NIMA-related Kinase Research Articles

Inhibition of the YAP-MMB interaction and targeting NEK2 as potential therapeutic strategies for YAP-driven cancers.

YAP activation in cancer is linked to poor outcomes, making it an attractive therapeutic target. Previous research focused on blocking the interaction of YAP with TEAD transcription factors. Here, we took a different approach by disrupting YAP's binding to the transcription factor B-MYB using MY-COMP, a fragment of B-MYB containing the YAP binding domain fused to a nuclear localization signal. MY-COMP induced cell cycle defects, nuclear abnormalities, and polyploidization. In an AKT and YAP-driven liver cancer model, MY-COMP significantly reduced liver tumorigenesis, highlighting the importance of the YAP-B-MYB interaction in tumor development. MY-COMP also perturbed the cell cycle progression of YAP-dependent uveal melanoma cells but not of YAP-independent cutaneous melanoma cell lines. It counteracted YAP-dependent expression of MMB-regulated cell cycle genes, explaining the observed effects. We also identified NIMA-related kinase (NEK2) as a downstream target of YAP and B-MYB, promoting YAP-driven transformation by facilitating centrosome clustering and inhibiting multipolar mitosis.

Osm-5p- driven fluorophores are differentially expressed in ccpp-1Δ and nekl-4Δ mutant ciliated neurons.

Intraflagellar transport (IFT) involves the coordinated transport of molecular motors and other proteins and is required for ciliogenesis and ciliary maintenance. The C. elegans IFT protein OSM-5 /IFT88 is expressed in a majority of the ciliated neurons in the animal, and osm-5 mutants exhibit structurally defective cilia. The osm-5 promoter is commonly used to express genetic constructs in the ciliated neurons. In this study, we show that brightness of osm-5p- driven constructs is altered in mutants of the tubulin deglutamylase ccpp-1 and the NIMA-related kinase nekl-4 . This raises the possibility that osm-5 expression levels may be regulated by ccpp-1 and nekl-4 .

Quercetin-induced pyroptosis in colon cancer through NEK7-mediated NLRP3 inflammasome-GSDMD signaling pathway activation.

Pyroptosis, a gasdermin-mediated lytic cell death, is a new hotspot topic in cancer research, and induction of tumor pyroptosis has emerged as a new target in cancer management. Quercetin (Que), a natural substance, demonstrates promising anticancer action. However, further information is required to fully comprehend the function and mechanism of Que in pyroptosis in colon cancer. This study revealed the underlying mechanism of Que-induced pyroptosis in colon cancer in vitro and in vivo. Que inhibited colon cancer cell growth through gasdermin D (GSDMD)-mediated pyroptosis. Depletion of GSDMD, rather than gasdermin E (GSDME), reversed the cytotoxic effects of Que on colon cancer cells. Que treatment upregulated NIMA-related kinase 7 (NEK7) protein expression, thus facilitating the assembly of the NLRP3 inflammasome and cleavage of GSDMD. NEK7 silencing resulted in colon cancer cell growth in vitro and in vivo. Mechanistically, NEK7 depression restrained the activation of the NLRP3 inflammasome-GSDMD pathway, thus attenuating pyroptosis triggered by Que in colon cancer cells. Furthermore, lower NEK7 and NLRP3 expression levels indicated colon cancer progression. Our results unveiled a novel pattern of anti-colon cancer activity of Que, and activation of NEK7-mediated pyroptosis is potentially a promising therapeutic target for colon cancer, which provides novel experimental proof for the clinical application of Que.

Computational insights into NIMA-related kinase 6: unraveling mutational effects on structure and function.

The NEK6 (NIMA-related kinase 6) serine/threonine kinase is a pivotal player in a multitude of cellular processes, including the regulation of the cell cycle and the response to DNA damage. Its significance extends to disease pathogenesis, as changes in NEK6 activity have been linked to the development of cancer. Non-synonymous single nucleotide polymorphisms (nsSNPs) in NEK6 have been linked to cancer as they alter the protein's native structure and function. The association between NEK6 activity and cancer development has prompted researchers to explore the effects of genetic variations within the NEK6 gene. Therefore, we utilized advanced computational tools to analyze 155 high-confidence nsSNPs in the NEK6 gene. From this analysis, 21 nsSNPs were identified as potentially harmful, raising concerns about their impact on NEK6 activity and cancer risk. These 21 mutations were then examined for structural alterations, and eight of nsSNPs (I51M, V76A, I134N, Y152D, R171Q, V186G, L237R, and C285S) were found to destabilize the protein. Among the destabilizing mutations screened, a specific mutation, R171Q, stood out due to its conserved nature. To understand its impact on the protein and conformation, all-atom molecular dynamics simulations (MDS) for 100ns were performed for both Wildtype NEK6 (WT-NEK6) and R171Q. The simulations revealed that the R171Q variant was unstable and led to significant conformational changes in NEK6. This study provides valuable insights into NEK6 dysfunction caused by single amino acid alterations, offering a novel understanding of the molecular mechanisms underlying NEK6-related cancer progression.

CircNEK6 promotes the progression of pancreatic ductal adenocarcinoma through targeting miR-503/CCND1 axis

PurposeThe present study aimed to reveal the function and underlying molecular mechanism of circRNA NIMA related kinase 6 (circNEK6) in promoting the progression of pancreatic ductal adenocarcinoma (PDAC). MethodsThe differentially expressed circRNAs in three paired PDAC tissues and adjacent tissues were identified by RNA sequencing. CircNEK6 was screened out to further explore its relationship with the prognosis of PDAC patients. The target microRNAs and mRNAs of circNEK6 were analyzed through online databases and detected by quantitative real-time polymerase chain reaction. Cell counting kit-8 assay, clone formation assay, transwell assay, flow cytometry and western blot were used to explore the function of circNEK6 on the biological behaviors of PDAC cells. The in vivo antitumor effect of circNEK6 silencing on PDAC was investigated by nude mouse xenograft models. Results203 differentially expressed circRNAs including circNEK6 were identified between paired PDAC tissues and adjacent tissues, and the expression level of circNEK6 was negatively correlated with the prognosis of PDAC patients. The results of in vitro experiments showed that knockdown of circNEK6 repressed the proliferation, migration and invasion, but induced the apoptosis of PDAC cells. Moreover, circNEK6 silencing inhibited tumor growth and prolonged the survival time of PDAC-bearing mice. Mechanistically, miR-503/cyclin D1 (CCND1) axis was predicted and confirmed as the target of circNEK6. ConclusionsCircNEK6 serves as a competing endogenous RNA of CCND1 by absorbing miR-503, which might be treated as a novel and potential target for PDAC treatment.

Non-classical functions of nuclear pore proteins in ciliopathy.

Nucleoporins (NUPs) constitute integral nuclear pore protein (NPC) elements. Although traditional NUP functions have been extensively researched, evidence of additional vital non-NPC roles, referred to herein as non-classical NUP functions, is also emerging. Several NUPs localise at the ciliary base. Indeed, Nup188, Nup93 or Nup205 knockdown results in cilia loss, impacting cardiac left-right patterning in models and cell lines. Genetic variants of Nup205 and Nup188 have been identified in patients with congenital heart disease and situs inversus totalis or heterotaxy, a prevalent human ciliopathy. These findings link non-classical NUP functions to human diseases. This mini-review summarises pivotal NUP interactions with NIMA-related kinases or nephronophthisis proteins that regulate ciliary function and explores other NUPs potentially implicated in cilia-related disorders. Overall, elucidating the non-classical roles of NUPs will enhance comprehension of ciliopathy aetiology.

Caspase 6 promotes innate immune activation by functional crosstalk between RIPK1-IκBα axis in liver inflammation

BackgroundCaspase 6 is an essential regulator in innate immunity, inflammasome activation and host defense. We aimed to characterize the causal mechanism of Caspase 6 in liver sterile inflammatory injury.MethodsHuman liver tissues were harvested from patients undergoing ischemia-related hepatectomy to evaluate Caspase 6 expression. Subsequently, we created Caspase 6-knockout (Caspase 6KO) mice to analyze roles and molecular mechanisms of macrophage Caspase 6 in murine models of liver ischemia/reperfusion (IR) injury.ResultsIn human liver biopsies, Caspase 6 expression was positively correlated with more severe histopathological injury and higher serum ALT/AST level at one day postoperatively. Moreover, Caspase 6 was mainly elevated in macrophages but not hepatocytes in ischemic livers. Unlike in controls, the Caspase 6-deficient livers were protected against IR injury, as evidenced by inhibition of inflammation, oxidative stress and iron overload. Disruption of macrophage NF-κB essential modulator (NEMO) in Caspase 6-deficient livers deteriorated liver inflammation and ferroptosis. Mechanistically, Caspase 6 deficiency spurred NEMO-mediated IκBα phosphorylation in macrophage. Then phosphorylated-inhibitor of NF-κBα (p-IκBα) co-localized with receptor-interacting serine/ threonine-protein kinase 1 (RIPK1) in the cytoplasm to degradate RIPK1 under inflammatory conditions. The disruption of RIPK1-IκBα interaction preserved RIPK1 degradation, triggering downstream apoptosis signal-regulating kinase 1 (ASK1) phosphorylation and inciting NIMA-related kinase 7/NOD-like receptor family pyrin domain containing 3 (NEK7/NLRP3) activation in macrophages. Moreover, ablation of macrophage RIPK1 or ASK1 diminished NEK7/NLRP3-driven inflammatory response and dampened hepatocyte ferroptosis by reducing HMGB1 release from macrophages.ConclusionsOur findings underscore a novel mechanism of Caspase 6 mediated RIPK1-IκBα interaction in regulating macrophage NEK7/NLRP3 function and hepatocytes ferroptosis, which provides therapeutic targets for clinical liver IR injury.Graphical DRFRodggEVQV_MBi1PPYnNVideo

METTL3 Regulates the m6A Modification of NEK7 to Inhibit the Formation of Osteoarthritis.

Osteoarthritis (OA) is a common degenerative joint disease. The occurrence of OA slowly destroys the soft tissue structure of the patient's joint. Severe cases could lead to disability. Current studies had shown that inhibition of chondrocytes pyroptosis could slow down the progression of OA. Our work aimed to explore the specific mechanisms and ways of regulating this process. In this work, the level of N6-methyladenosine (m6A) in clinical tissues was detected by ribonucleic acid (RNA) m6A dot blot. qRT-PCR (quantitative real-time polymerase chain reaction) was used to detect the messenger RNA (mRNA) expression level of m6A modified enzyme in clinical tissues. MTT (3-(4,5)-dimethylthiahiazo(-z-y1)-3,5-di-phenytetrazoliumromid) and flow cytometry were used to detect the effect of sh-METTL3 (methyltransferase like 3) and NIMA-related kinase 7 (NEK7) transfection on chondrocytes pyroptosis in OA. Western blot was used to detect the protein expression levels of pyroptosis-related proteins. ELISA (enzyme-linked immunosorbent assay) was used to measure the protein concentration of inflammatory cytokines. The SRAMP online database was used to predict the m6A site of NEK7. HE staining was used to assess the progression of OA in mice. The level of m6A in clinical samples of OA patients was higher, and METTL3 was significantly higher expressed in clinical samples of OA patients. We provided evidence that low expression of METTL3 inhibited chondrocytes pyroptosis. In addition, Rescue experiments and in vivo experiments had shown that METTL3 in combination with NEK7 inhibited the progression of OA by promoting chondrocytes pyroptosis. METTL3 regulates m6A modification of NEK7 and inhibits OA progression.

Loss of function of the ALS-associated NEK1 kinase disrupts microtubule homeostasis and nuclear import.

Loss-of-function variants in NIMA-related kinase 1 (NEK1) constitute a major genetic cause of amyotrophic lateral sclerosis (ALS), accounting for 2 to 3% of all cases. However, how NEK1 mutations cause motor neuron (MN) dysfunction is unknown. Using mass spectrometry analyses for NEK1 interactors and NEK1-dependent expression changes, we find functional enrichment for proteins involved in the microtubule cytoskeleton and nucleocytoplasmic transport. We show that α-tubulin and importin-β1, two key proteins involved in these processes, are phosphorylated by NEK1 in vitro. NEK1 is essential for motor control and survival in Drosophila models in vivo, while using several induced pluripotent stem cell (iPSC)-MN models, including NEK1 knockdown, kinase inhibition, and a patient mutation, we find evidence for disruptions in microtubule homeostasis and nuclear import. Notably, stabilizing microtubules with two distinct classes of drugs restored NEK1-dependent deficits in both pathways. The capacity of NEK1 to modulate these processes that are critically involved in ALS pathophysiology renders this kinase a formidable therapeutic candidate.

Aurora A polyubiquitinates the BRCA1-interacting protein OLA1 to promote centrosome maturation

The BRCA1-interacting protein Obg-like ATPase 1 (OLA1) functions in centriole duplication. In this study, we show the role of the mitotic kinase Aurora A in the reduction of centrosomal OLA1. Aurora A binds to and polyubiquitinates OLA1, targeting it for proteasomal degradation. NIMA-related kinase 2 (NEK2) phosphorylates the T124 residue of OLA1, increases binding of OLA1 to Aurora A and OLA1 polyubiquitination by Aurora A, and reduces centrosomal OLA1 in G2 phase. The kinase activity of Aurora A suppresses OLA1 polyubiquitination. The decrease in centrosomal OLA1 caused by Aurora A-mediated polyubiquitination promotes the recruitment of pericentriolar material proteins in G2 phase. The E3 ligase activity of Aurora A is critical for centrosome amplification induced by its overexpression. The results suggest a dual function of Aurora A as an E3 ubiquitin ligase and a kinase in the regulation of centrosomal OLA1, which is essential for proper centrosome maturation in G2 phase.

Transcriptional pausing induced by ionizing radiation enables the acquisition of radioresistance in nasopharyngeal carcinoma.

Lesions on the DNA template can impact transcription via distinct regulatory pathways. Ionizing radiation (IR) as the mainstay modality for many malignancies elicits most of the cytotoxicity by inducing a variety of DNA damages in the genome. How the IR treatment alters the transcription cycle and whether it contributes to the development of radioresistance remain poorly understood. Here, we report an increase in the paused RNA polymerase II (RNAPII), as indicated by the phosphorylation at serine 5 residue of its C-terminal domain, in recurrent nasopharyngeal carcinoma (NPC) patient samples after IR treatment and cultured NPC cells developing IR resistance. Reducing the pool of paused RNAPII by either inhibiting TFIIH-associated CDK7 or stimulating the positive transcription elongation factor b, a CDK9-CycT1 heterodimer, attenuates IR resistance of NPC cells. Interestingly, the poly(ADP-ribosyl)ation of CycT1, which disrupts its phase separation, is elevated in the IR-resistant cells. Mutation of the major poly(ADP-ribosyl)ation sites of CycT1 decreases RNAPII pausing and restores IR sensitivity. Genome-wide chromatin immunoprecipitation followed by sequencing analyses reveal that several genes involved in radiation response and cell cycle control are subject to the regulation imposed by the paused RNAPII. Particularly, we identify the NIMA-related kinase NEK7 under such regulation as a new radioresistance factor, whose downregulation results in the increased chromosome instability, enabling the development of IR resistance. Overall, our results highlight a novel link between the alteration in the transcription cycle and the acquisition of IR resistance, opening up new opportunities to increase the efficacy of radiotherapy and thwart radioresistance in NPC.

Screening of novel biomarkers for breast cancer based on WGCNA and multiple machine learning algorithms.

Breast cancer (BC) ranks first in incidence among women, with approximately 2 million new cases per year. Therefore, it is essential to investigate emerging targets for BC patients' diagnosis and prognosis. We analyzed gene expression data from 99 normal and 1,081 BC tissues in The Cancer Genome Atlas (TCGA) database. Differentially expressed genes (DEGs) were identified using "limma" R package, and relevant modules were chosen through Weighted Gene Coexpression Network Analysis (WGCNA). Intersection genes were obtained by matching DEGs to WGCNA module genes. Functional enrichment studies were performed on these genes using Gene Ontology (GO), Disease Ontology (DO), and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases. Biomarkers were screened via Protein-Protein Interaction (PPI) networks and multiple machine-learning algorithms. The Gene Expression Profiling Interactive Analysis (GEPIA), The University of ALabama at Birmingham CANcer (UALCAN), and Human Protein Atlas (HPA) databases were employed to examine mRNA and protein expression of eight biomarkers. Kaplan-Meier mapper tool assessed their prognostic capabilities. Key biomarkers were analyzed via single-cell sequencing, and their relationship with immune infiltration was examined using Tumor Immune Estimation Resource (TIMER) database and "xCell" R package. Lastly, drug prediction was conducted based on the identified biomarkers. We identified 1,673 DEGs and 542 important genes through differential analysis and WGCNA, respectively. Intersection analysis revealed 76 genes, which play significant roles in immune-related viral infection and IL-17 signaling pathways. DIX domain containing 1 (DIXDC1), Dual specificity phosphatase 6 (DUSP6), Pyruvate dehydrogenase kinase 4 (PDK4), C-X-C motif chemokine ligand 12 (CXCL12), Interferon regulatory factor 7 (IRF7), Integrin subunit alpha 7 (ITGA7), NIMA related kinase 2 (NEK2), and Nuclear receptor subfamily 3 group C member 1 (NR3C1) were selected as BC biomarkers using machine-learning algorithms. NEK2 was the most critical gene for diagnosis. Prospective drugs targeting NEK2 include etoposide and lukasunone. Our study identified DIXDC1, DUSP6, PDK4, CXCL12, IRF7, ITGA7, NEK2, and NR3C1 as potential diagnostic biomarkers for BC, with NEK2 having the highest potential to aid in diagnosis and prognosis in clinical settings.

NIMA-related kinase 6 as an effective target inhibits the hepatocarcinogenesis and progression of hepatocellular carcinoma

BackgroundNIMA-related kinase 6 (NEK 6) is over-expressed in some tumor cell lines and tissues. However, its expression in hepatocellular carcinoma (HCC) and its correlation with clinical features remain unclear. MethodsTotal RNA from HCC liver tissues, other liver specimens, and hepatic cell lines was extracted and QPCR was adopted to detect NEK6 expression. The correlation between NEK6 expression and the clinical characteristics of HCC was analyzed. Scratch assay, Transwell assay, and tumor-formation assay were used to evaluate the effects of NEK6 on the HCC progression in vitro and in vivo. ResultsThe expression of NEK6 was up-regulated in HCC tissues and HCC cell lines: Li-7 and HepG2. The overexpression of NEK6 was correlated with hepatitis B virus infection and tumor diameter (P = 0.045). When down-regulated the expression of NEK6, both the migration and invasion capabilities of Li-7 and HepG2 cells and the growth of xenograft tumors were suppressed. (P < 0.05). ConclusionsNEK6 expression was up-regulated in HCC and correlated with the progression, suggesting it might be a valuable biomarker and a potential therapeutic target for HCC.

Total tanshinones protect against acute lung injury through the PLCγ2/NLRP3 inflammasome signaling pathway

Ethnopharmacological relevanceSalvia miltiorrhiza Bunge is a widely used traditional Chinese medicine with anticholinesterase, antitumor, and anti-inflammatory. Total Tanshinones (TTN), the most significant active ingredient of Salvia miltiorrhiza Bunge, exerts anti-inflammatory activity. However, the protective mechanism of total Tanshinones on acute lung injury (ALI) still needs to be explored. Aim of this studyIn this study, the underlying mechanisms of TTN to treat with ALI were investigated in vitro and in vivo. Materials and methodsCell experiments established an in vitro model of LPS-induced J774A.1 and MH-S macrophages to verify the mechanism. The levels of inflammatory cytokines (TNF-α, IL-6 and IL-1β) were estimated by ELISA. The changes of ROS, Ca2+ and NO were detected by flow cytometry. The expression levels of proteins related to the NLRP3 inflammasome were determined by Western blotting. The effect of TTN on NLRP3 inflammasome activation was examined by immunofluorescence analysis of caspase-1 p20. Male BALB/c mice were selected to establish the ALI model. The experiment was randomly divided into six groups: control, LPS, LPS + si-NC, LPA + si-Nek7, LPS + TTN, and DEX. Pathological alterations were explored by H&E staining. The expression levels of proteins related to the NLRP3 inflammasome were analyzed by Western blotting. ResultsTTN decreased pro-inflammatory cytokines levels like TNF-α, IL-6, IL-1β, NO, and ROS in alveolar macrophages. TTN bound to NIMA-related kinase 7 (NEK7), a new therapeutic protein to modulate NLRP3 inflammasome and PLCγ2-PIP2 signaling pathway. In ALI mice, LPS enhanced IL-1β levels in the serum, lung tissues, and bronchoalveolar lavage fluid (BALF),which were reversed by TTN. TTN decreased cleaved-caspase-1 and NLRP3 expressions in lung tissues. When Nek7 was knocked down in mice by siRNA, the syndrome of ALI in mice was significantly suppressed, of which the effect was similar to that of TTN. ConclusionsThis research demonstrates that TTN alleviated ALI by binding to NEK7 in vitro and in vivo to modulate NLRP3 inflammasome activation and PLCγ2-PIP2 signaling pathways.

Conserved NIMA kinases regulate multiple steps of endocytic trafficking.

Human NIMA-related kinases have primarily been studied for their roles in cell cycle progression (NEK1/2/6/7/9), checkpoint-DNA-damage control (NEK1/2/4/5/10/11), and ciliogenesis (NEK1/4/8). We previously showed that Caenorhabditis elegans NEKL-2 (NEK8/9 homolog) and NEKL-3 (NEK6/7 homolog) regulate apical clathrin-mediated endocytosis (CME) in the worm epidermis and are essential for molting. Here we show that NEKL-2 and NEKL-3 also have distinct roles in controlling endosome function and morphology. Specifically, loss of NEKL-2 led to enlarged early endosomes with long tubular extensions but showed minimal effects on other compartments. In contrast, NEKL-3 depletion caused pronounced defects in early, late, and recycling endosomes. Consistently, NEKL-2 was strongly localized to early endosomes, whereas NEKL-3 was localized to multiple endosomal compartments. Loss of NEKLs also led to variable defects in the recycling of two resident cargoes of the trans-Golgi network (TGN), MIG-14/Wntless and TGN-38/TGN38, which were missorted to lysosomes after NEKL depletion. In addition, defects were observed in the uptake of clathrin-dependent (SMA-6/Type I BMP receptor) and independent cargoes (DAF-4/Type II BMP receptor) from the basolateral surface of epidermal cells after NEKL-2 or NEKL-3 depletion. Complementary studies in human cell lines further showed that siRNA knockdown of the NEKL-3 orthologs NEK6 and NEK7 led to missorting of the mannose 6-phosphate receptor from endosomes. Moreover, in multiple human cell types, depletion of NEK6 or NEK7 disrupted both early and recycling endosomal compartments, including the presence of excess tubulation within recycling endosomes, a defect also observed after NEKL-3 depletion in worms. Thus, NIMA family kinases carry out multiple functions during endocytosis in both worms and humans, consistent with our previous observation that human NEKL-3 orthologs can rescue molting and trafficking defects in C. elegans nekl-3 mutants. Our findings suggest that trafficking defects could underlie some of the proposed roles for NEK kinases in human disease.

Abstract 878: In silico insights from NEK10 modeling, its interactome, and protein-protein interactions

Abstract Cancer is the second-leading cause of death worldwide and by 2040, it is projected that the global burden is expected to grow to 27.5 million new cases. Proteins that directly regulate cell cycle progression are attractive targets in cancer therapy since aberrant regulation of the cell cycle, especially at checkpoints, allows the cells to accumulate DNA errors and continue to grow/divide. A subset of protein kinases functionally involved in regulating the cell cycle and its checkpoints are the eleven members of the human NIMA-related kinases (NEKs). NEK10 is the most divergent member of the NEK family and is linked to melanoma, breast cancer, and various ciliopathies. It is unique in having a centrally positioned coiled-coil flanked catalytic domain, in contrast to the N-terminal catalytic domains of other NEKs. Despite its unique domain architecture and biological relevance, very little is known about its complete structural information and structure-function relationships. To address this gap in knowledge we have modeled and characterized the full-length NEK10 protein, including its unique catalytic domain flanked by two coiled-coil domains, a region with armadillo-type repeats, an ATP binding site, and two unreported putative sites of interaction with ubiquitin (UBA domains). Additionally, we present a comprehensive interactome of NEK10. From this interactome we selected 2 unique and previously unreported NEK10 interactions with the known cancer-causing proteins, Mitogen-activated protein kinase kinase kinase 1 (MAP3K1) and Heat Shock Factor Binding Protein 1 (HSPB1). MAP3K1, a serine/threonine kinase and ubiquitin ligase that plays a pivotal role in integrating cellular responses to mitogenic/metabolic stimuli and mutated in a significant number of cancers, most prominently in luminal breast cancer, contains a protein kinase domain, a PHD finger with E3 ubiquitin ligase activity, and scaffold regions that mediate protein-protein interactions. Our docking analysis shows that I693 of NEK10 located within its kinase domain interacts with well-known phosphosite S275 of MAP3K1. We speculate a scenario in which, upon UV irradiation, NEK10 phosphorylates and activates MAP3K1, which in turn phosphorylates MAP2K1/2-ERK1/2 promoting cell survival. HSPB1, is a molecular chaperone, characterized by a common alpha-crystallin domain in its C-terminus and its overexpression is linked with poor clinical outcome in gastric, uterine, breast, prostate, ovarian, kidney and head/neck cancers. Our docking analysis suggests that the distinctive armadillo repeats of NEK10 may be responsible for its interaction with HSPB1 where H301 of NEK10 forms a salt bridge with I179 of HSPB1, within a known protein-protein hotspot of HSPB1. Our results establish the framework for elucidating the detailed molecular mechanisms of NEK10 interactions with other proteins to further explore its potential as a therapeutic target. Citation Format: Andriele Silva Eichner, Igor Semchenkov Pastukhov, Shaneen Singh. In silico insights from NEK10 modeling, its interactome, and protein-protein interactions [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 878.

Phosphate NIMA-Related Kinase 2-Dependent Epigenetic Pathways in Dorsal Root Ganglion Neurons Mediates Paclitaxel-Induced Neuropathic Pain.

The microtubule-stabilizing drug paclitaxel (PTX) is an important chemotherapeutic agent for cancer treatment and causes peripheral neuropathy as a common side effect that substantially impacts the functional status and quality of life of patients. The mechanistic role for NIMA-related kinase 2 (NEK2) in the progression of PTX-induced neuropathic pain has not been established. Adult male Sprague-Dawley rats intraperitoneally received PTX to induce neuropathic pain. The protein expression levels in the dorsal root ganglion (DRG) of animals were measured by biochemical analyses. Nociceptive behaviors were evaluated by von Frey tests and hot plate tests. PTX increased phosphorylation of the important microtubule dynamics regulator NEK2 in DRG neurons and induced profound neuropathic allodynia. PTX-activated phosphorylated NEK2 (pNEK2) increased jumonji domain-containing 3 (JMJD3) protein, a histone demethylase protein, to specifically catalyze the demethylation of the repressive histone mark H3 lysine 27 trimethylation (H3K27me3) at the Trpv1 gene, thereby enhancing transient receptor potential vanilloid subtype-1 (TRPV1) expression in DRG neurons. Moreover, the pNEK2-dependent PTX response program is regulated by enhancing p90 ribosomal S6 kinase 2 (RSK2) phosphorylation. Conversely, intrathecal injections of kaempferol (a selective RSK2 activation antagonist), NCL 00017509 (a selective NEK2 inhibitor), NEK2-targeted siRNA, GSK-J4 (a selective JMJD3 inhibitor), or capsazepine (an antagonist of TRPV1 receptor) into PTX-treated rats reversed neuropathic allodynia and restored silencing of the Trpv1 gene, suggesting the hierarchy and interaction among phosphorylated RSK2 (pRSK2), pNEK2, JMJD3, H3K27me3, and TRPV1 in the DRG neurons in PTX-induced neuropathic pain. pRSK2/JMJD3/H3K27me3/TRPV1 signaling in the DRG neurons plays as a key regulator for PTX therapeutic approaches.

Structural Mechanisms of NLRP3 Inflammasome Assembly and Activation.

As an important sensor in the innate immune system, NLRP3 detects exogenous pathogenic invasions and endogenous cellular damage and responds by forming the NLRP3 inflammasome, a supramolecular complex that activates caspase-1. The three major components of the NLRP3 inflammasome are NLRP3, which captures the danger signals and recruits downstream molecules; caspase-1, which elicits maturation of the cytokines IL-1β and IL-18 and processing of gasdermin D to mediate cytokine release and pyroptosis; and ASC (apoptosis-associated speck-like protein containing a caspase recruitment domain), which functions as a bridge connecting NLRP3 and caspase-1. In this article, we review the structural information that has been obtained on the NLRP3 inflammasome and its components or subcomplexes, with special focus on the inactive NLRP3 cage, the active NLRP3-NEK7 (NIMA-related kinase 7)-ASC inflammasome disk, and the PYD-PYD and CARD-CARD homotypic filamentous scaffolds of the inflammasome. We further implicate structure-derived mechanisms for the assembly and activation of the NLRP3 inflammasome.

Phosphorylation of the prolyl isomerase Cyclophilin A regulates its localisation and release from the centrosome during mitosis

ABSTRACT The centrosome acts as a protein platform from which proteins are deployed to function throughout the cell cycle. Previously, we have shown that the prolyl isomerase Cyclophilin A (CypA) localizes to the centrosome in interphase and re-localizes to the midbody during mitosis where it functions in cytokinesis. In this study, investigation of CypA by SDS-PAGE during the cell cycle reveals that it undergoes a mobility shift during mitosis, indicative of a post-translational modification, which may correlate with its subcellular re-localization. Due to the lack of a phospho-specific antibody, we used site-directed mutagenesis to demonstrate that the previously identified serine 77 phosphorylation site within CypA is important for control of CypA centrosome localization. Furthermore, CypA is shown to interact with the mitotic NIMA-related kinase 2 (Nek2) during interphase and mitosis, while also interacting with the Nek2-antagonist PP1 during interphase but not during mitosis, suggesting a potential role for the Nek2-PP1 complex in CypA phospho-regulation. In support of this, Nek2 is capable of phosphorylating CypA in vitro. Overall, this work reveals that phosphorylation of CypA at serine 77 is important for its release from the centrosome during mitosis and may be regulated by the activity of Nek2 and PP1 during the cell cycle.

Synthesis, biological evaluation and in silico investigations of benzotriazole derivatives as potential inhibitors of NIMA related kinase.

In the current study, a novel compound, bis(3-(2H-benzo[d][1,2,3]triazol-2-yl)-2-(prop-2-yn-1-yloxy)-5-(2,4,4-trimethylpentan-2-yl)phenyl)methane (TAJ1), has been synthesized by the reaction of 6,6'-methylenebis(2-(2H-benzo[d][1,2,3]triazol-2-yl)-4-(2,4,4-trimethylpentan-2-yl)phenol) (1), propargyl bromide (2) and potassium carbonate. Spectroscopic (FTIR, 1H-NMR, 13C-NMR) and single-crystal assays proved the structure of the synthesized sample. XRD analysis confirmed the structure of the synthesized compound, showing that it possesses two aromatic parts linked via a -CH2 carbon with a bond angle of 108.40°. The cell line activity reported a percent growth reduction for different cell types (HeLa cells, MCF-7 cells, and Vero cells) under various treatment conditions (TAJ1, cisplatin, and doxorubicin) after 24 hours and 48 hours. The percent growth reduction represents a decrease in cell growth compared to a control condition. Furthermore, density functional theory (DFT) calculations were utilized to examine the frontier molecular orbitals (FMOs) and overall chemical reactivity descriptors of TAJ1. The molecule's chemical reactivity and stability were assessed by determining the HOMO-LUMO energy gap. TAJ1 displayed a HOMO energy level of -0.224 eV, a LUMO energy level of -0.065 eV, and a HOMO-LUMO gap of 0.159 eV. Additionally, molecular docking analysis was performed to assess the binding affinities of TAJ1 with various proteins. The compound TAJ1 showed potent interactions with NEK2, exhibiting -10.5 kcal mol-1 binding energy. Although TAJ1 has demonstrated interactions with NEK7, NEK9, TP53, NF-KAPPA-B, and caspase-3 proteins, suggesting its potential as a therapeutic agent, it is important to evaluate the conformational stability of the protein-ligand complex. Hence, molecular dynamics simulations were conducted to assess this stability. To analyze the complex, root mean square deviation (RMSD) and root mean square fluctuation analyses were performed. The results of these analyses indicate that the top hits obtained from the virtual screening possess the ability to act as effective NEK2 inhibitors. Therefore, further investigation of the inhibitory potential of these identified compounds using in vitro and in vivo approaches is recommended.