corticotrophs (AtT-20 cells). The ability of NES-1 and NLP to modulate POMC mRNA and protein in AtT-20 cells was assessed by qPCR and Western blot, respectively. Moreover, cell-signaling molecules mediating the effect of NES-1 and NLP on POMC synthesis in mouse tumoral corticotrophs were studied using pharmacological blockers. Mouse tumoral corticotrophs showed immunoreactivity for both NUCB1/NLP and NUCB2/NES-1. Both NES-1 and NLP exerted a stimulatory effect on POMC transcript abundance and protein expression in a dose- and time-dependent manner. This effect was comparable to corticotropin-releasing factor (CRF, positive control) stimulation of POMC. Incubation of mouse tumoral corticotrophs with NES-1 or NLP upregulated the phosphorylation of protein kinase A (PKA) and cAMP-response element-binding protein (CREB). The stimulatory effect of these peptides on POMC transcript abundance and protein expression was blocked by the PKA inhibitor, H89, and an adenylate cyclase inhibitor, 2',3'-dideoxyadenosine (DDA). These pharmacological studies indicate that NES-1 and NLP act through the cAMP/PKA/CREB cellular pathway to stimulate POMC synthesis. Our results provide molecular evidence to support a stimulatory role for nucleobindin-derived peptides on POMC synthesis from corticotrophs. Collectively, this research indicates that corticotrophs produce NUCBs, and the encoded peptides NES-1 and NLP could elicit a direct action to stimulate the pituitary stress hormone. This stimulatory effect is mediated by an uncharacterized G protein-coupled receptor (GPCR) that utilizes the cAMP/PKA/CREB pathway.

A Change in Domain Cooperativity Drives the Function of Calnuc.

With the increasing incidence of neurodegenerative disorders, there is an urgent need to understand the protein folding process. Examining the folding process of multidomain proteins remains a prime challenge, as their complex conformational dynamics make them highly susceptible to misfolding and/or aggregation. The presence of multiple domains in a protein can lead to interaction between the partially folded domains, thereby driving misfolding and/or aggregation. Calnuc is one such multidomain protein for which Ca2+ binding plays a pivotal role in governing its structural dynamics and stability and, presumably, in directing its interactions with other proteins. We demonstrate differential structural dynamics between the Ca2+-free and Ca2+-bound forms of calnuc. In the absence of Ca2+, full-length calnuc displays equilibrium structural transitions with four intermediate states, reporting a sum of the behavioral properties of its individual domains. Fragment-based studies illustrate the sequential events of structure adoption proceeding in the following order: EF domain followed by the NT and LZ domains in the apo state. On the other hand, Ca2+ binding increases domain cooperativity and enables the protein to fold as a single unit. Single-tryptophan mutant proteins, designed in a domain-dependent manner, confirm an increase in the number of interdomain interactions in the Ca2+-bound form as compared to the Ca2+-free state of the protein, thereby providing insight into its folding process. The attenuated domain crosstalk in apo-calnuc is likely to influence and regulate its physiologically important intermolecular interactions.

Calcium ions modulate the structure of the intrinsically disordered Nucleobindin-2 protein.

Nucleobindin-2 (Nucb2) is a widely expressed multi-domain protein. Nucb2 participates in many physiological processes, i.e. calcium level maintenance, feeding regulation in the hypothalamus, emotion and stress regulation, and many others. To date, this protein has not been structurally characterized. We describe the first comparative structural analysis of two homologs, a Gallus gallus and a Homo sapiens Nucb2. The in silico analysis suggested that apo-Nucb2s contain a mosaic-like structure, consisting of intertwined disordered and ordered regions. Surprisingly, the hydrogen-deuterium exchange mass spectrometry results revealed that Nucb2 is divided into two parts: an N-terminal half with a stable mosaic-like structure and a disordered C-terminal half. However, the presence of Ca2+ induces the formation of a mosaic-like structure in the C-terminal half of the Nucb2s. The Ca2+ also affects the tertiary and quaternary structure of Nucb2s. The presence of Ca2+ leads to an overall compaction of the Nucb2 molecule, resulting in structural change that is propagated along the molecule, which in turn affects the quaternary structure of the protein. Intrinsic disorder, and the mosaic-like Ca2+ dependent structure of Nucb2s, might be seen as the molecular factors responsible for their multifunctionality. Thus, Nucb2s might function as the versatile Ca2+ sensor involved in signal transduction.

Genetic analyses uncover pleiotropic compensatory roles for Drosophila Nucleobindin-1 in inositol trisphosphate-mediated intracellular calcium homeostasis.

Nucleobindin-1 is an EF-hand calcium-binding protein with a distinctive profile, predominantly localized to the Golgi in insect and wide-ranging vertebrate cell types, alike. Its putative involvements in intracellular calcium (Ca2+) homeostasis have never been phenotypically characterized in any model organism. We have analyzed an adult-viable mutant that completely disrupts the G protein α-subunit binding and activating (GBA) motif of Drosophila Nucleobindin-1 (dmNUCB1). Such disruption does not manifest any obvious fitness-related, morphological/developmental, or behavioral abnormalities. A single copy of this mutation or the knockdown of dmnucb1 in restricted sets of cells variously rescues pleiotropic mutant phenotypes arising from impaired inositol 1,4,5-trisphosphate receptor (IP3R) activity (in turn depleting cytoplasmic Ca2+ levels across diverse tissue types). Additionally, altered dmNUCB1 expression or function considerably reverses lifespan and mobility improvements effected by IP3R mutants, in a Drosophila model of amyotrophic lateral sclerosis. Homology modeling-based analyses further predict a high degree of conformational conservation in Drosophila, of biochemically validated structural determinants in the GBA motif that specify in vertebrates, the unconventional Ca2+-regulated interaction of NUCB1 with Gαi subunits. The broad implications of our findings are hypothetically discussed, regarding potential roles for NUCB1 in GBA-mediated, Golgi-associated Ca2+ signaling, in health and disease.