Multiple sequences orchestrate subcellular trafficking of neuronal PAS domain–containing protein 4 (NPAS4)

Abstract

Neuronal Per-Arnt-Sim (PAS) domain–containing protein 4 (NPAS4) is a basic helix–loop–helix (bHLH)-PAS transcription factor first discovered in neurons in the neuronal layer of the mammalian hippocampus and later discovered in pancreatic β-cells. NPAS4 has been proposed as a therapeutic target not only for depression and neurodegenerative diseases associated with synaptic dysfunction but also for type 2 diabetes and pancreas transplantation. The ability of bHLH-PAS proteins to fulfil their function depends on their intracellular trafficking, which is regulated by specific sequences, i.e. the nuclear localization signal (NLS) and the nuclear export signal (NES). However, until now, no study examining the subcellular localization signals of NPAS4 has been published. We show here that Rattus norvegicus NPAS4 was not uniformly localized in the nuclei of COS-7 and N2a cells 24 h after transfection. Additionally, cytoplasmic localization of NPAS4 was leptomycin B-sensitive. We demonstrate that NPAS4 possesses a unique arrangement of localization signals. Its bHLH domain contains an overlapping NLS and NES. We observed that its PAS-2 domain contains an NLS, an NES, and a second, proximally located, putative NLS. Moreover, the C terminus of NPAS4 contains two active NESs that overlap with a putative NLS. Our data indicate that glucose concentration could be one of the factors influencing NPAS4 localization. The presence of multiple localization signals and the differentiated localization of NPAS4 suggest a precise, multifactor-dependent regulation of NPAS4 trafficking, potentially crucial for its ability to act as a cellular stress sensor and transcription factor.

Subcellular Localization Signals of bHLH-PAS Proteins: Their Significance, Current State of Knowledge and Future Perspectives

“…In silico analysis suggested the presence of NLSs in the bHLH domain, the PAS-2 domain and the C-terminal region of NPAS4. Accordingly, putative NESs were expected to be located in the bHLH domain, the PAS-2 domain and the C-terminal region of NPAS4 [21]. For the purpose of this review, we have repeated all predictions (Table 1, Supplementary Materials 1, Supplementary Materials Summary), which are generally still consistent.…”

“…We performed detailed in vivo experiments, which revealed the presence of two overlapping signals: NLS (10-52aa) and NES (26-45aa) in bHLH domain. Simultaneously in the region adjacent to PAS-2 domain and within this domain in close proximity are located: NLS (158-191aa), NES (227-242aa) and putative NLS (285-316aa) [21]. We demonstrated that C-terminal region of NPAS4 contains overlapping NES (591-600aa) and putative NLS (593-622aa).…”

“…NPAS4 was shown to induce autophagy in rat primary cortical neurons and to degrade tau proteins involved in the pathogenesis of Alzheimer’s disease and other tauopathies [85]. The discrepancies in localization studies concerning NPAS4, led us to perform a detailed characterization of the subcellular localization motifs in NPAS4 sequence [21]. NPAS4 localized in the nucleus or the cytoplasm of COS-7 and N2a cells.…”

“…Previously, we have performed systematic research of NLS and NES motifs in Drosophila melonagaster Methoprene tolerant protein (MET) [19] and its paralog—germ cell-expressed (GCE) protein [20]. Then, we carried out similar analyzes for mammalian NPAS4 protein [21]. We have determined an interesting pattern of overlaying signals with opposing activity in bHLH and PAC domains as well as in C-termini.…”

The Participation of the Intrinsically Disordered Regions of the bHLH-PAS Transcription Factors in Disease Development

“…For SIM2 C-terminal region cytoplasmic localization was documented [111]. Finally, we demonstrated previously the presence of overlapping NES and NLS in the C-terminal region of NPAS4 [112]. PTMs like phosphorylation, especially close to the NLS/NES were shown to regulate the intracellular distribution of proteins via activation/deactivation of the localization motifs [113].…”