SIRT3 regulates mitophagy in liver fibrosis through deacetylation of PINK1/NIPSNAP1.

Damaged mitochondria, a key factor in liver fibrosis, can be removed by the mitophagy pathway to maintain homeostasis of the intracellular environment to alleviate the development of fibrosis. PINK1 (PTEN-induced kinase 1) and NIPSNAP1 (nonneuronal SNAP25-like protein 1), which cooperatively regulate mitophagy, have been predicted to include the sites of lysine acetylation related to SIRT3 (mitochondrial deacetylase sirtuin 3). Our study aimed to discuss whether SIRT3 deacetylates PINK1 and NIPSNAP1 to regulate mitophagy in liver fibrosis. Carbon tetrachloride (CCl4 )-induced liver fibrosis as an in vivo model and LX-2 cells as activated cells were used to simulate liver fibrosis. SIRT3 expression was significantly decreased in mice in response to CCl4 , and SIRT3 knockout in vivo significantly deepened the severity of liver fibrosis, as indicated by increased α-SMA and Col1a1 levels both in vivo and in vitro. SIRT3 overexpression decreased α-SMA and Col1a1 levels. Furthermore, SIRT3 significantly regulated mitophagy in liver fibrosis, as demonstrated by LC3-Ⅱ/Ⅰ and p62 expression and colocalization between TOM20 and LAMP1. Importantly, PINK1 and NIPSNAP1 expression was also decreased in liver fibrosis, and PINK1 and NIPSNAP1 overexpression significantly improved mitophagy and attenuated ECM production. Furthermore, after simultaneously interfering with PINK1 or NIPSNAP1 and overexpressing SIRT3, the effect of SIRT3 on improving mitophagy and alleviating liver fibrosis was disrupted. Mechanistically, we show that SIRT3, as a mitochondrial deacetylase, specifically regulates the acetylation of PINK1 and NIPSNAP1 to mediate the mitophagy pathway in liver fibrosis. SIRT3-mediated PINK1 and NIPSNAP1 deacetylation is a novel molecular mechanism in liver fibrosis.

Nipsnap1-A regulatory factor required for long-term maintenance of non-shivering thermogenesis.

OBJECTIVE: The activation of non-shivering thermogenesis (NST) has strong potential to combat obesity and metabolic disease. The activation of NST however is extremely temporal and the mechanisms surrounding how the benefits of NST are sustained once fully activated, remain unexplored. The objective of this study is to investigate the role of 4-Nitrophenylphosphatase Domain and Non-Neuronal SNAP25-Like 1 (Nipsnap1) in NST maintenance, which is a critical regulator identified in this study. METHODS: The expression of Nipsnap1 was profiled by immunoblotting and RT-qPCR. We generated Nipsnap1 knockout mice (N1-KO) and investigated the function of Nipsnap1 in NST maintenance and whole-body metabolism using whole body respirometry analyses. We evaluate the metabolic regulatory role of Nipsnap1 using cellular and mitochondrial respiration assay. RESULTS: Here, we show Nipsnap1 as a critical regulator of long-term thermogenic maintenance in brown adipose tissue (BAT). Nipsnap1 localizes to the mitochondrial matrix and increases its transcript and protein levels in response to both chronic cold and ß3 adrenergic signaling. We demonstrated that these mice are unable to sustain activated energy expenditure and have significantly lower body temperature in the face of an extended cold challenge. Furthermore, when mice are exposed to the pharmacological ß3 agonist CL 316, 243, the N1-KO mice exhibit significant hyperphagia and altered energy balance. Mechanistically, we demonstrate that Nipsnap1 integrates with lipid metabolism and BAT-specific ablation of Nipsnap1 leads to severe defects in beta-oxidation capacity when exposed to a cold environmental challenge. CONCLUSION: Our findings identify Nipsnap1 as a potent regulator of long-term NST maintenance in BAT.

NIPSNAP1 directs dual mechanisms to restrain senescence in cancer cells.

BACKGROUND: Although the executive pathways of senescence are known, the underlying control mechanisms are diverse and not fully understood, particularly how cancer cells avoid triggering senescence despite experiencing exacerbated stress conditions within the tumor microenvironment. METHODS: Mass spectrometry (MS)-based proteomic screening was used to identify differentially regulated genes in serum-starved hepatocellular carcinoma cells and RNAi employed to determine knockdown phenotypes of prioritized genes. Thereafter, gene function was investigated using cell proliferation assays (colony-formation, CCK-8, Edu incorporation and cell cycle) together with cellular senescence assays (SA-ß-gal, SAHF and SASP). Gene overexpression and knockdown techniques were applied to examine mRNA and protein regulation in combination with luciferase reporter and proteasome degradation assays, respectively. Flow cytometry was applied to detect changes in cellular reactive oxygen species (ROS) and in vivo gene function examined using a xenograft model. RESULTS: Among the genes induced by serum deprivation, NIPSNAP1 was selected for investigation. Subsequent experiments revealed that NIPSNAP1 promotes cancer cell proliferation and inhibits P27-dependent induction of senescence via dual mechanisms. Firstly, NIPSNAP1 maintains the levels of c-Myc by sequestering the E3 ubiquitin ligase FBXL14 to prevent the proteasome-mediated turnover of c-Myc. Intriguingly, NIPSNAP1 levels are restrained by transcriptional repression mediated by c-Myc-Miz1, with repression lifted in response to serum withdrawal, thus identifying feedback regulation between NIPSNAP1 and c-Myc. Secondly, NIPSNAP1 was shown to modulate ROS levels by promoting interactions between the deacetylase SIRT3 and superoxide dismutase 2 (SOD2). Consequent activation of SOD2 serves to maintain cellular ROS levels below the critical levels required to induce cell cycle arrest and senescence. Importantly, the actions of NIPSNAP1 in promoting cancer cell proliferation and preventing senescence were recapitulated in vivo using xenograft models. CONCLUSIONS: Together, these findings reveal NIPSNAP1 as an important mediator of c-Myc function and a negative regulator of cellular senescence. These findings also provide a theoretical basis for cancer therapy where targeting NIPSNAP1 invokes cellular senescence.

NLRX1/FUNDC1/NIPSNAP1-2 axis regulates mitophagy and alleviates intestinal ischaemia/reperfusion injury.

OBJECTIVES: Mitophagy is considered to be a key mechanism in the pathogenesis of intestinal ischaemic reperfusion (IR) injury. NOD-like receptor X1 (NLRX1) is located in the mitochondria and is highly expressed in the intestine, and is known to modulate ROS production, mitochondrial damage, autophagy and apoptosis. However, the function of NLRX1 in intestinal IR injury is unclear. MATERIALS AND METHODS: NLRX1 in rats with IR injury or in IEC-6 cells with hypoxia reoxygenation (HR) injury were measured by Western blotting, real-time PCR and immunohistochemistry. The function of NLRX1-FUNDC1-NIPSNAP1/NIPSNAP2 axis in mitochondrial homeostasis and cell apoptosis were assessed in vitro. RESULTS: NLRX1 is significantly downregulated following intestinal IR injury. In vivo studies showed that rats overexpressing NLRX1 exhibited resistance against intestinal IR injury and mitochondrial dysfunction. These beneficial effects of NLRX1 overexpression were dependent on mitophagy activation. Functional studies showed that HR injury reduced NLRX1 expression, which promoted phosphorylation of FUN14 domain-containing 1 (FUNDC1). Based on immunoprecipitation studies, it was evident that phosphorylated FUNDC1 could not interact with the mitophagy signalling proteins NIPSNAP1 and NIPSNAP2 on the outer membrane of damaged mitochondria, which failed to launch the mitophagy process, resulting in the accumulation of damaged mitochondria and epithelial apoptosis. CONCLUSIONS: NLRX1 regulates mitophagy via FUNDC1-NIPSNAP1/NIPSNAP2 signalling pathway. Thus, this study provides a potential target for the development of a therapeutic strategy for intestinal IR injury.

NIPSNAP1 and NIPSNAP2 act as "eat me" signals to allow sustained recruitment of autophagy receptors during mitophagy.

Removal of damaged mitochondria is vital for cellular homeostasis especially in non-dividing cells, like neurons. Damaged mitochondria that cannot be repaired by the ubiquitin-proteasomal system are cleared by a form of selective autophagy known as mitophagy. Following damage, mitochondria become labelled with 'eat-me' signals that selectively determine their degradation. Recently, we identified the mitochondrial matrix proteins, NIPSNAP1 (nipsnap homolog 1) and NIPSNAP2 as 'eat-me' signals for damaged mitochondria. NIPSNAP1 and NIPSNAP2 accumulate on the mitochondrial outer membrane following mitochondrial depolarization, recruiting autophagy receptors and adaptors, as well as human Atg8 (autophagy-related 8)-family proteins to facilitate mitophagy. The NIPSNAPs allow a sustained recruitment of SQSTM1-like receptors (SLRs) to ensure efficient mitophagy. Zebrafish lacking Nipsnap1 show decreased mitophagy in the brain coupled with increased ROS production, loss of dopaminergic neurons and strongly reduced locomotion.

NIPSNAP1 and NIPSNAP2 Act as "Eat Me" Signals for Mitophagy.

The clearance of damaged or dysfunctional mitochondria by selective autophagy (mitophagy) is important for cellular homeostasis and prevention of disease. Our understanding of the mitochondrial signals that trigger their recognition and targeting by mitophagy is limited. Here, we show that the mitochondrial matrix proteins 4-Nitrophenylphosphatase domain and non-neuronal SNAP25-like protein homolog 1 (NIPSNAP1) and NIPSNAP2 accumulate on the mitochondria surface upon mitochondrial depolarization. There, they recruit proteins involved in selective autophagy, including autophagy receptors and ATG8 proteins, thereby functioning as an "eat me" signal for mitophagy. NIPSNAP1 and NIPSNAP2 have a redundant function in mitophagy and are predominantly expressed in different tissues. Zebrafish lacking a functional Nipsnap1 display reduced mitophagy in the brain and parkinsonian phenotypes, including loss of tyrosine hydroxylase (Th1)-positive dopaminergic (DA) neurons, reduced motor activity, and increased oxidative stress.

Nocistatin sensitizes TRPA1 channels in peripheral sensory neurons.

The ability of sensory neurons to detect potentially harmful stimuli relies on specialized molecular signal detectors such as transient receptor potential (TRP) A1 ion channels. TRPA1 is critically implicated in vertebrate nociception and different pain states. Furthermore, TRPA1 channels are subject to extensive modulation and regulation - processes which consequently affect nociceptive signaling. Here we show that the neuropeptide Nocistatin sensitizes TRPA1-dependent calcium influx upon application of the TRPA1 agonist mustard oil (MO) in cultured sensory neurons of dorsal root ganglia (DRG). Interestingly, TRPV1-mediated cellular calcium responses are unaffected by Nocistatin. Furthermore, Nocistatin-induced TRPA1-sensitization is likely independent of the Nocistatin binding partner 4-Nitrophenylphosphatase domain and non-neuronal SNAP25-like protein homolog 1 (NIPSNAP1) as assessed by siRNA-mediated knockdown in DRG cultures. In conclusion, we uncovered the sensitization of TRPA1 by Nocistatin, which may represent a novel mechanism how Nocistatin can modulate pain.

Involvement of NIPSNAP1, a neuropeptide nocistatin-interacting protein, in inflammatory pain.

BACKGROUND: Chronic pain associated with inflammation is an important clinical problem, and the underlying mechanisms remain poorly understood. 4-Nitrophenylphosphatase domain and nonneuronal SNAP25-like protein homolog (NIPSNAP) 1, an interacting protein with neuropeptide nocistatin, is implicated in the inhibition of tactile pain allodynia. Although nocistatin inhibits some inflammatory pain responses, whether NIPSNAP1 affects inflammatory pain appears to be unclear. Here, we examined the nociceptive behavioral response of NIPSNAP1-deficient mice and the expression of NIPSNAP1 following peripheral inflammation to determine the contribution of NIPSNAP1 to inflammatory pain. RESULTS: Nociceptive behavioral response increased in phase II of the formalin test, particularly during the later stage (26-50 min) in NIPSNAP1-deficient mice, although the response during phase I (0-15 min) was not significantly different between the deficient and wild-type mice. Moreover, phosphorylation of extracellular signal-related kinase was enhanced in the spinal dorsal horn of the deficient mice. The prolonged inflammatory pain induced by carrageenan and complete Freund's adjuvant was exacerbated in NIPSNAP1-deficient mice. NIPSNAP1 mRNA was expressed in small- and medium-sized neurons of the dorsal root ganglion and motor neurons of the spinal cord. In the formalin test, NIPSNAP1 mRNA was slightly increased in dorsal root ganglion but not in the spinal cord. In contrast, NIPSNAP1 mRNA levels in dorsal root ganglion were significantly decreased during 24-48 h after carrageenan injection. Prostaglandin E2, a major mediator of inflammation, stimulated NIPSNAP1 mRNA expression via the cAMP-protein kinase A signaling pathway in isolated dorsal root ganglion cells. CONCLUSIONS: These results suggest that changes in NIPSNAP1 expression may contribute to the pathogenesis of inflammatory pain.

Pain regulation by nocistatin-targeting molecules: G protein-coupled-receptor and nocistatin-interacting protein.

Nociceptin/orphanin FQ (N/OFQ) and nocistatin (NST) are neuropeptides produced from the same precursor protein. N/OFQ is involved in a broad range of central functions including pain, learning, memory, anxiety, and feeding. However, NST has opposite effects on various central functions evoked by N/OFQ. The regulation of their receptors may be important for these opposite functions of NST and N/OFQ. Although N/OFQ binds to a specific N/OFQ receptor, the target molecule of NST remains unclear. Some biological effects of NST are mediated by a G protein-coupled receptor. Furthermore, using high-performance affinity nanobeads, we recently identified a 4-nitrophenylphosphatase domain and nonneuronal SNAP25-like protein homolog 1 (NIPSNAP1) as a protein that interacts with NST in the mouse spinal cord. The inhibition of N/OFQ-evoked tactile pain allodynia by NST is mediated by NIPSNAP1. This review focuses on the molecular mechanisms of pain regulation by the target molecules of NST including a G protein-coupled receptor and NIPSNAP1.

Chromosomal structural variations during progression of a prostate epithelial cell line to a malignant metastatic state inactivate the NF2, NIPSNAP1, UGT2B17, and LPIN2 genes.

Prostate cancer is the second highest cause of male cancer deaths in the United States. A significant number of tumors advance to a highly invasive and metastatic stage, which is typically resistant to traditional cancer therapeutics. In order to identify chromosomal structural variants that may contribute to prostate cancer progression we sequenced the genomes of a HPV-18 immortalized nonmalignant human prostate epithelial cell line, RWPE1, and compared it to its malignant, metastatic derivative, WPE1-NB26. There were a total of 34 large (> 1 Mbp) and 38 small copy number variants (<100 kbp) in WPE1-NB26 that were not present in the precursor cell line. We also identified and validated 46 structural variants present in the two cell lines, of which 23 were unique to WPE1-NB26. Structural variants unique to the malignant cell line inactivated: (1) the neurofibromin2 (NF2) gene, a known tumor suppressor; (2) its neighboring gene NIPSNAP1, another putative tumor suppressor that inhibits TRPV6, an anti-apoptotic oncogene implicated in prostate cancer progression; (3) UGT2B17, a gene that inactivates dihydrotestosterone, a known activator of prostate cancer progression; and (4) LPIN2, a phosphatidic acid phosphatase and a co-factor of PGC1a that is important for lipid metabolism and for suppressing autoinflammation. Our results illustrate the value of comparing the genomes of defined related pairs of cell lines to discover chromosomal structural variants that may contribute to cancer progression.