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1.
Nat Commun ; 15(1): 3901, 2024 May 09.
Article in English | MEDLINE | ID: mdl-38724505

ABSTRACT

Activation of the NF-κB pathway is strictly regulated to prevent excessive inflammatory and immune responses. In a well-known negative feedback model, IκBα-dependent NF-κB termination is a delayed response pattern in the later stage of activation, and the mechanisms mediating the rapid termination of active NF-κB remain unclear. Here, we showed IκBα-independent rapid termination of nuclear NF-κB mediated by CLK2, which negatively regulated active NF-κB by phosphorylating the RelA/p65 subunit of NF-κB at Ser180 in the nucleus to limit its transcriptional activation through degradation and nuclear export. Depletion of CLK2 increased the production of inflammatory cytokines, reduced viral replication and increased the survival of the mice. Mechanistically, CLK2 phosphorylated RelA/p65 at Ser180 in the nucleus, leading to ubiquitin‒proteasome-mediated degradation and cytoplasmic redistribution. Importantly, a CLK2 inhibitor promoted cytokine production, reduced viral replication, and accelerated murine psoriasis. This study revealed an IκBα-independent mechanism of early-stage termination of NF-κB in which phosphorylated Ser180 RelA/p65 turned off posttranslational modifications associated with transcriptional activation, ultimately resulting in the degradation and nuclear export of RelA/p65 to inhibit excessive inflammatory activation. Our findings showed that the phosphorylation of RelA/p65 at Ser180 in the nucleus inhibits early-stage NF-κB activation, thereby mediating the negative regulation of NF-κB.


Subject(s)
Cytoplasm , NF-KappaB Inhibitor alpha , NF-kappa B , Protein-Tyrosine Kinases , Transcription Factor RelA , Animals , Phosphorylation , NF-KappaB Inhibitor alpha/metabolism , NF-KappaB Inhibitor alpha/genetics , Mice , Transcription Factor RelA/metabolism , Humans , Protein-Tyrosine Kinases/metabolism , Protein-Tyrosine Kinases/genetics , NF-kappa B/metabolism , Cytoplasm/metabolism , Proteolysis , Cell Nucleus/metabolism , Virus Replication , HEK293 Cells , Signal Transduction , Mice, Inbred C57BL , Cytokines/metabolism , Active Transport, Cell Nucleus , Protein Serine-Threonine Kinases
2.
Dev Cell ; 59(3): 384-399.e5, 2024 Feb 05.
Article in English | MEDLINE | ID: mdl-38198890

ABSTRACT

Different types of cells uptake fatty acids in response to different stimuli or physiological conditions; however, little is known about context-specific regulation of fatty acid uptake. Here, we show that muscle injury induces fatty acid uptake in muscle stem cells (MuSCs) to promote their proliferation and muscle regeneration. In humans and mice, fatty acids are mobilized after muscle injury. Through CD36, fatty acids function as both fuels and growth signals to promote MuSC proliferation. Mechanistically, injury triggers the translocation of CD36 in MuSCs, which relies on dynamic palmitoylation of STX11. Palmitoylation facilitates the formation of STX11/SNAP23/VAMP4 SANRE complex, which stimulates the fusion of CD36- and STX11-containing vesicles. Restricting fatty acid supply, blocking fatty acid uptake, or inhibiting STX11 palmitoylation attenuates muscle regeneration in mice. Our studies have identified a critical role of fatty acids in muscle regeneration and shed light on context-specific regulation of fatty acid sensing and uptake.


Subject(s)
Fatty Acids , Lipoylation , Muscle, Skeletal , Qa-SNARE Proteins , Regeneration , Animals , Humans , Mice , Biological Transport , CD36 Antigens/metabolism , Cell Membrane/metabolism , Fatty Acids/metabolism , Muscle, Skeletal/injuries , Muscle, Skeletal/physiology , Qa-SNARE Proteins/metabolism
3.
J Clin Biochem Nutr ; 73(2): 116-123, 2023 Sep.
Article in English | MEDLINE | ID: mdl-37700850

ABSTRACT

A widespread degenerative condition of the aorta, abdominal aortic aneurysm (AAA), severely endangers the health of middle-aged and elderly people. SPARC related modular calcium binding2 (SMOC2) is upregulated in the carotid arteries of rats with atherosclerotic lesions, but its function in AAA is still unknown. Therefore, the aim of this research was to evaluate the function of SMOC2 in AAA. The results showed that in the AAA tissues, SMOC2 expression was upregulated compared with healthy controls. Overexpression of SMOC2 promoted vascular smooth muscle cells (VSMCs) proliferation, migration, and extracellular matrix (ECM) degradation. In contrast, silence of SMOC2 inhibited VSMCs proliferation, migration, and ECM degradation. Overexpression of SMOC2 promoted BMP and TGF-ß1 expression and silence of SMOC2 had an opposite effect. Besides, inhibition of BMP or TGF-ß1 suppressed VSMCs cell proliferation, migration, and ECM degradation. Moreover, inhibition BMP or TGF-ß1 reversed the promotive effects of SMOC2 overexpression on VSMCs proliferation, migration, and ECM degradation. SMOC2 may affecte the formation of AAA by upregulating BMP and TGF-ß1 to regulate the proliferation, migration, and ECM degradation of VSMCs.

4.
Heart Surg Forum ; 26(3): E292-E302, 2023 Jun 29.
Article in English | MEDLINE | ID: mdl-37401427

ABSTRACT

Emboli caused by cardiac myxomas mostly occur in the cardiovascular or cerebrovascular systems and rarely in the lower extremity vasculature. We introduce the rare case of a patient with left atrial myxoma (LAM) whose right lower extremity (RLE) suffered from acute ischemia due to tumor fragments, along with a review of the relevant literature, and highlight the clinical characteristics of LAM. An 81-year-old female presented with acute ischemia of RLE. Color Doppler ultrasound showed no blood flow signal far from the RLE femoral artery. Computed tomography angiography showed an occlusion of the right common femoral artery. A transthoracic echocardiogram revealed a left atrial mass. Femoral artery embolectomy was performed under local anesthesia, followed by thoracotomy with tumor resection under general anesthesia on postoperative day seven. The tumor was pathologically confirmed as an atrial myxoma. A literature search of the PubMed database returned 58 cases of limb ischemia due to LAM, and the conclusions drawn from the statistical analysis were that emboli from LAM occurred most commonly in the aortoiliac and bilateral lower limb vasculature and were rarely associated with upper extremity and atrial fibrillation. Multisystem embolism is characteristic of cardiac myxoma. The removed embolus should be examined pathologically for signs of a cardiac myxoma. Lower-limb embolisms should be promptly diagnosed and treated to avoid osteofascial compartment syndrome.


Subject(s)
Arterial Occlusive Diseases , Atrial Fibrillation , Embolism , Heart Neoplasms , Myxoma , Peripheral Vascular Diseases , Female , Humans , Aged, 80 and over , Atrial Fibrillation/complications , Ischemia/diagnosis , Ischemia/etiology , Ischemia/surgery , Embolism/diagnosis , Embolism/etiology , Embolism/surgery , Lower Extremity/blood supply , Heart Neoplasms/complications , Heart Neoplasms/diagnosis , Heart Neoplasms/surgery , Heart Atria/surgery , Peripheral Vascular Diseases/complications , Myxoma/complications , Myxoma/diagnosis , Myxoma/surgery
5.
J Endovasc Ther ; : 15266028231165725, 2023 Apr 12.
Article in English | MEDLINE | ID: mdl-37050852

ABSTRACT

PURPOSE: Isolated infected iliac artery aneurysms (IIIAAs) are extremely rare, life-threatening, and intractable. This study aimed to evaluate the outcomes of endovascular treatment in patients with IIIAAs. METHODS: A retrospective study was conducted for all patients who underwent endovascular treatment for IIIAAs between June 2012 and June 2022 in 3 hospitals. The clinical data and follow-up outcomes were reviewed and assessed. RESULTS: Fifteen patients were included in this study. The median age was 69 years, 12 patients (80%) were men, and 8 (53%) had hypertension. Most of the patients presented with abdominal or lumbar pain (87%) and fever (60%). The offending pathogen was identified in 11 patients (73%). Fifteen patients had a total of 16 IIIAAs, with 12 (75%) involving the common iliac artery. The immediate technical success rate was 100%, and the 30-day mortality was 7%. Infection-related complications occurred in 2 patients (13%) during hospitalization who were treated by open surgery at a later stage. The median follow-up was 23 months (range: 6-80 months, mean: 32 ± 25 months). Aneurysm recurrence was identified in one patient (7%) 5 months after endovascular repair. It was managed by endovascular stent-graft repair with percutaneous catheter drainage. No patients died during the follow-up period. CONCLUSION: Endovascular treatment is feasible, safe, and effective for patients with IIIAAs, achieving acceptable clinical outcomes. Infection surveillance with essential reintervention should be considered for potential infection-related complications. CLINICAL IMPACT: This study first reported that 15 patients underwent endovascular treatment for primary isolated infected iliac artery aneurysms (IIIAAs). It showed a good early and midterm outcomes. This is the first and largest multi-center study and the first literature review of IIIAAs. It provides an evidence that endovascular treatment is feasible, safe, and effective to treat IIIAAs. It suggests endovascular treatment is a promising alternative or a bridge to conventional open surgery for IIIAAs. This may promote endovascular therapy in the management of IIIAAs. It would help clinicians to make an appropriate treatment choice for IIIAAs.

6.
Cell Death Differ ; 30(2): 500-514, 2023 02.
Article in English | MEDLINE | ID: mdl-36481789

ABSTRACT

The Cancer/Testis Antigen (CTA) genes comprise a group of genes whose expression under physiological conditions is restricted to the testis but is activated in many human cancers. Depending on the particular expression pattern, the CTA genes are speculated to play a role in spermatogenesis, but evidence is limited thus far. Here, we reported patients with a hemizygous nonsense mutation in cancer-testis antigen 55 (CT55) suffering from male infertility with extreme disruption in sperm production, morphology, and locomotion. Specifically, the insufficiency of sperm individualization, excessive residue of unnecessary cytoplasm, and defects in acrosome development were evident in the spermatozoa of the patients. Furthermore, mouse models with depletion of Ct55 showed accelerated infertility with age, mimicking the defects in sperm individualization, unnecessary cytoplasm removal, and meanwhile exhibiting the disrupted cumulus-oocyte complex penetration. Mechanistically, our functional experiments uncovered CT55 as a new autophagic manipulator to regulate spermatogenesis via selectively interacting with LAMP2 and GABARAP (which are key regulators in the autophagy process) and further fine-tuning their expression. Therefore, our findings revealed CT55 as a novel CTA gene involved in spermatogenesis due to its unprecedented autophagy activity.


Subject(s)
Antigens, Nuclear , Infertility, Male , Neoplasms , Animals , Humans , Male , Mice , Infertility, Male/genetics , Infertility, Male/metabolism , Neoplasms/metabolism , Semen/metabolism , Spermatogenesis/genetics , Spermatozoa/metabolism , Testis/metabolism , Antigens, Nuclear/metabolism
7.
Cell Metab ; 35(1): 200-211.e9, 2023 01 03.
Article in English | MEDLINE | ID: mdl-36309010

ABSTRACT

Despite its central importance in cellular metabolism, many details remain to be determined regarding subcellular lactate metabolism and its regulation in physiology and disease, as there is sensitive spatiotemporal resolution of lactate distribution, and dynamics remains a technical challenge. Here, we develop and characterize an ultrasensitive, highly responsive, ratiometric lactate sensor, named FiLa, enabling the monitoring of subtle lactate fluctuations in living cells and animals. Utilizing FiLa, we demonstrate that lactate is highly enriched in mammalian mitochondria and compile an atlas of subcellular lactate metabolism that reveals lactate as a key hub sensing various metabolic activities. In addition, FiLa sensors also enable direct imaging of elevated lactate levels in diabetic mice and facilitate the establishment of a simple, rapid, and sensitive lactate assay for point-of-care clinical screening. Thus, FiLa sensors provide powerful, broadly applicable tools for defining the spatiotemporal landscape of lactate metabolism in health and disease.


Subject(s)
Diabetes Mellitus, Experimental , Animals , Mice , Diabetes Mellitus, Experimental/metabolism , Mitochondria/metabolism , Lactic Acid/metabolism , Mammals
8.
Int J Immunopathol Pharmacol ; 36: 3946320221111117, 2022.
Article in English | MEDLINE | ID: mdl-35731855

ABSTRACT

OBJECTIVE: Formononetin is a bioactive isoflavone that has numerous medicinal benefits. We explored the feasibility and its mechanism of formononetin on treating acute deep vein thrombosis (DVT) in rats. MATERIALS AND METHODS: Inferior vena cava (IVC) stenosis was performed to establish the DVT rat model. First, different doses of formononetin were used to observe the feasibility of formononetin on treating DVT. In sham and DVT groups, rats were orally treated with vehicle. In the remaining groups, formononetin (10 mg/kg, 20 mg/kg, and 40 mg/kg) was orally treated once a day for 7 days at 24 h after IVC. After 7 days, the levels of thrombosis and inflammation related factors in plasma were measured. The expression of endothelial nitric oxide synthase (eNOS) was analyzed by western blot and immunofluorescence. Molecular docking was used to evaluate the interaction between the formononetin and eNOS. Further, the NOS inhibitor (L-NAME) was used to explore the mechanism of formononetin for DVT. RESULT: After treatment with formononetin, the average weights of thrombosis were decreased, and the levels of thrombosis and inflammation related factors were also significantly decreased. Additionally, phosphorylation of eNOS was increased with the formononetin administration. There is a good activity of formononetin to eNOS (total score = -6.8). However, the effects of 40 mg/kg formononetin were concealed by the NOS inhibitor (L-NAME). CONCLUSION: Formononetin reduces vascular endothelium injury induced by DVT through increasing eNOS in rats, which provides a potential drug for treatment of venous thrombosis.


Subject(s)
Isoflavones , Venous Thrombosis , Animals , Endothelium, Vascular/metabolism , Enzyme Inhibitors/pharmacology , Inflammation/metabolism , Isoflavones/metabolism , Isoflavones/pharmacology , Isoflavones/therapeutic use , Molecular Docking Simulation , NG-Nitroarginine Methyl Ester/pharmacology , Nitric Oxide/metabolism , Nitric Oxide Synthase Type III/metabolism , Nitric Oxide Synthase Type III/pharmacology , Nitric Oxide Synthase Type III/therapeutic use , Rats , Rats, Sprague-Dawley , Venous Thrombosis/drug therapy
9.
J Mol Histol ; 53(1): 1-11, 2022 Feb.
Article in English | MEDLINE | ID: mdl-34813022

ABSTRACT

Abdominal aortic aneurysm (AAA) has been associated with the dysfunction of vascular smooth muscle cells (VSMCs) and extracellular matrix (ECM) remodelling. Runt-related transcription factor 3 (RUNX3) has been reported to be up-regulated in aneurysmal aorta samples compared with normal aorta. However, its function in VSMCs and the mechanism of function remains unknown. Therefore, our study aimed to investigate the role of RUNX3 in ECM remodelling and VSMC function, and further explore the underlying mechanism. Our results verified that RUNX3 was increased in aortic samples of AAA compared with healthy controls. Overexpression vectors of RUNX3 (ov-RUNX3) and siRNA of RUNX3 (si-RUNX3) were transfected into Human aortic smooth muscle cells (HAoSMCs). The results indicated that ov-RUNX3 promoted cell proliferation, migration, and MMP-2/3/9 secretion, and suppressed TIMP-1, collagen I/III, SM22, MYH11 and CNN1 expression in HAoSMCs. The silencing of RUNX3 has the opposite effect. Furthermore, we found that RUNX3 targets TGF-ß1 and suppressed its transcription. The silencing of TGF-ß1 increased cell proliferation, migration and MMP-2/3/9 expression, and inhibited TIMP-1, Collagen I/III, SM22, MYH11 and CNN1 expression. In addition, TGF-ß1 reversed the effect of RUNX3 overexpression on HAoSMCs. Hence, our study indicated that RUNX3 promotes cell proliferation, migration, and ECM remodelling through suppressing TGF-ß1.


Subject(s)
Aortic Aneurysm, Abdominal/genetics , Core Binding Factor Alpha 3 Subunit/genetics , Muscle, Smooth, Vascular/metabolism , Transforming Growth Factor beta1/metabolism , Up-Regulation/physiology , Adult , Aged , Aorta/cytology , Aortic Aneurysm, Abdominal/metabolism , Blotting, Western , Cell Movement/physiology , Cell Proliferation/physiology , Cells, Cultured , Enzyme-Linked Immunosorbent Assay , Extracellular Matrix Proteins/metabolism , Female , Humans , Male , Middle Aged , Plasmids/genetics , RNA, Small Interfering/genetics , Real-Time Polymerase Chain Reaction , Transfection
10.
J Laparoendosc Adv Surg Tech A ; 31(4): 462-467, 2021 Apr.
Article in English | MEDLINE | ID: mdl-33595365

ABSTRACT

Aim: Emergent biliary drainage is necessary due to acute symptoms of choledochal cysts. Percutaneous biliary drainage (PBD), laparoscopic bile duct drainage (LBD), and laparoscopic cholecystostomy (LC) are the three most common drainage treatments. So far, there is no comparative study on these three approaches, which all have been applied in our hospital. This article compares the drainage effects of these three approaches and illustrates their respective merits and demerits, with the aim of providing a reference for clinical application. Materials and Methods: We conducted a retrospective study of 20 children who underwent biliary drainage before their definitive surgery due to acute symptoms of choledochal cysts between June 2008 and May 2015. Among them, 6 underwent PBD, 8 underwent LBD, and 6 underwent LC. Results: Preoperative abdominal pain, fever, and jaundice symptoms were effectively relieved by the three approaches. There were no significant differences in terms of the recovery of liver functions. The average interval and duration of procedures of three groups were PBD (25.00 ± 4.47 minutes and 262.50 ± 35.74 minutes), LBD (84.37 ± 24.99 minutes and 283.75 ± 39.62 minutes), and LC (50.83 ± 13.57 minutes and 218.33 ± 28.58 minutes), respectively. Conclusions: LC has advantages of a comparatively simple operation and no foreign body sensation (external drain) in the hepatic duct, which is beneficial for relieving inflammation of the common bile duct, and thus is suitable for majority of patients needing external bile drainage. Meanwhile, PBD and LBD also have their respective applicable patients.


Subject(s)
Choledochal Cyst/surgery , Drainage/methods , Hepatic Duct, Common/surgery , Laparoscopy/methods , Abdominal Pain/surgery , Child , Child, Preschool , Female , Humans , Infant , Jaundice , Male , Retrospective Studies , Ultrasonography
11.
Mol Cell ; 80(4): 607-620.e12, 2020 11 19.
Article in English | MEDLINE | ID: mdl-33113344

ABSTRACT

Aberrant mitophagy has been implicated in a broad spectrum of disorders. PINK1, Parkin, and ubiquitin have pivotal roles in priming mitophagy. However, the entire regulatory landscape and the precise control mechanisms of mitophagy remain to be elucidated. Here, we uncover fundamental mitophagy regulation involving PINK1 and a non-canonical role of the mitochondrial Tu translation elongation factor (TUFm). The mitochondrion-cytosol dual-localized TUFm interacts with PINK1 biochemically and genetically, which is an evolutionarily conserved Parkin-independent route toward mitophagy. A PINK1-dependent TUFm phosphoswitch at Ser222 determines conversion from activating to suppressing mitophagy. PINK1 modulates differential translocation of TUFm because p-S222-TUFm is restricted predominantly to the cytosol, where it inhibits mitophagy by impeding Atg5-Atg12 formation. The self-antagonizing feature of PINK1/TUFm is critical for the robustness of mitophagy regulation, achieved by the unique kinetic parameters of p-S222-TUFm, p-S65-ubiquitin, and their common kinase PINK1. Our findings provide new mechanistic insights into mitophagy and mitophagy-associated disorders.


Subject(s)
Drosophila melanogaster/growth & development , Mitochondria/pathology , Mitochondrial Proteins/metabolism , Mitophagy , Peptide Elongation Factor Tu/metabolism , Protein Kinases/metabolism , Animals , Cytosol/metabolism , Drosophila melanogaster/genetics , Drosophila melanogaster/metabolism , Female , HeLa Cells , Humans , Male , Mitochondria/genetics , Mitochondria/metabolism , Mitochondrial Proteins/genetics , Peptide Elongation Factor Tu/genetics , Phosphorylation , Protein Interaction Domains and Motifs , Protein Kinases/genetics , Protein Transport , Ubiquitin-Protein Ligases/genetics , Ubiquitin-Protein Ligases/metabolism
12.
Aging Cell ; 19(9): e13210, 2020 09.
Article in English | MEDLINE | ID: mdl-32749068

ABSTRACT

How complex interactions of genetic, environmental factors and aging jointly contribute to dopaminergic degeneration in Parkinson's disease (PD) is largely unclear. Here, we applied frequent gene co-expression analysis on human patient substantia nigra-specific microarray datasets to identify potential novel disease-related genes. In vivo Drosophila studies validated two of 32 candidate genes, a chromatin-remodeling factor SMARCA4 and a biliverdin reductase BLVRA. Inhibition of SMARCA4 was able to prevent aging-dependent dopaminergic degeneration not only caused by overexpression of BLVRA but also in four most common Drosophila PD models. Furthermore, down-regulation of SMARCA4 specifically in the dopaminergic neurons prevented shortening of life span caused by α-synuclein and LRRK2. Mechanistically, aberrant SMARCA4 and BLVRA converged on elevated ERK-ETS activity, attenuation of which by either genetic or pharmacological manipulation effectively suppressed dopaminergic degeneration in Drosophila in vivo. Down-regulation of SMARCA4 or drug inhibition of MEK/ERK also mitigated mitochondrial defects in PINK1 (a PD-associated gene)-deficient human cells. Our findings underscore the important role of epigenetic regulators and implicate a common signaling axis for therapeutic intervention in normal aging and a broad range of age-related disorders including PD.


Subject(s)
DNA Helicases/genetics , Dopaminergic Neurons/physiology , Epigenesis, Genetic/genetics , MAP Kinase Signaling System/genetics , Nuclear Proteins/genetics , Transcription Factors/genetics , Aged , Aging , Animals , Disease Models, Animal , Humans
13.
Biosci Biotechnol Biochem ; 84(9): 1788-1798, 2020 Sep.
Article in English | MEDLINE | ID: mdl-32448038

ABSTRACT

Many phenolic compounds, derived from lignin during the pretreatment of lignocellulosic biomass, could obviously inhibit the activity of cellulolytic and hemicellulolytic enzymes. Acetosyringone (AS) is one of the phenolic compounds produced from lignin degradation. In this study, we investigated the inhibitory effects of AS on xylanase activity through kinetic experiments. The results showed that AS could obviously inhibit the activity of xylanase in a reversible and noncompetitive binding manner (up to 50% activity loss). Inhibitory kinetics and constants of xylanase on AS were conducted by the HCH-1 model (ß = 0.0090 ± 0.0009 mM-1). Furthermore, intrinsic and 8-anilino-1-naphthalenesulfonic (ANS)-binding fluorescence results showed that the tertiary structure of AS-mediated xylanase was altered. These findings provide new insights into the role of AS in xylanase activity. Our results also suggest that AS was an inhibitor of xylanase and targeting AS was a potential strategy to increase xylose production.


Subject(s)
Acetophenones/pharmacology , Endo-1,4-beta Xylanases/antagonists & inhibitors , Enzyme Inhibitors/pharmacology , Polysaccharides/metabolism , Hydrolysis/drug effects , Kinetics
14.
Nucleic Acids Res ; 48(10): e57, 2020 06 04.
Article in English | MEDLINE | ID: mdl-32232370

ABSTRACT

Site-specific DNA double-strand breaks have been used to generate knock-in through the homology-dependent or -independent pathway. However, low efficiency and accompanying negative impacts such as undesirable indels or tumorigenic potential remain problematic. In this study, we present an enhanced reduced-risk genome editing strategy we named as NEO, which used either site-specific trans or cis double-nicking facilitated by four bacterial recombination factors (RecOFAR). In comparison to currently available approaches, NEO achieved higher knock-in (KI) germline transmission frequency (improving from zero to up to 10% efficiency with an average of 5-fold improvement for 8 loci) and 'cleaner' knock-in of long DNA fragments (up to 5.5 kb) into a variety of genome regions in zebrafish, mice and rats. Furthermore, NEO yielded up to 50% knock-in in monkey embryos and 20% relative integration efficiency in non-dividing primary human peripheral blood lymphocytes (hPBLCs). Remarkably, both on-target and off-target indels were effectively suppressed by NEO. NEO may also be used to introduce low-risk unrestricted point mutations effectively and precisely. Therefore, by balancing efficiency with safety and quality, the NEO method reported here shows substantial potential and improves the in vivo gene-editing strategies that have recently been developed.


Subject(s)
Bacterial Proteins/metabolism , Gene Editing/methods , Animals , DNA Breaks, Double-Stranded , DNA-Binding Proteins/metabolism , Female , Gene Knock-In Techniques , Genomics , Homologous Recombination , Humans , INDEL Mutation , Macaca fascicularis , Mice , Rats, Sprague-Dawley , Rec A Recombinases/metabolism , Zebrafish/genetics
15.
Cell Cycle ; 18(24): 3472-3490, 2019 Dec.
Article in English | MEDLINE | ID: mdl-31713447

ABSTRACT

Protein kinase CK2 alpha (CK2α) is involved in the development of multiple malignancies. Overexpression of Y-box binding protein 1 (YBX1) is related to tumor proliferation, drug resistance, and poor prognosis. Studies have demonstrated that both CK2 and YBX1 could regulate the PI3K/AKT pathway. In addition, we predicted that CK2 might be the upstream kinase of YBX1 through the Human Protein Reference Database (HPRD). Herein, we hypothesize that CK2 may interact with YBX1 and they regulate the PI3K/AKT signaling pathway together. Expressions of CK2α and YBX1 in cancer cell lines were evaluated by immunoblotting. The results showed that CK2α could regulate the expression of YBX1 at the transcriptional level, which is dependent on its enzymatic activity. Synergistic effects of PI3K/AKT pathway inactivation could be observed through combined inhibition of CK2α and YBX1, and YBX1 was required for CK2α-induced PI3K/AKT pathway activation. Further results demonstrated that CK2α could interact with YBX1 and PI3K/AKT antagonist decreased cell resistance to doxorubicin induced by co-activation of CK2α and YBX1. These results indicated that combined inhibition of CK2α and YBX1 showed synergistic effects in inactivating the PI3K/AKT signaling pathway and may be one of the mechanisms involved in tumor growth and migration.


Subject(s)
Doxorubicin/pharmacology , Drug Resistance, Neoplasm/genetics , Neoplasms/drug therapy , Y-Box-Binding Protein 1/genetics , Casein Kinase II/antagonists & inhibitors , Casein Kinase II/genetics , Cell Movement/drug effects , Cell Proliferation/drug effects , Gene Expression Regulation, Neoplastic/drug effects , HeLa Cells , Hep G2 Cells , Humans , Neoplasms/genetics , Neoplasms/pathology , Oncogene Protein v-akt/genetics , Phosphatidylinositol 3-Kinases/genetics , Protein Binding/genetics , Signal Transduction/genetics , Y-Box-Binding Protein 1/antagonists & inhibitors
16.
Cell Rep ; 26(1): 209-221.e5, 2019 01 02.
Article in English | MEDLINE | ID: mdl-30605677

ABSTRACT

Fatty acid uptake is the first step in fatty acid utilization, but it remains unclear how the process is regulated. Protein palmitoylation is a fatty acyl modification that plays a key regulatory role in protein targeting and trafficking; however, its function in regulating fatty acid metabolism is unknown. Here, we show that two of the Asp-His-His-Cys (DHHC) motif-containing palmitoyl acyltransferases, DHHC4 and DHHC5, regulate fatty acid uptake. DHHC4 and DHHC5 function at different subcellular localizations to control the palmitoylation, plasma membrane localization, and fatty acid uptake activity of the scavenger receptor CD36. Depletion of either DHHC4 or DHHC5 in cells disrupts CD36-dependent fatty acid uptake. Furthermore, both Dhhc4-/- and adipose-specific Dhhc5 knockout mice show decreased fatty acid uptake activity in adipose tissues and develop severe hypothermia upon acute cold exposure. These findings demonstrate a critical role of DHHC4 and DHHC5 in regulating fatty acid uptake.


Subject(s)
Acyltransferases/metabolism , CD36 Antigens/metabolism , Fatty Acids/metabolism , Lipoylation , Membrane Proteins/metabolism , 3T3-L1 Cells , Adipose Tissue/metabolism , Amino Acid Sequence , Animals , Biological Transport , Cell Membrane/metabolism , HEK293 Cells , Humans , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Transfection
17.
Biochem Biophys Res Commun ; 502(2): 250-254, 2018 07 12.
Article in English | MEDLINE | ID: mdl-29803677

ABSTRACT

Adenosine kinase (ADK) plays an important role in the growth and development of organisms. A convenient, quick, reliable, sensitive and low-cost assay for ADK activity is of great significance. Here, we found the reaction system with bromothymol blue as the pH indicator had a maximum absorption peak at 614 nm. The absorbance change in 614 nm was positively correlated with the generated hydrogen ions in the reaction catalyzed by ADK. Then, we demonstrated this assay was feasible for ADK activity. Further, we analyzed the effects of buffer, bromothymol blue concentrations on the sensitivity of the assay, and investigated the sensitivity of ADK contents and adenosine concentration on the assay. Finally, we calculated the Km and Vmax of ADK from Bombyx mori with this assay. Our results suggested this assay was quick, convenient, reliable, sensitive and economic for the activity of ADK. It is an excellent alternative for the conventional ADK assays.


Subject(s)
Adenosine Kinase/analysis , Colorimetry/methods , Adenosine Kinase/chemistry , Adenosine Kinase/metabolism , Animals , Bombyx/enzymology , Bromphenol Blue , Bromthymol Blue , Buffers , Colorimetry/statistics & numerical data , Coloring Agents , Feasibility Studies , Hydrogen-Ion Concentration , Kinetics , Spectrophotometry
18.
Arch Insect Biochem Physiol ; 98(3): e21463, 2018 Jul.
Article in English | MEDLINE | ID: mdl-29569264

ABSTRACT

Alpha-tubulin N-acetyltransferase 1 (ATAT1) is an acetyltransferase specific to α-tubulin and performs important functions in many cellular processes. Bombyx mori is an economic insect and also known as a model lepidoptera insect. In this study, we cloned a B. mori ATAT1 gene (BmATAT1) (Gen Bank accession number: XP_004932777.1). BmATAT1 contained an open reading frame (ORF) of 1,065 bp encoding 355 amino acids (aa). Expression profiling of BmATAT1 protein showed that the expression levels of BmATAT1 at different developmental stages and different tissues in fifth-instar larvae differ. BmATAT1 was highly expressed at the egg stage and in the head of the fifth-instar larvae. Subcellular localization showed that BmATAT1 was distributed in the cytoplasm and nucleus. Furthermore, BmATAT1 may lead to time-dependent induction of cell cycle arrest in the G2/M phase by flow cytometry analysis. Interestingly, using site-specific mutation, immunoprecipitation, and Western blotting, we further found a BmATAT1 acetylated site at K156, suggesting that this acetyltransferase could be regulated by acetylation itself.


Subject(s)
Acetyltransferases/metabolism , Bombyx/enzymology , Tubulin/metabolism , Animals , Cell Cycle , Insect Proteins/metabolism , Larva/enzymology
19.
Nat Methods ; 14(7): 720-728, 2017 Jul.
Article in English | MEDLINE | ID: mdl-28581494

ABSTRACT

Reduced nicotinamide adenine dinucleotide phosphate (NADPH) is essential for biosynthetic reactions and antioxidant functions; however, detection of NADPH metabolism in living cells remains technically challenging. We develop and characterize ratiometric, pH-resistant, genetically encoded fluorescent indicators for NADPH (iNap sensors) with various affinities and wide dynamic range. iNap sensors enabled quantification of cytosolic and mitochondrial NADPH pools that are controlled by cytosolic NAD+ kinase levels and revealed cellular NADPH dynamics under oxidative stress depending on glucose availability. We found that mammalian cells have a strong tendency to maintain physiological NADPH homeostasis, which is regulated by glucose-6-phosphate dehydrogenase and AMP kinase. Moreover, using the iNap sensors we monitor NADPH fluctuations during the activation of macrophage cells or wound response in vivo. These data demonstrate that the iNap sensors will be valuable tools for monitoring NADPH dynamics in live cells and gaining new insights into cell metabolism.


Subject(s)
Gene Expression Regulation/physiology , Luminescent Proteins/metabolism , NADP/metabolism , AMP-Activated Protein Kinases/genetics , AMP-Activated Protein Kinases/metabolism , Animals , Cell Survival , Glucose , Homeostasis , Humans , Luminescent Proteins/chemistry , Luminescent Proteins/genetics , Mice , Models, Molecular , Oxidative Stress , Protein Binding , Protein Conformation , Protein Domains , Protein Engineering
20.
Int J Biol Macromol ; 105(Pt 3): 1654-1662, 2017 Dec.
Article in English | MEDLINE | ID: mdl-27940338

ABSTRACT

Alpha-ketoglutaric acid (AKG) is naturally found in organisms and is a well-known intermediate in the production of ATP or GTP in the Krebs cycle. We elucidated the effects of AKG on tyrosinase activity and conformation via methods of inhibition kinetics integrated with molecular dynamics (MD) simulations. AKG was found to be a reversible inhibitor of tyrosinase (IC50=15±0.5mM) and induced parabolic slope mixed-type inhibition. Based on our newly established equation, the dissociation constant (Kislope) was determined to be 7.93±0.31mM. The spectrofluorimetry studies showed that AKG mainly induced regional changes in the active site of tyrosinase, which reflects the flexibility of the active site. The computational docking and molecular dynamics (MD) simulations further demonstrated that AKG could interact with several residues near the substrate-binding site located in the tyrosinase active site pocket. Our study provides insight into the mechanism by which energy-producing intermediates such as AKG inhibit tyrosinase through its ketone groups. Also, AKG could be a potential natural antipigmentation agent due to its non-toxic property.


Subject(s)
Ketoglutaric Acids/pharmacology , Molecular Dynamics Simulation , Monophenol Monooxygenase/chemistry , Monophenol Monooxygenase/metabolism , Catalytic Domain/drug effects , Ketoglutaric Acids/metabolism , Kinetics , Molecular Docking Simulation
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