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Qian Cai,1 Sirui Chen,2 Yingqun Zhu,1 Zhe Li1 1Department of Respiratory and Critical Care Medicine, The Third Hospital of Changsha, Changsha City, Hunan Province, 410015, People’s Republic of China; 2Department of Emergency Medicine, The Third Hospital of Changsha, Changsha City, Hunan Province, 410015, People’s Republic of ChinaCorrespondence: Sirui Chen, Department of Emergency Medicine, The Third Hospital of Changsha, No. 176, Laodong West Road, Tianxin District, Changsha City, Hunan Province, 410015, People’s Republic of China, Tel +86-0731-85171616, Email realcsr@163.comBackground: Chronic obstructive pulmonary disease (COPD) is a persistent chronic bronchitis disease, and its potential biomarkers have not been fully expounded. This study aims to explore the role of Guanine nucleotide binding protein like-3-like (GNL3L) in COPD induced by cigarette smoking (CS) in vivo.Methods: Two microarray datasets of COPD were selected to screen differentially expressed genes (DEGs). A protein–protein interaction network was constructed to find hub genes. The COPD model was conducted using CS/LPS-induced mouse and cigarette smoke extract induced human bronchial epithelial cells. The pathological changes of lung tissue in mice were observed by hematoxylin–eosin staining and mean linear intercept. Cell viability was measured by CCK8 assay. Oxidative stress-related indicators, inflammatory factors, and ATM/p53 related-proteins were assessed using ELISA and Western blot.Results: In this study, there were 110 common DEGs identified from the two datasets (GSE5058 and GSE38974). The key gene GNL3L was the optimal indicator to distinguish between samples with COPD and healthy controls. Through the in vivo and in vitro experiments, GNL3L knockdown significantly improved the pathological features of CS/LPS-induced COPD mice, promoted cell viability, inhibited inflammation (IL-1β, IL-8, and TNF-α), oxidative stress (MDA, SOD, and CAT), and ATM/p53 related-proteins (ATM, p53, and p21).Conclusion: GNL3L is a novel biomarker of COPD, and knockdown of GNL3L participates in the progression of COPD by inhibiting ATM/p53 pathway.Keywords: COPD, ATM/p53 pathway, hub genes, GNL3L

Guanine nucleotide-binding protein-like 3-like (GNL3L), a conserved GTP-binding nucleolar protein, participates in regulating cell proliferation, and associates with tumorigenesis and poor prognosis in several kind of cancers. However, the role of GNL3L in modulating autophagy remains unclear. Here, we verified that GNL3L was higher expressed in esophageal cancer (ESCA) biopsies than that in the corresponding normal biopsies by a human ESCA tissue array. Utilizing immunoblotting and real-time PCR assays, we analyzed the expression of GNL3L in several ESCA cell lines, and it was highly expressed in KYSE410 cells and rarely expressed in KYSE150 cells, respectively. GNL3L overexpression promoted cell viability and cell proliferation in KYSE150 cells. On the contrary, silencing of GNL3L resulted in opposite phenotypes in KYSE410 cells. Furthermore, GNL3L level correlated with autophagic flux and influenced the levels of autophagy core proteins. Meanwhile, GNL3L also affected the AMPK signaling pathway, which is a pivotal signaling pathway for autophagy regulation. In the GNL3L-silenced cells, the AMPK agonist AICAR partly rescued the autophagic flux. Inversely, both pharmacologically and genetically deprivation of AMPK attenuated the autophagic flux induced by GNL3L overexpression. Moreover, AMPK activity alteration influenced the effect of GNL3L in regulating cell proliferation. Collectively, these findings suggest that GNL3L positively regulates cell proliferation and autophagy in ESCA cells via regulating the AMPK signaling, making itself a promising therapeutic target for ESCA. [ABSTRACT FROM AUTHOR]

Simple Summary: Guanine nucleotide-binding protein-like 3-like (GNL3L) is a novel GTP-binding nucleolar protein. In this study, we analyzed the expression, prognosis, and immune roles of GNL3L in pan-cancer from multiple omics analyses. The final results showed that GNL3L is differentially expressed in a variety of cancers, plays a prognostic role, and has good immune value. Moreover, GNL3L may affect the occurrence of cancer through processes such as ribonucleoprotein, ribosomal RNA processing, and cell proliferation. At the same time, we established an esophageal cancer (ESCA) prediction model with strong predictive ability and proved that GNL3L can significantly affect the proliferation ability of esophageal cancer cells through clone formation assays. In conclusion, GNL3L is an important biomarker. Guanine nucleotide-binding protein-like 3-like protein (GNL3L) is a novel, evolutionarily conserved, GTP-binding nucleolar protein. This study aimed to investigate the expression, prognosis, and immune value of GNL3L in pan-cancer from multiple omics analyses. Firstly, the expression and prognostic value of GNL3L in pan-cancer were discussed using the TIMER2 database, the GEPIA database, the cBioportal database, COX regression analysis, and enrichment analysis. The association of GNL3L with tumor mutational burden (TMB), tumor microsatellite instability (MSI), mismatch repair (MMR) genes, and immune cells was then analyzed. Finally, an esophageal cancer (ESCA) prediction model was established, and GNL3L clone formation assays were performed. The final results showed that GNL3L is differentially expressed in the vast majority of cancers, is associated with the prognosis of various cancers, and may affect cancer occurrence through processes such as ribonucleoprotein, ribosomal RNA processing, and cell proliferation. At the same time, it was found that the correlation between GNL3L and TMB, MSI, MMR, and various immune cells is significant. The established ESCA prediction model had a strong predictive ability, and GNL3L could significantly affect the proliferation of esophageal cancer cells. In conclusion, GNL3L may serve as an important prognostic biomarker and play an immunomodulatory role in tumors. [ABSTRACT FROM AUTHOR]

Nucleostemin (NS) is a vertebrate gene preferentially expressed in stem and cancer cells, which acts to regulate cell cycle progression, genome stability and ribosome biogenesis. NS and its paralogous gene, GNL3-like (GNL3L), arose in the vertebrate clade after a duplication event from their orthologous gene, G protein Nucleolar 3 (GNL3). Research on invertebrate GNL3, however, has been limited. To gain a greater understanding of the evolution and functions of the GNL3 gene, we have performed studies in the hydrozoan cnidarian Hydractinia symbiolongicarpus, a colonial hydroid that continuously generates pluripotent stem cells throughout its life cycle and presents impressive regenerative abilities. We show that Hydractinia GNL3 is expressed in stem and germline cells. The knockdown of GNL3 reduces the number of mitotic and S-phase cells in Hydractinia larvae of different ages. Genome editing of Hydractinia GNL3 via CRISPR/Cas9 resulted in colonies with reduced growth rates, polyps with impaired regeneration capabilities, gonadal morphological defects, and low sperm motility. Collectively, our study shows that GNL3 is an evolutionarily conserved stem cell and germline gene involved in cell proliferation, animal growth, regeneration and sexual reproduction in Hydractinia, and sheds new light into the evolution of GNL3 and of stem cell systems.

The mammalian nucleolar proteins nucleostemin and GNL3-like (GNL3L) are encoded by paralogous genes that arose from an ancestral invertebrate gene, GNL3. Invertebrate GNL3 has been implicated in ribosome biosynthesis, as has its mammalian descendent, GNL3L. The paralogous mammalian nucleostemin protein has, instead, been implicated in cell renewal. Here, we found that depletion of nucleostemin in a human breast carcinoma cell line triggers prompt and significant DNA damage in S-phase cells without perturbing the initial step of ribosomal (r)RNA synthesis and only mildly affects the total ribosome production. By contrast, GNL3L depletion markedly impairs ribosome production without inducing appreciable DNA damage. These results indicate that, during vertebrate evolution, GNL3L retained the role of the ancestral gene in ribosome biosynthesis, whereas the paralogous nucleostemin acquired a novel genome-protective function. Our results provide a coherent explanation for what had seemed to be contradictory findings about the functions of the invertebrate versus vertebrate genes and are suggestive of how the nucleolus was fine-tuned for a role in genome protection and cell-cycle control as the vertebrates evolved. [ABSTRACT FROM AUTHOR]

Abstract: A variety of G-proteins and GTPases are known to be involved in nucleolar function. We describe here a new evolutionarily conserved putative human GTPase, guanine nucleotide binding protein-like 3-like (GNL3L). Genes encoding proteins related to GNL3L are present in bacteria and yeast to metazoa and suggests its critical role in development. Conserved domain search analysis revealed that the GNL3L contains a circularly permuted G-motif described by a G5-G4-G1-G2-G3 pattern similar to the HSR1/MMR1 GTP-binding protein subfamily. Highly conserved and critical residues were identified from a three-dimensional structural model obtained for GNL3L using the crystal structure of an Ylqf GTPase from Bacillus subtilis. We demonstrate here that GNL3L is transported into the nucleolus by a novel lysine-rich nucleolar localization signal (NoLS) residing within 1–50 amino acid residues. NoLS identified here is necessary and sufficient to target the heterologous proteins to the nucleolus. We show for the first time that the lysine-rich targeting signal interacts with the nuclear transport receptor, importin-beta and transports GNL3L into the nucleolus. Interestingly, depletion of intracellular GTP blocks GNL3L accumulation into the nucleolar compartment. Furthermore, mutations within the G-domains alter the GTP binding ability of GNL3L and abrogate wild-type nucleolar retention even in the presence of functional NoLS, suggesting that the efficient nucleolar retention of GNL3L involves activities of both basic NoLS and GTP-binding domains. Collectively, these data suggest that GNL3L is composed of distinct modules, each of which plays a specific role in molecular interactions for its nucleolar retention and subsequent function(s) within the nucleolus. [Copyright &y& Elsevier]

Abstract: Nucleostemin was first identified in neural stem cells and has become a focus of research in cell cycle control, tumorigenesis and cellular senescence. As the biology of nucleostemin begins to be unveiled in multiple species, an ensuing task is to resolve the apparent differences between the functions of mammalian and invertebrate nucleostemin and its homologues, an issue of pressing interest given the role of nucleostemin in stem cell self-renewal and tissue regeneration. A genome-wide search reveals that nucleostemin and its closest homologue, GNL3L, only emerge as separate genes in vertebrates and possess conserved protein sequences as evolution proceeded to the Mammalia. The invertebrate orthologue of nucleostemin and GNL3L resembles GNL3L more than it does nucleostemin in function, raising the idea that nucleostemin acquires new properties while GNL3L inherits an evolutionarily fixed role, and that the birth of nucleostemin may signify the appearance of new functional features in the vertebrate lineage. [Copyright &y& Elsevier]

Guanine nucleotide binding protein-like 3 (GNL3L) is the closest homologue of a stem cell-enriched factor nucleostemin in vertebrates. They share the same yeast orthologue, Grn1p, but only GNL3L can rescue the growth-deficient phenotype in Grn1-null yeasts. To determine the unique function of GNL3L, we identified estrogen-related receptor γ (ERRγ) as a GNL3L-specific binding protein. GNL3L and ERRγ are coexpressed in the eye, kidney and muscle, and co-reside in the nucleoplasm. The interaction between GNL3L and ERRγ requires the intermediate domain of GNL3L and the AF2-domain of ERRγ. Gain-of- and loss-of-function experiments show that GNL3L can inhibit the transcriptional activities of ERR genes in a cell-based reporter system, which does not require the nucleolar localization of GNL3L. We further demonstrate that GNL3L is able to reduce the steroid receptor coactivator (SRC) binding and the SRC-mediated transcriptional coactivation of ERRγ. This work reveals a novel mechanism that negatively regulates the transcriptional function of ERRγ by GNL3L through coactivator competition. [ABSTRACT FROM AUTHOR]