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At least 19 recordsLinked to original sources
PubMed2025

Programmed ribosomal frameshifting during PLEKHM2 mRNA decoding generates a constitutively active proteoform that supports myocardial function.

Programmed ribosomal frameshifting is a process where a proportion of ribosomes change their reading frame on an mRNA. While frameshifting is commonly used by viruses, very few phylogenetically conserved examples are known in nuclear encoded genes. Here, we report a +1 frameshifting event during decoding of the human gene PLEKHM2 that provides access to a second internally overlapping ORF. The new carboxyl-terminal domain of this frameshift protein forms an α helix, which relieves PLEKHM2 from autoinhibition and allows it to move to the tips of cells without activation by ARL8. Reintroducing both the canonically translated and frameshifted protein are necessary to restore normal contractile function of PLEKHM2 knockout cardiomyocytes, demonstrating the necessity of frameshifting for normal cardiac activity.

Frameshifting, Ribosomal↗
PubMed1977

1-naphthyl acetate esterases in fluids and tissues of jaw cysts.

The activity and electrophoretic mobility of 1-naphthyl acetate esterases in cystic fluids and cystic tissues of ameloblastomas, follicular and apical cysts were examined. The cystic fluids showed lower activities than sera but had very similar patterns on the electrophoretogram. The activity levels of the three kinds of cystic fluids were not statistically significantly different. The fluid esterases may have originated from serum but they were not produced by the cystic lining tissue. Ameloblastoma tissues showed the highest activity per wet weight and per mg protein of the three kinds of cyst lesions (P less than 0.05). On the electrophoretogram, the esterase-I activity constituted 41% of the total activity in ameloblastomas, whereas in follicular cysts and apical cysts the esterase-I activity constituted 32% and 24% of the total activity, respectively.

Ameloblastoma↗
PubMed2026

Hearing loss associated with CDC42 in mice and humans (Takenouchi-Kosaki syndrome): CDC42 and RHOQ synergistically function in cochlear hair cells.

CDC42 is involved in multiple signaling pathways, including actin organization and polarity. We previously reported progressive sensorineural hearing loss (SNHL) in inner ear hair cell (HC)-specific Cdc42-knockout (Atoh1-Cre+/-;Cdc42flox/flox) mice. However, the phenotype was milder than expected, suggesting possible redundancy with other Rho-family GTPases. Thereafter, Takenouchi-Kosaki syndrome (TKS), caused by de novo CDC42 mutations and manifesting as SNHL, was reported, in which the p.Y64C mutation was speculated to be constitutively active. However, the relationship between CDC42 status and hearing phenotypes in TKS remains unclear. Using cell models, mouse models, and patient data, we propose that impaired and/or dysregulated cycling between GDP/inactive and GTP/active forms, through either loss-of-function or constitutive activation, can lead to SNHL. Furthermore, to test redundancy, we generated HC-specific Cdc42;RhoQ double-knockout (Atoh1-Cre+/-;Cdc42flox/flox;RhoQflox/flox) mice, which revealed synergistic roles of CDC42 and RHOQ in cochlear HCs. Supporting this synergy, MDCK cells with CDC42 and RHOQ double knockdown showed greater phospho-cofilin, a key regulator of actin turnover, elevation than single knockdowns.

CDC42↗
PubMed1977

The pressor activity of burimamide: a relationship between chemical constitution and pressor activity of burimamide and related histamine H2-receptor antagonists.

Burimamide, a histamine H2-receptor antagonist, has been shown to cause pressor responses in pithed rats. The response can be prevented by prior removal of the adrenal glands or by pretreatment with the alpha-adrenoceptor antagonist, phentolamine, 5 mg/kg, suggesting that the pressor response to burimamide is due to release of catecholamines from the adrenal glands. The pressor activity of burimamide has been compared with that of metiamide and two close chemical analogues, methylburimamide and thiaburimamide, in order to identify which chemical features of the compounds are necessary for this activity. Methylburimamide was the most potent pressor agent, followed by burimamide, metiamide and thiaburimamide. The pressor effects (and presumably catecholamine-releasing activities) appear to be related to the basicities of the compounds. We conclude that the release of catecholamines by these histamine H2-receptor antagonists is probably due to their cationic (imidazolium) forms.

Animals↗
PubMedDate not supplied

Increased endocytosis with lysosomal activation in skeletal muscle of dystrophic mouse.

Endocytosis in dystrophic muscles was studied by a combination of biochemical, radiochemical, and light and electron microscopic techniques. It was observed that the uptake of horseradish peroxidase (HRP) and 3H-Inulin in vitro was increased in leg skeletal muscles from dystrophic mice compared with littermate controls. Endocytosis of HRP in vivo was also increased in dystrophic muscles. When HRP was administered intravenously, light microscopic examination of the muscles showed that the macromolecular tracer was present not only in the extracellular space but also as intracellular deposits in several dystropic muscle fibers. Ultrastructural examination of these fibers showed HRP to be present in membrane limited bodies of variable size, some of which likely represented secondary lysosomes, located preferentially close to the A-I junction. HRP was also found inside vacuoles which were sometimes in close vicinity to autophagic vacuoles. Primary uptake vesicles containing HRP appeared to originate from the sarcolemma and the transverse tubules. Biochemical determination of lysosomal enzyme activities revealed elevated levels of both cathepsin D and N-acetylglucosaminidase in dystrophic muscles as compared with controls. The results suggest an increased endocytic activity in dystrophic muscles with distribution of exogenous marcromolecular tracers into endocytic vesicles and lysosomal structures. The hypothesis is put forward that endocytic activity constitutes an important mechanism of lysosomal activation in dystrophic muscles.

Acetylglucosaminidase↗
PubMed2018

Long non-coding RNA metallothionein 1 pseudogene 3 promotes p2y12 expression by sponging miR-126 to activate platelet in diabetic animal model.

Platelet hyperaggregation and hypercoagulation are associated with increase of thrombogenic risk, especially in patients with type 2 diabetes (T2D). High activity of P2Y12 receptor is found in T2D patients, exposing such patients to a prothrombotic condition. P2Y12 is a promising target for antiplatelet, but due to P2Y12 receptor constitutive activation, the clinical practical phenomena such as "clopidogrel resistance" are commonly occurring. In this study, we investigate the role of lncRNA on platelet activation. By lncRNA array, we screened thousands of differentially expressed lncRNA in megakaryocytes from T2D patients and confirmed that lncRNA metallothionein 1 pseudogene 3 (MT1P3) was significantly upregulated in megakaryocytes from T2D patients than in healthy controls. And we further investigate the biofunction of MT1P3 on platelet activation and the regulatory mechanism on p2y12. MT1P3 was positively correlated with p2y12 mRNA levels and promoted p2y12 expression by sponging miR-126. Knockdown of MT1P3 by siRNA reduced p2y12 expression, inhibiting platelet activation and aggregation in diabetes animal model. In conclusion, our findings identify MT1P3 as a key regulator in platelet activation by increasing p2y12 expression through sponging miR-126 under T2D condition. These findings may provide a new insight for managing platelet hyperactivity-related diseases.

Animals↗
PubMed2026

Development of receptor and oncogene-responsive luciferase reporter vectors that are activated via kinase signaling to AP-1.

This study aimed to generate lentiviral vectors carrying an array of AP-1 motifs driving luciferase gene expression as reporters of mitogen-activated protein kinase (MAPK) activity. We created a series of vectors based on LeGO-iG that were used to generate stably transduced leukemia cell lines. A vector termed LEGO-AP1×6-GM55 containing an array of 6 AP-1 sites linked to the minimal CSF2 promoter was sufficient to support high levels of MAPK-inducible luciferase activity in leukemic cell lines that was suppressed by MAPK inhibitors. The inclusion of a putative chromatin priming element encompassing RUNX and ETS motifs increased the activity of these vectors. The additional inclusion of the full-length mouse CSF2 promoter, or the human DUSP5 promoter further increased the MAPK-dependent activity of these vectors in leukemic cells. These vectors support moderate levels of constitutive activity in cells carrying mutations that activate the RAS/RAF/MEK MAPK signaling pathway, and high-level activity after direct activation of MAPK signaling. They also respond to T-cell receptor activation via MAPK and Ca2+ signaling pathways. This resource will now make it easier to track receptor or oncogene-inducible MAPK activity in cultured cells, and potentially in tumors, close to real-time.

Humans↗
PubMed2025

Remote Regulation by VirB, the Transcriptional Anti-Silencer of Shigella Virulence Genes, Provides Mechanistic Information.

Classical models of bacterial transcription show regulators binding close to promoter elements to exert their effect. However, the scope for long-range regulation exists, especially by nucleoid structuring proteins, like H-NS. Here, long-range regulation by VirB, a transcriptional regulator that alleviates H-NS-mediated silencing of key virulence genes in Shigella species, is explored in vivo to test the limits of long-range regulation and provide further mechanistic insight. VirB-dependent regulation of the well-characterized icsP promoter persists if its cognate site is repositioned 1 kb, 3.3 kb, and even 4.7 kb further upstream than its native position in a plasmid reporter. VirB-dependent regulation diminishes with binding site distance. While increasing cellular VirB pools elevated promoter activity in all constructs with wild-type VirB binding sites, it did not generate a disproportionate increase in promoter activity from remote sites relative to the native site. Since VirB occludes a constitutively active promoter (PT5) when docked adjacent to its -35 element, we next moved the VirB binding site far outside the promoter region. We discovered that VirB still interfered with promoter activity. These findings and those generated from molecular roadblocks engineered around a distally located VirB-binding site are reconciled with the various models of transcriptional regulation by VirB.

Gene Expression Regulation, Bacterial↗
PubMed2026

Phosphoproteomics identification of ERK-dependent activation of Rps6kb1 in cardiac hypertrophy.

Cardiomyocyte growth is tightly controlled by multiple signaling pathways. Identification of master kinases in this process is essential in exploring potential targets for the treatment of pathological cardiac hypertrophy and heart failure. Here we identified the mTOR-independent activation of ribosomal protein S6 kinase b1 (Rps6kb1) during cardiomyocyte growth. By utilizing phosphoproteomics in primary neonatal rat ventricular myocytes, we revealed Rps6kb1 as one of most activated kinases under growth stimulation. We further demonstrated the role of Rps6kb1 phosphorylation in pathological cardiac hypertrophy and heart failure. We showed that the phosphorylation of multiple sites in Rps6kb1, including T367 in the kinase domain and S418/T421/S424 in the C-terminal domain, is not directly regulated by the activity of mTOR but coupled with the activation of the MEK1/ERK axis. In mice, cardiomyocyte-specific deletion of Rps6kb1 significantly inhibited both constitutively active ERK- and pressure overload-induced cardiac hypertrophy. In contrast, cardiomyocyte-specific overexpression of wild-type Rps6kb1, rather than the phosphorylation-defective mutant, elevated cardiac hypertrophy and augmented pressure overload-induced heart failure. In conclusion, our findings reveal that the MEK/ERK axis primes Rps6kb1 activation through phosphorylation of 2 separate domains of Rps6kb1, which may play an essential role in cardiac hypertrophy and heart failure under hemodynamic stress.

Animals↗
PubMedDate not supplied

[Effect of precipitating antibodies to pancreatic kallikrein on arginine esterase contact activation in human and rabbit blood].

Effect of precipitating antibodies against pig pancreatic kallikrein on the BAEE-esterase activity was studied in human blood plasma and in rabbit blood serum, activated by kaolin. An inhibitory effect of the antibodies on the increased BAEE-esterase activity was observed within the first minute of incubation after treatment with kaolin. The inhibition of the BAEE-esterase activity constituted 65.1% in human blood plasma and 40.9% in rabbit serum. Possible mechanism is discussed on the inhibition of contact activation of arginine esterase in human and rabbit blood by antibodies against pancreatic kallikrein.

Animals↗
PubMed2026

Oncogene activation mechanism determines the limits of targeted protein degradation.

Protein degrader drugs such as PROTACs are being advanced as therapeutics targeted against oncogenic proteins. During tumorigenesis, oncogenic proteins can become constitutively activated via mechanisms including gene amplification, which increases protein production, and point mutations, which can extend protein half-life. Few experimental studies have addressed how disease-associated changes in target protein homeostasis influence PROTAC activity. We developed orthogonal methods to increase production or enhance stability of β-catenin, an important oncoprotein and target for degrader therapeutics, and used the dTAG system to evaluate the consequences for PROTAC activity. Stabilizing oncogenic missense mutations increase protein expression up to 5-fold but do not alter the PROTAC-imposed minimal steady-state level. In contrast, transcriptional upregulation increases both pre- and post-treatment target levels, revealing a synthesis-dependent ceiling on achievable depletion. Our results highlight distinct constraints on PROTAC activity arising from different mechanisms of oncogene activation, with potential implications for preclinical modeling, drug resistance and personalized medicine.

Humans↗
PubMed2026

The human cytomegalovirus vGPCR UL33 is essential for efficient lytic replication in epithelial cells.

UNLABELLED: Human cytomegalovirus (HCMV) is a β-herpesvirus that is ubiquitous in the human population. HCMV has the largest genome of the human herpesviruses and encodes an array of genes that affect pathogenesis in different cell types. Given the ability of HCMV to replicate in a range of cell types, investigators have begun to identify viral proteins required for cell type-specific replication. There are four proteins encoded by HCMV that are homologous to G protein-coupled receptors (GPCRs); these viral GPCRs (vGPCRs) are UL33, UL78, US27, and US28. In this study, we find that deletion of all four vGPCR genes severely attenuates HCMV replication in primary human salivary gland epithelial cells and ARPE-19 retinal epithelial cells, as evidenced by decreases in viral gene expression and virus production. Deletion of UL33 from the HCMV genome also results in a failure to efficiently replicate in epithelial cells, and this defect is manifested by decreased levels of viral gene expression and virus production. We find that, similar to US28, UL33 constitutively activates Gαq signaling to high levels in epithelial cells. We also find that UL33 transcription is more complicated than originally believed, and there is the potential for the virus to utilize various 5' UTRs to create novel UL33 proteins that are all capable of constitutive Gαq signaling. Taken together, these studies provide novel molecular and biochemical data regarding UL33 expression, subcellular localization, and signaling, and indicate that UL33 activity is essential for efficient HCMV replication in cells of epithelial origin. IMPORTANCE: Human cytomegalovirus (HCMV) replicates in a number of cell types and tissues in vivo, and the viral genes involved in cell type-specific replication are just beginning to be elucidated. The HCMV-encoded viral G protein-coupled receptors (vGPCRs) UL33, UL78, US27, and US28 are proving to play important roles in multiple aspects of HCMV replication, including the establishment and maintenance of latency. Here, we demonstrate that the HCMV vGPCRs and UL33, in particular, play an important role in driving lytic replication in cells of epithelial origin, including those derived from the salivary gland. This work expands on potential functions of the vGPCRs, will drive future studies to understand mechanistically how they affect tropism, and provides a new target for future therapeutics.

Gαq↗
PubMed2025

Dynamic phosphorylation of Hcm1 promotes fitness in chronic stress.

Cell survival depends upon the ability to adapt to changing environments. Environmental stressors trigger an acute stress response program that rewires cell physiology, downregulates proliferation genes and pauses the cell cycle until the cell adapts. After the acute response is resolved, cells resume cycling but at a reduced rate. The importance of cell cycle changes for survival in chronic stress is not clear. Here, we show that dynamic phosphorylation of the yeast cell cycle-regulatory transcription factor Hcm1 is required to maintain fitness in chronic stress. Hcm1 is activated by cyclin dependent kinase (CDK) during S-phase and is inactivated by the phosphatase calcineurin (CN) in response to stressors that signal through increases in cytosolic Ca2+. Cells expressing a constitutively active, phosphomimetic Hcm1 mutant exhibit a reduction in fitness in stress, suggesting Hcm1 inactivation promotes survival. However, a comprehensive analysis of Hcm1 phosphomutants revealed that Hcm1 activity is also important to survive stress, and that all mutants with fixed phosphorylation states are less fit in stress. Moreover, our data suggests that pulses of Hcm1 activity are necessary to maximize target gene expression in stress. These findings demonstrate that expression levels of Hcm1 target genes influence fitness in stress and suggest that the dynamic phosphorylation of cell cycle regulators plays a crucial role in promoting survival in stressful environments.

Phosphorylation↗
PubMed2024

The Human Cytomegalovirus vGPCR UL33 is Essential for Efficient Lytic Replication in Epithelial Cells.

Human cytomegalovirus (HCMV) is a β-herpesvirus which is ubiquitous in the human population. HCMV has the largest genome of all known human herpesviruses, and thus encodes a large array of proteins that affect pathogenesis in different cell types. Given the large genome and the ability of HCMV to replicate in a range of cells, investigators have begun to identify viral proteins required for cell type-specific replication. There are four proteins encoded in the HCMV genome that are homologous to human G protein-coupled receptors (GPCRs); these viral-encoded GPCRs (vGPCRs) are UL33, UL78, US27, and US28. In the current study, we find that deletion of all four vGPCR genes from a clinical isolate of HCMV severely attenuates lytic replication in both primary human salivary gland epithelial cells, as well as ARPE-19 retinal epithelial cells as evidenced by significant decreases in immediate early gene expression and virus production. Deletion of UL33 from the HCMV genome also results in a failure to efficiently replicate in epithelial cells, and this defect is manifested by decreased levels of immediate early, early, and late gene expression, as well as reduced viral production. We find that similar to US28, UL33 constitutively activates Gαq-dependent PLC-β signaling to high levels in these epithelial cells. We also find that UL33 transcription is more complicated than originally believed, and there is the potential for the virus to utilize various 5' UTRs to create novel UL33 proteins that are all capable of constitutive Gαq signaling. Taken together, these studies suggest that UL33 driven signaling is important for lytic HCMV replication in cells of epithelial origin.

Journal Article↗
PubMed2025

TCF25 serves as a nutrient sensor to orchestrate metabolic adaptation and cell death by enhancing lysosomal acidification under glucose starvation.

Cells adapt to nutrient limitation by activating catabolic and inhibiting anabolic pathways, yet prolonged stress may lead to cell death. How cells orchestrate metabolic adaptation and cell death to nutrient stress is poorly understood. We conduct a genome-wide CRISPR-Cas9 screen to identify regulators in glucose-starvation-induced cell death and find a group of genes in lysosomal pathway is enriched following glucose starvation. We focus on one candidate gene, Transcriptional Factor 25 (TCF25). We find TCF25 enhances lysosomal acidification by targeting V-ATPase, promoting autophagy and ATP generation under glucose starvation. However, prolonged glucose starvation constitutively activates ferritinophagy via TCF25, increasing lysosomal membrane permeability (LMP) and leading to lysosome-dependent cell death (LDCD). Knocking out TCF25 or V-ATPase components prevents cell death. Furthermore, TCF25 deficiency protects mice from hepatic ischemia-reperfusion injury. Our findings identify TCF25 as a crucial nutrient sensor that regulates lysosomal activity, offering potential therapeutic targets for metabolic and ischemic disorders.

Lysosomes↗
PubMedDate not supplied

Characteristics of a microsomal cytochrome P-448-mediated reaction. Ethoxyresorufin O-de-ethylation.

Certain characteristics of ethoxyresorufin O-de-ethylation, as catalyzed by microsomes of liver, lung, and intestine of control and pretreated rats and hamsters, were studied. The results support previous suggestions that the reaction is catalyzed primarily by a 3-methyl-cholanthrene (MC)-inducible mono-oxygenase which has a 448-nm absorption maximum in the reduced-CO difference spectrum. Ethoxyresorufin exhibited a type I binding spectrum with liver microsomes from MC-induced rats, but there was no clear interaction with microsomes from control or phenobarbital (PB)-induced rats. Maximum MC-induction of type I binding and de-ethylase activity coincided with the appearance of a cytochrome P-450 spectrum whose absorption maximum was shifted to 448 nm. Low concentrations of alpha-naphthoflavone (ANF) or benzo[a]pyrene (BP) inhibited the de-ethylation with liver microsomes of MC-treated rats (150 approximately 10(-9)M) but not those of control rats. A kinetic analysis of BP inhibition of this reaction showed it to be competitive. Inhibition of the MC-induced liver microsomal reaction by low concentrations of BP or ANF diminished rapidly with time. MC-induced rat liver microsomal de-ethylation of ethoxyresorufin was less sensitive than the PB-induced reaction to inhibition by metyrapone or SKF 525-A (I50 approximately 10(-6) - 10(-4)M). However, microsomes from rat liver, lung, and intestine had very low constitutive activities (less than 0.1 nmol/min/mg of protein). MC greatly induced the de-ethylation reaction in liver (200 X), intestine (40 X) and lung (10 X). De-ethylation of ethoxyresorufin in microsomes from control and MC-induced rat lung was inhibited by low concentrations of either ANF or BP (I50 approximately 10(-8) M). Control hamster liver microsomes were several times more active in de-ethylation than control rat liver microsomes, but MC-induction of hamster liver was only 1/10 of that in rat liver. Control hamster lung activity was similar to that of control rat lung, but was not appreciably induced by MC.

Animals↗