Search Results (12)

The whitefly Bemisia tabaci is a globally invasive pest that threatens crop production through feeding and virus transmission. In this study, we identified genes encoding enzymes in the chitin metabolism pathway of B. tabaci—β-N-acetylglucosaminidase (BtNAG), N-acetylglucosamine kinase (BtNAGK), phosphoacetylglucosamine mutase (BtPAGM), UDP-N-acetylglucosamine pyrophosphorylase (BtUAP), and glucosamine-6-phosphate N-acetyltransferase (BtGNA)—using bioinformatic analysis. Quantitative reverse-transcription PCR (RT-qPCR) analyses revealed distinct stage-specific expression patterns for these genes. We used the nanomaterial star polycation (SPc) to deliver gene-specific double-stranded RNA (dsRNA) targeting these genes to fourth instar B. tabaci nymphs, which resulted in significant mortality and developmental defects upon gene silencing. Notably, the fusion dsRNA targeting three genes—BtNAG1, BtNAGK, and BtUAP—achieved approximately 80% nymph mortality, 70% inhibition of adult emergence, and an earlier onset of gene silencing. These findings provide evidence that nanomaterial-assisted delivery of dsRNA can significantly enhance RNAi effects in hemipteran pests and that dsRNA targeting chitin metabolic genes may be an effective strategy for RNAi-based control of B. tabaci. Full article

Background/Objectives: Lung cancer remains a global health concern, with substantial variation in patient survival. Despite advances in detection and treatment, the genetic basis for the divergent outcomes is not understood. We investigated germline polymorphisms that modulate overall survival in 1464 surgically resected lung adenocarcinoma patients. Methods: A multivariable Cox proportional hazard model was used to assess the association of more than seven million polymorphisms with overall survival at the 60-month follow-up, considering age, sex, pathological stage, decade of surgery and principal components as covariates. Genes in which variants were identified were studied in silico to investigate functional roles. Results: Six germline variants passed the genome-wide significance threshold. These single nucleotide polymorphisms were mapped to non-coding (intronic) regions on chromosomes 2, 3, and 5. The minor alleles of rs13000315, rs151212827, and rs190923216 (chr. 2, 3 and 5, respectively) were found to be independent negative prognostic factors. All six variants have been reported to regulate the expression of nine genes, seven of which are protein-coding, in different tissues. Survival-associated variants on chromosomes 2 and 3 were already reported to regulate the expression of NT5DC2 and NAGK, with high expression associated with the minor alleles. High NT5DC2 and NAGK expression in lung adenocarcinoma tissue was already shown to correlate with poor overall survival. Conclusions: This study highlights a potential regulatory role of the identified polymorphisms in influencing outcome and suggests a mechanistic link between these variants, gene expression regulation, and lung adenocarcinoma prognosis. Validation and functional studies are warranted to elucidate the mechanisms underlying these associations. Full article

The clinical severity of multiple sclerosis (MS), an autoimmune disorder of the central nervous system, is thought to be determined by environmental and genetic factors that have not yet been identified. In a recent genome-wide association study (GWAS), a single nucleotide polymorphism (SNP), rs10191329, has been associated with MS severity in two large independent cohorts of patients. Different approaches were followed by the authors to prioritize the genes that are transcriptionally regulated by such an SNP. It was concluded that the identified SNP regulates a group of proximal genes involved in brain resilience and cognitive abilities rather than immunity. Here, by conducting an alternative strategy for gene prioritization, we reached the opposite conclusion. According to our re-analysis, the main target of rs10191329 is N-Acetylglucosamine Kinase (NAGK), a metabolic gene recently shown to exert major immune functions via the regulation of the nucleotide-binding oligomerization domain-containing protein 2 (NOD2) pathway. To gain more insights into the immunometabolic functions of NAGK, we analyzed the currently known list of NAGK protein partners. We observed that NAGK integrates a dense network of human proteins that are involved in glucose metabolism and are highly expressed by classical monocytes. Our findings hold potentially major implications for the understanding of MS pathophysiology. Full article

New protein-fragment complementation assays (PCA) have successfully been developed to characterize protein–protein interactions in vitro and in vivo. Notably, the NanoBiT technology, employing fragment complementation of NanoLuc luciferase, stands out for its high sensitivity, wide dynamic range, and straightforward read out. Previously, we explored the in vitro protein interaction dynamics of the PII signalling protein using NanoBiT, revealing significant modulation of luminescence signals generated by the interaction between PII and its receptor protein NAGK by 2-oxoglutarate levels. In the current work, we investigated this technology in vivo, to find out whether recombinantly expressed NanoBiT constructs using the NanoLuc large fragment fused to PII and PII-interaction partners NAGK or PipX-fused to the NanoLuc Small BiT are capable of detecting the metabolic fluctuations in Escherichia coli. Therefore, we devised an assay capable of capturing the metabolic responses of E. coli cells, demonstrating real-time metabolic perturbation upon nitrogen upshift or depletion treatments. In particular, the PII-NAGK NanoBitT sensor pair reported these changes in a highly sensitive manner. Full article

N-Acetyl-L-glutamate kinase (NAGK) catalyzes the rate-limiting step in the ornithine/arginine biosynthesis pathway in eukaryotic and bacterial oxygenic phototrophs. NAGK is the most highly conserved target of the PII signal transduction protein in Cyanobacteria and Archaeplastida (red algae and Chlorophyta). However, there is still much to be learned about how NAGK is regulated in vivo. The use of unicellular green alga Chlamydomonas reinhardtii as a model system has already been instrumental in identifying several key regulation mechanisms that control nitrogen (N) metabolism. With a combination of molecular-genetic and biochemical approaches, we show the existence of the complex CrNAGK control at the transcriptional level, which is dependent on N source and N availability. In growing cells, CrNAGK requires CrPII to properly sense the feedback inhibitor arginine. Moreover, we provide primary evidence that CrPII is only partly responsible for regulating CrNAGK activity to adapt to changing nutritional conditions. Collectively, our results suggest that in vivo CrNAGK is tuned at the transcriptional and post-translational levels, and CrPII and additional as yet unknown factor(s) are integral parts of this regulation. Full article

N-acetylglucosamine kinase (NAGK) has been identified as an anchor protein that facilitates neurodevelopment with its non-canonical structural role. Similarly, small nuclear ribonucleoprotein polypeptide N (SNRPN) regulates neurodevelopment and cognitive ability. In our previous study, we revealed the interaction between NAGK and SNRPN in the neuron. However, the precise role in neurodevelopment is elusive. In this study, we investigate the role of NAGK and SNRPN in the axodendritic development of neurons. NAGK and SNRPN interaction is significantly increased in neurons at the crucial stages of neurodevelopment. Furthermore, overexpression of the NAGK and SNRPN proteins increases axodendritic branching and neuronal complexity, whereas the knockdown inhibits neurodevelopment. We also observe the interaction of NAGK and SNRPN with the dynein light-chain roadblock type 1 (DYNLRB1) protein variably during neurodevelopment, revealing the microtubule-associated delivery of the complex. Interestingly, NAGK and SNRPN proteins rescued impaired axodendritic development in an SNRPN depletion model of Prader–Willi syndrome (PWS) patient-derived induced pluripotent stem cell neurons. Taken together, these findings are crucial in developing therapeutic approaches for neurodegenerative diseases. Full article

The PII protein is an evolutionary, highly conserved regulatory protein found in both bacteria and higher plants. In bacteria, it modulates the activity of several enzymes, transporters, and regulatory factors by interacting with them and thereby regulating important metabolic hubs, such as carbon/nitrogen homeostasis. More than two decades ago, the PII protein was characterized for the first time in plants, but its physiological role is still not sufficiently resolved. To gain more insights into the function of this protein, we investigated the interaction behavior of AtPII with candidate proteins by BiFC and FRET/FLIM in planta and with GFP/RFP traps in vitro. In the course of these studies, we found that AtPII interacts in chloroplasts with itself as well as with known interactors such as N-acetyl-L-glutamate kinase (NAGK) in dot-like aggregates, which we named PII foci. In these novel protein aggregates, AtPII also interacts with yet unknown partners, which are known to be involved in plastidic protein degradation. Further studies revealed that the C-terminal component of AtPII is crucial for the formation of PII foci. Altogether, the discovery and description of PII foci indicate a novel mode of interaction between PII proteins and other proteins in plants. These findings may represent a new starting point for the elucidation of physiological functions of PII proteins in plants. Full article

O-linked-N-acetylglucosaminylation (O-GlcNAcylation) performed by O-GlcNAc transferase (OGT) is a nutrient-responsive post-translational modification (PTM) via the hexosamine biosynthetic pathway (HBP). Various transcription factors (TFs) are O-GlcNAcylated, affecting their activities and significantly contributing to cellular processes ranging from survival to cellular differentiation. Given the pleiotropic functions of O-GlcNAc modification, it has been studied in various fields; however, the role of O-GlcNAcylation during osteoclast differentiation remains to be explored. Kinetic transcriptome analysis during receptor activator of nuclear factor-kappaB (NF-κB) ligand (RANKL)-mediated osteoclast differentiation revealed that the nexus of major nutrient metabolism, HBP was critical for this process. We observed that the critical genes related to HBP activation, including Nagk, Gfpt1, and Ogt, were upregulated, while the global O-GlcNAcylation was increased concomitantly during osteoclast differentiation. The O-GlcNAcylation inhibition by the small-molecule inhibitor OSMI-1 reduced osteoclast differentiation in vitro and in vivo by disrupting the translocation of NF-κB p65 and nuclear factor of activated T cells c1 (NFATc1) into the nucleus by controlling their PTM O-GlcNAcylation. Furthermore, OSMI-1 had a synergistic effect with bone target therapy on osteoclastogenesis. Lastly, knocking down Ogt with shRNA (shOgt) mimicked OSMI-1’s effect on osteoclastogenesis. Targeting O-GlcNAcylation during osteoclast differentiation may be a valuable therapeutic approach for osteoclast-activated bone diseases. Full article

An enzyme of the mammalian amino-sugar metabolism pathway, N-acetylglucosamine kinase (NAGK), that synthesizes N-acetylglucosamine (GlcNAc)-6-phosphate, is reported to promote dynein functions during mitosis, axonal and dendritic growth, cell migration, and selective autophagy, which all are unrelated to its enzyme activity. As non-enzymatic structural functions can be altered by genetic variation, we made an effort in this study aimed at deciphering the pathological effect of nonsynonymous single-nucleotide polymorphisms (nsSNPs) in NAGK gene. An integrated computational approach, including molecular dynamics (MD) simulation and protein–protein docking simulation, was used to identify the damaging nsSNPs and their detailed structural and functional consequences. The analysis revealed the four most damaging variants (G11R, G32R, G120E, and A156D), which are highly conserved and functional, positioned in both small (G11R and G32R) and large (G120E and A156D) domains of NAGK. G11R is located in the ATP binding region, while variants present in the large domain (G120E and A156D) were found to induce substantial alterations in the structural organizations of both domains, including the ATP and substrate binding sites. Furthermore, all variants were found to reduce binding energy between NAGK and dynein subunit DYNLRB1, as revealed by protein–protein docking and MM-GBSA binding energy calculation supporting their deleteriousness on non-canonical function. We hope these findings will direct future studies to gain more insight into the role of these variants in the loss of NAGK function and their role in neurodevelopmental disorders. Full article

Recently, we showed that N-acetylglucosamine kinase (NAGK), an enzyme of amino sugar metabolism, interacts with dynein light chain roadblock type 1 (DYNLRB1) and promotes the functions of dynein motor. Here, we report that NAGK interacts with nuclear distribution protein C (NudC) and lissencephaly 1 (Lis1) in the dynein complex. Yeast two-hybrid assays, pull-down assays, immunocytochemistry, and proximity ligation assays revealed NAGK–NudC–Lis1–dynein complexes around nuclei, at the leading poles of migrating HEK293T cells, and at the tips of migratory processes of cultured rat neuroblast cells. The exogenous expression of red fluorescent protein (RFP)-tagged NAGK accelerated HEK293T cell migration during in vitro wound-healing assays and of neurons during in vitro neurosphere migration and in utero electroporation assays, whereas NAGK knockdown by short hairpin RNA (shRNA) delayed migration. Finally, a small NAGK peptide derived from the NudC interacting domain in in silico molecular docking analysis retarded the migrations of HEK293T and SH-SY5Y cells. These data indicate a functional interaction between NAGK and dynein–NudC–Lis1 complex at the nuclear envelope is required for the regulation of cell migration. Full article

Glyphodes pyloalis Walker (Lepidoptera: Pyralididae) is a serious pest in the sericulture industry, which has caused damage and losses in recent years. With the widespread use of insecticides, the insecticide resistance of G. pyloalis has becomes increasingly apparent. In order to find other effective methods to control G. pyloalis, this study performed a transcriptome analysis of the midgut, integument, and whole larvae. Transcriptome data were annotated with KEGG and GO, and they have been shown to be of high quality by RT-qPCR. The different significant categories of differentially expressed genes between the midgut and the integument suggested that the transcriptome data could be used for next analysis. With the exception of Dda9 (GpCDA5), 19 genes were involved in chitin metabolism, most of which had close protein–protein interactions. Among them, the expression levels of 11 genes, including GpCHSA, GpCDA1, GpCDA2, GpCDA4, GPCHT1, GPCHT2a, GPCHT3a, GPCHT7, GpTre1, GpTre2, and GpRtv were higher in the integument than in the midgut, while the expression levels of the last eight genes, including GpCHSB, GpCDA5, GpCHT2b, GpCHT3b, GpCHT-h, GpPAGM, GpNAGK, and GpUAP, were higher in the midgut than in the integument. Moreover, 282 detoxification-related genes were identified and can be divided into 10 categories, including cytochrome P450, glutathione S-transferase, carboxylesterase, nicotinic acetylcholine receptor, aquaporin, chloride channel, methoprene-tolerant, serine protease inhibitor, sodium channel, and calcium channel. In order to further study the function of chitin metabolism-related genes, dsRNA injection knocked down the expression of GpCDA1 and GpCHT3a, resulting in the significant downregulation of its downstream genes. These results provide an overview of chitin metabolism and detoxification of G. pyloalis and lay the foundation for the effective control of this pest in the sericulture industry. Full article

N-acetylglutamate synthase (NAGS) catalyzes the production of N-acetylglutamate (NAG) from acetyl-CoA and l-glutamate. In microorganisms and plants, the enzyme functions in the arginine biosynthetic pathway, while in mammals, its major role is to produce the essential co-factor of carbamoyl phosphate synthetase 1 (CPS1) in the urea cycle. Recent work has shown that several different genes encode enzymes that can catalyze NAG formation. A bifunctional enzyme was identified in certain bacteria, which catalyzes both NAGS and N-acetylglutamate kinase (NAGK) activities, the first two steps of the arginine biosynthetic pathway. Interestingly, these bifunctional enzymes have higher sequence similarity to vertebrate NAGS than those of the classical (mono-functional) bacterial NAGS. Solving the structures for both classical bacterial NAGS and bifunctional vertebrate-like NAGS/K has advanced our insight into the regulation and catalytic mechanisms of NAGS, and the evolutionary relationship between the two NAGS groups. Full article