Search Results (104)

Background: Personalized anti-tumor therapy that activates the immune response has demonstrated clinical benefits in various cancers. However, its efficacy against testicular cancer (TC) remains uncertain. This study aims to identify suitable patients for anti-tumor immunotherapy and to uncover potential therapeutic targets in TC for the development of tailored anti-tumor immunotherapy. Methods: Consensus clustering analysis was conducted to delineate immune subtypes, while weighted gene co-expression network analysis (WGCNA), least absolute shrinkage and selection operator (LASSO) regression, and support vector machine (SVM) algorithms were employed to evaluate the potential efficacy of anti-tumor immunotherapy. Candidate immunotherapy targets were systematically identified through multi-gene panel analyses and subsequently validated using molecular biology assays. A prioritized target emerging from cellular screening was further evaluated for its capacity to potentiate anti-tumor immunity. The therapeutic efficacy of this candidate was rigorously confirmed through a comprehensive suite of immunological experiments. Results: Following systematic screening of five candidate genes (WNT11, FAM181B, GRK5, FSCN1, and ECHS1), GRK5 emerged as a promising therapeutic target for immunotherapy based on its distinct functional and molecular associations with immune evasion mechanisms. Cellular functional assays revealed that GRK5 knockdown significantly attenuated the malignant phenotype of testicular cancer cells, as evidenced by reduced proliferative capacity and invasive potential. Complementary immunological validation established that specific targeting of GRK5 with the selective antagonist GRK5-IN-2 disrupts immune evasion pathways in testicular cancer, as quantified by T-cell-mediated cytotoxicity. Conclusions: These findings position GRK5 as a critical modulator of tumor-immune escape, warranting further preclinical exploration of GRK5-IN-2 as a candidate immunotherapeutic agent. Full article

Neuropathic pain is a chronic condition resulting from injury or dysfunction in the somatosensory nervous system, which leads to persistent pain and a significant impairment of quality of life. Research has highlighted the complex molecular mechanisms that underlie neuropathic pain and has begun to delineate the roles of microRNAs (miRNAs) in modulating pain pathways. miRNAs, which are small non-coding RNAs that regulate gene expression post-transcriptionally, have been shown to influence key cellular processes, including neuroinflammation, neuronal excitability, and synaptic plasticity. These processes contribute to the persistence of neuropathic pain, and miRNAs have emerged as critical regulators of pain behaviors by modulating signaling pathways that control pain sensitivity. miRNAs can influence neuropathic pain by targeting genes that encode protein kinases involved in pain signaling. This review focuses on miRNAs that have been demonstrated to modulate neuropathic pain behavior through their effects on protein kinases or their immediate upstream regulators. The relationship between miRNAs and neuropathic pain behaviors is characterized as either an upregulation or a downregulation of miRNA levels that leads to a reduction in neuropathic pain. In the case of miRNA upregulation resulting in an alleviation of neuropathic pain behaviors, protein kinases exhibit a positive correlation with neuropathic pain, whereas decreased protein kinase levels correlate with diminished neuropathic pain behaviors. The only exception is GRK2, which shows an inverse correlation with neuropathic pain. In the case of miRNA downregulation resulting in a reduction in neuropathic pain behaviors, protein kinases display mixed relationships to neuropathic pain, with some kinases exhibiting positive correlation, while others exhibit negative correlation. By exploring how protein kinases mediate miRNA modulation of neuropathic pain, valuable insight may be gained into the pathophysiology of neuropathic pain, offering potential therapeutic targets for developing more effective strategies for pain management. Full article

Upon activation of certain G protein-coupled receptors, Mdm2 promotes the ubiquitination of both GRK2 and arrestin3. Similar to arrestin3, GRK2 ubiquitination was associated with its endocytic activity and proteasomal degradation. Ubiquitination of GRK2 was essential for arrestin3 ubiquitination, and vice versa. Cellular components involved in arrestin3 ubiquitination, including Gβγ, clathrin, and 14-3-3η, were also necessary for GRK2 ubiquitination. Additionally, the arrestin-biased signaling pathway contributed to the ubiquitination of both GRK2 and arrestin3. By employing Mdm2-knockdown cells alongside GRK2 and arrestin3 mutants deficient in ubiquitination sites, as well as receptors lacking phosphorylation sites, we established that the ubiquitinated forms of GRK2 and arrestin3 facilitate clathrin-dependent endocytosis, whereas non-ubiquitinated GRK2 and arrestin3 are responsible for caveolar and a distinct third endocytic pathway, respectively. In the context of clathrin-mediated endocytosis, arrestin3’s interaction with clathrin and GRK2’s interaction with the β2-adaptin subunit of adaptor protein complex 2 were critical. These findings suggest that GRK2 and arrestin3 ubiquitination are mutually dependent, with their ubiquitination states determining their roles in distinct endocytic pathways. Full article

During the early growth stages of fish larvae, there are significant challenges to their viability, so improving their visual environment is essential to promoting their growth and survival. Following the successful knockout of thyroid hormone receptor beta 2 (thrb2) using Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 technology, there was an increase in the expression of UV opsin (short-wave-sensitive 1, sws1), while the expression of other cone opsins was significantly decreased. Further analysis of the retinal structure demonstrated that the thrb2 knockout resulted in an increased lens thickness and a decreased thickness of the ganglion cell layer (GCL), outer plexiform layer (OPL), and outer nuclear layer (ONL) in the retina. The slowing down of swimming speed under light conditions in thrb2^−/− may be related to the decreased expression of phototransduction-related genes such as G protein-coupled receptor kinase 7a (grk7a), G protein-coupled receptor kinase 7b (grk7b), and phosphodiesterase 6c (pde6c). Notably, thrb2^−/− larvae exhibited a significant increase in the amount and proportion of first feeding, and their growth rate significantly exceeded that of wild-type controls during the week after feeding. This observation suggests that although the development of the retina may be somewhat affected, thrb2^−/− larvae show positive changes in feeding behaviour and growth rate, which may be related to their enhanced ability to adapt to their environment. These results provide novel insights into the function of the thrb2 gene in the visual system and behaviour and may have implications in areas such as fish farming and genetic improvement. Full article

G protein-coupled receptors (GPCRs) represent a family of membrane proteins that regulate several cellular processes. Among the GPCRs, G protein-coupled receptor kinases (GRKs) regulate downstream signaling pathways and receptor desensitization. GRK2 has gained significant interest due to its cardiovascular physiology and pathological involvement. GRK2’s presence in cardiac tissue and its influence on cardiac function, β-adrenergic signaling, and myocardial remodeling underlies its involvement in cardiovascular diseases such as heart failure and ischemia. GRK2’s canonical role is receptor desensitization, but emerging evidence suggests its involvement in mitochondrial dynamics and bioenergetics, influencing processes such as oxidative phosphorylation, reactive oxygen species production, and apoptosis. Moreover, GRK2’s localization within mitochondria suggests a direct role in regulating mitochondrial health and function. Notably, while GRK2 inhibition seems to be a therapeutic approach to heart failure, its precise role in mitochondrial dynamics and pathology needs further investigation. This review explores the complex relationship between mitochondrial function and GRK2 and clarifies the implications for cardiovascular health. Cardiovascular medicine might greatly benefit from future studies that focus on understanding the processes behind GRK2–mitochondrial crosstalk to develop personalized therapies Full article

High temperatures present considerable challenges to global fish growth and production, yet the genetic basis of heat tolerance remains underexplored. This study combines quantitative trait locus (QTL) mapping and genome-wide association studies (GWAS) to examine the genetics of heat tolerance in Larimichthys polyactis. As a result, a genetic linkage map was constructed with 3237 bin markers spanning 24 linkage groups and totaling 1900.84 centimorgans, using genotyping-by-sequencing of a full-sib family comprising 120 progeny and their two parents. Based on this genetic linkage map, QTL mapping identified four QTLs associated with heat tolerance, which encompassed 18 single nucleotide polymorphisms and harbored 648 genes within the QTL intervals. The GWAS further disclosed 76 candidate genes related to heat tolerance, 56 of which overlapped with the QTL results. Enrichment analysis indicated that these genes are involved in immune response, development, lipid metabolism, and endocrine regulation. qPCR validation of 14 prioritized genes, which were simultaneously enriched in Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathways, confirmed significant upregulation of smpd5, polr3d, rab11fip2, and gfpt1, along with downregulation of gpat4 and grk5 after 6 h of heat stress. These findings demonstrate their responsiveness to elevated high temperatures. This meta-analysis of QTL mapping and GWAS has successfully identified functional genes related to heat tolerance, enhancing understanding of the genetic architecture underlying this critical trait in L. polyactis. It also provides a molecular breeding tool to improve genetic traits associated with heat tolerance in cultured L. polyactis. Full article

The prolonged overstimulation of β-adrenergic receptors (β-ARs), a member of the G protein-coupled receptor (GPCR) family, causes abnormalities in the density and functionality of the receptor and contributes to cardiac dysfunctions, leading to the development and progression of heart diseases, especially heart failure (HF). Despite recent advancements in HF therapy, mortality and morbidity rates continue to be high. Treatment with β-AR antagonists (β-blockers) has improved clinical outcomes and reduced overall hospitalization and mortality rates. However, several barriers in the management of HF remain, providing opportunities to develop new strategies that focus on the functions and signal transduction of β-ARs involved in the pathogenesis of HF. As β-AR can signal through multiple pathways influenced by different receptor subtypes, expression levels, and signaling components such as G proteins, G protein-coupled receptor kinases (GRKs), β-arrestins, and downstream effectors, it presents a complex mechanism that could be targeted in HF management. In this narrative review, we focus on the regulation of β-ARs at the receptor, G protein, and effector loci, as well as their signal transductions in the physiology and pathophysiology of the heart. The discovery of potential ligands for β-AR that activate cardioprotective pathways while limiting off-target signaling is promising for the treatment of HF. However, applying findings from preclinical animal models to human patients faces several challenges, including species differences, the genetic variability of β-ARs, and the complexity and heterogeneity of humans. In this review, we also summarize recent updates and future research on the regulation of β-ARs in the molecular basis of HF and highlight potential therapeutic strategies for HF. Full article

GRK2 and arrestin3, key players in the functional regulation of G protein-coupled receptors (GPCRs), are ubiquitinated by Mdm2, a nuclear protein. The agonist-induced increase in arrestin3 ubiquitination occurs in the nucleus, underscoring the crucial role of its nuclear translocation in this process. The ubiquitination of arrestin3 occurs in the nucleus, highlighting the pivotal role of its nuclear translocation in this process. In contrast, GRK2 cannot translocate into the nucleus; thus, facilitation of the cytosolic translocation of nuclear Mdm2 is required to ubiquitinate GRK2 in the cytosol. Among the explored cellular components and processes, arrestin, Gβγ, clathrin, and receptor phosphorylation were found to be required for the nuclear import of arrestin3, the ubiquitination of arrestin3 in the nucleus, nuclear export of Mdm2, and the ubiquitination of GRK2 in the cytosol. In conclusion, our findings demonstrate that agonist-induced ubiquitination of arrestin3 in the nucleus is interconnected with cytosolic GRK2 ubiquitination. Full article

Sleep deprivation (SD) is a recognized risk factor for atrial fibrillation (AF), yet the precise molecular and electrophysiological mechanisms behind SD-induced AF are unclear. This study explores the electrical and structural changes that contribute to AF in chronic partial SD. We induced chronic partial SD in Wistar rats using a modified multiple-platform method. Echocardiography demonstrated impaired systolic and diastolic function in the left ventricle (LV) of the SD rats. The SD rats exhibited an elevated heart rate and a higher low-frequency to high-frequency ratio in a heart-rate variability analysis. Rapid transesophageal atrial pacing led to a higher incidence of AF and longer mean AF durations in the SD rats. Conventional microelectrode recordings showed accelerated pulmonary vein (PV) spontaneous activity in SD rats, along with a heightened occurrence of delayed after-depolarizations in the PV and left atrium (LA) induced by tachypacing and isoproterenol. A Western blot analysis showed reduced expression of G protein-coupled receptor kinase 2 (GRK2) in the LA of the SD rats. Chronic partial SD impairs LV function, promotes AF genesis, and increases PV and LA arrhythmogenesis, potentially attributed to sympathetic overactivity and reduced GRK2 expression. Targeting GRK2 signaling may offer promising therapeutic avenues for managing chronic partial SD-induced AF. Future investigations are mandatory to investigate the dose–response relationship between SD and AF genesis. Full article

Sympathetic nervous system (SNS) hyperactivity is mediated by elevated catecholamine (CA) secretion from the adrenal medulla, as well as enhanced norepinephrine (NE) release from peripheral sympathetic nerve terminals. Adrenal CA production from chromaffin cells is tightly regulated by sympatho-inhibitory α₂-adrenergic (auto)receptors (ARs), which inhibit both epinephrine (Epi) and NE secretion via coupling to Gi/o proteins. α₂-AR function is, in turn, regulated by G protein-coupled receptor (GPCR)-kinases (GRKs), especially GRK2, which phosphorylate and desensitize them, i.e., uncouple them from G proteins. On the other hand, the short-chain free fatty acid (SCFA) receptor (FFAR)-3, also known as GPR41, promotes NE release from sympathetic neurons via the Gi/o-derived free Gβγ-activated phospholipase C (PLC)-β/Ca²⁺ signaling pathway. However, whether it exerts a similar effect in adrenal chromaffin cells is not known at present. In the present study, we examined the interplay of the sympatho-inhibitory α_2A-AR and the sympatho-stimulatory FFAR3 in the regulation of CA secretion from rat adrenal chromaffin (pheochromocytoma) PC12 cells. We show that FFAR3 promotes CA secretion, similarly to what GRK2-dependent α_2A-AR desensitization does. In addition, FFAR3 activation enhances the effect of the physiologic stimulus (acetylcholine) on CA secretion. Importantly, GRK2 blockade to restore α_2A-AR function or the ketone body beta-hydroxybutyrate (BHB or 3-hydroxybutyrate), via FFAR3 antagonism, partially suppress CA production, when applied individually. When combined, however, CA secretion from PC12 cells is profoundly suppressed. Finally, propionate-activated FFAR3 induces leptin and adiponectin secretion from PC12 cells, two important adipokines known to be involved in tissue inflammation, and this effect of FFAR3 is fully blocked by the ketone BHB. In conclusion, SCFAs can promote CA and adipokine secretion from adrenal chromaffin cells via FFAR3 activation, but the metabolite/ketone body BHB can effectively inhibit this action. Full article

Arrestins are known to be involved not only in the desensitization and internalization of G protein-coupled receptors but also in the G protein-independent activation of mitogen-activated protein (MAP) kinases, such as extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK), to regulate cell proliferation and inflammation. Our previous study revealed that the histamine H₁ receptor-mediated activation of ERK is dually regulated by G_q proteins and arrestins. In this study, we investigated the roles of G_q proteins and arrestins in the H₁ receptor-mediated activation of JNK in Chinese hamster ovary (CHO) cells expressing wild-type (WT) human H₁ receptors, the G_q protein-biased mutant S487TR, and the arrestin-biased mutant S487A. In these mutants, the Ser487 residue in the C-terminus region of the WT was truncated (S487TR) or mutated to alanine (S487A). Histamine significantly stimulated JNK phosphorylation in CHO cells expressing WT and S487TR but not S487A. Histamine-induced JNK phosphorylation in CHO cells expressing WT and S487TR was suppressed by inhibitors against H₁ receptors (ketotifen and diphenhydramine), G_q proteins (YM-254890), and protein kinase C (PKC) (GF109203X) as well as an intracellular Ca²⁺ chelator (BAPTA-AM) but not by inhibitors against G protein-coupled receptor kinases (GRK2/3) (cmpd101), β-arrestin2 (β-arrestin2 siRNA), and clathrin (hypertonic sucrose). These results suggest that the H₁ receptor-mediated phosphorylation of JNK is regulated by G_q-protein/Ca²⁺/PKC-dependent but GRK/arrestin/clathrin-independent pathways. Full article

Pathological cardiac remodeling is associated with cardiovascular disease and can lead to heart failure. Nuclear factor-kappa B (NF-κB) is upregulated in the hypertrophic heart. Moreover, the expression of the G-protein-coupled receptor kinase 2 (GRK2) is increased and linked to the progression of heart failure. The inhibitory effects of paroxetine on GRK2 have been established. However, its protective effect on IκBα/NFκB signaling has not been elucidated. This study investigated the cardioprotective effect of paroxetine in an animal model of cardiac hypertrophy (CH), focusing on its effect on GRK2-mediated NF-κB-regulated expression of prohypertrophic and profibrotic genes. Wistar albino rats were administered normal saline, paroxetine, or fluoxetine, followed by isoproterenol to induce CH. The cardioprotective effects of the treatments were determined by assessing cardiac injury, inflammatory biomarker levels, histopathological changes, and hypertrophic and fibrotic genes in cardiomyocytes. Paroxetine pre-treatment significantly decreased the HW/BW ratio (p < 0.001), and the expression of prohypertrophic and profibrotic genes Troponin-I (p < 0.001), BNP (p < 0.01), ANP (p < 0.001), hydroxyproline (p < 0.05), TGF-β1 (p < 0.05), and αSMA (p < 0.01) as well as inflammatory markers. It also markedly decreased pIκBα, NFκB(p105) subunit expression (p < 0.05) and phosphorylation. The findings suggest that paroxetine prevents pathological cardiac remodeling by inhibiting the GRK2-mediated IκBα/NF-κB signaling pathway. Full article

Reproductive traits hold considerable economic importance in pig breeding and production. However, candidate genes underpinning the reproductive traits are still poorly identified. In the present study, we executed a genome-wide association study (GWAS) and runs of homozygosity (ROH) analysis using the PorcineSNP50 BeadChip array for 585 Yorkshire pigs. Results from the GWAS identified two genome-wide significant and eighteen suggestive significant single nucleotide polymorphisms (SNPs) associated with seven reproductive traits. Furthermore, we identified candidate genes, including ELMO1, AOAH, INSIG2, NUP205, LYPLAL1, RPL34, LIPH, RNF7, GRK7, ETV5, FYN, and SLC30A5, which were chosen due to adjoining significant SNPs and their functions in immunity, fertilization, embryonic development, and sperm quality. Several genes were found in ROH islands associated with spermatozoa, development of the fetus, mature eggs, and litter size, including INSL6, TAF4B, E2F7, RTL1, CDKN1C, and GDF9. This study will provide insight into the genetic basis for pig reproductive traits, facilitating reproduction improvement using the marker-based selection methods. Full article

The D2 dopamine receptor (D2R) signals through both G proteins and β-arrestins to regulate important physiological processes, such as movement, reward circuitry, emotion, and cognition. β-arrestins are believed to interact with G protein-coupled receptors (GPCRs) at the phosphorylated C-terminal tail or intracellular loops. GPCR kinases (GRKs) are the primary drivers of GPCR phosphorylation, and for many receptors, receptor phosphorylation is indispensable for β-arrestin recruitment. However, GRK-mediated receptor phosphorylation is not required for β-arrestin recruitment to the D2R, and the role of GRKs in D2R–β-arrestin interactions remains largely unexplored. In this study, we used GRK knockout cells engineered using CRISPR-Cas9 technology to determine the extent to which β-arrestin recruitment to the D2R is GRK-dependent. Genetic elimination of all GRK expression decreased, but did not eliminate, agonist-stimulated β-arrestin recruitment to the D2R or its subsequent internalization. However, these processes were rescued upon the re-introduction of various GRK isoforms in the cells with GRK2/3 also enhancing dopamine potency. Further, treatment with compound 101, a pharmacological inhibitor of GRK2/3 isoforms, decreased β-arrestin recruitment and receptor internalization, highlighting the importance of this GRK subfamily for D2R–β-arrestin interactions. These results were recapitulated using a phosphorylation-deficient D2R mutant, emphasizing that GRKs can enhance β-arrestin recruitment and activation independently of receptor phosphorylation. Full article

Visual perception plays a crucial role in foraging, avoiding predators, mate selection, and communication. The regulation of color vision is largely dependent on opsin, which is the first step in the formation of the visual transduction cascade in photoreceptor cells. Short-wave-sensitive 1 (sws1) is a visual pigment that mediates short-wavelength light transduction in vertebrates. The depletion of sws1 resulted in increased M-opsin in mice. However, there is still no report on the visual function of sws1 in teleost fish. Here, we constructed the sws1 knockout medaka using CRISPR/Cas9 technology. The 6 dph (days post-hatching) medaka sws1^−/− larvae exhibited significantly decreased food intake and total length at the first feeding stage, and the mRNA levels of orexigenic genes (npy and agrp) were significantly upregulated after feeding. The swimming speed was significantly reduced during the period of dark-light transition stimulation in the sws1-mutant larvae. Histological analysis showed that the thickness of the lens was reduced, whereas the thickness of the ganglion cell layer (GCL) was significantly increased in sws1^−/− medaka larvae. Additionally, the deletion of sws1 decreased the mRNA levels of genes involved in phototransduction (gnb3b, grk7a, grk7b, and pde6c). We also observed increased retinal cell apoptosis and oxidative stress in sws1 knockout medaka larvae. Collectively, these results suggest that sws1 deficiency in medaka larvae may impair visual function and cause retinal cell apoptosis, which is associated with the downregulation of photoconduction expression and oxidative stress. Full article