Exercise accelerates recruitment of CD8+ T cell to promotes anti-tumor immunity in lung cancer via epinephrine.

BACKGROUND AND PURPOSE: In recent years, there has been extensive research on the role of exercise as an adjunctive therapy for cancer. However, the potential mechanisms underlying the anti-tumor therapy of exercise in lung cancer remain to be fully elucidated. As such, our study aims to confirm whether exercise-induced elevation of epinephrine can accelerate CD8+ T cell recruitment through modulation of chemokines and thus ultimately inhibit tumor progression. METHOD: C57BL/6 mice were subcutaneously inoculated with Lewis lung cancer cells (LLCs) to establish a subcutaneous tumor model. The tumor mice were randomly divided into different groups to performed a moderate-intensity exercise program on a treadmill for 5 consecutive days a week, 45 min a day. The blood samples and tumor tissues were collected after exercise for IHC, RT-qPCR, ELISA and Western blot. In addition, another group of mice received daily epinephrine treatment for two weeks (0.05 mg/mL, 200 µL i.p.) (EPI, n = 8) to replicate the effects of exercise on tumors in vivo. Lewis lung cancer cells were treated with different concentrations of epinephrine (0, 5, 10, 20 µM) to detect the effect of epinephrine on chemokine levels via ELISA and RT-qPCR. RESULTS: This study reveals that both pre- and post-cancer exercise effectively impede the tumor progression. Exercise led to an increase in EPI levels and the infiltration of CD8+ T cell into the lung tumor. Exercise-induced elevation of EPI is involved in the regulation of Ccl5 and Cxcl10 levels further leading to enhanced CD8+ T cell infiltration and ultimately inhibiting tumor progression. CONCLUSION: Exercise training enhance the anti-tumor immunity of lung cancer individuals. These findings will provide valuable insights for the future application of exercise therapy in clinical practice.

MEN1 deficiency stabilizes PD-L1 and promotes tumor immune evasion of lung cancer.

Multiple Endocrine Neoplasia 1 gene (MEN1), which is known to be a tumor suppressor gene in lung tissues, encodes a 610 amino acid protein menin. Previous research has proven that MEN1 deficiency promotes the malignant progression of lung cancer. However, the biological role of this gene in the immune microenvironment of lung cancer remains unclear. In this study, we found that programmed cell death-ligand 1 (PD-L1) is upregulated in lung-specific KrasG12D mutation-induced lung adenocarcinoma in mice, after Men1 deficiency. Simultaneously, CD8+ and CD3+ T cells are depleted, and their cytotoxic effects are suppressed. In vitro, PD-L1 is inhibited by the overexpression of menin. Mechanistically, we found that MEN1 inactivation promotes the deubiquitinating activity of COP9 signalosome subunit 5 (CSN5) and subsequently increases the level of PD-L1.

High mobility group box 1 mediates inflammatory responses in malignant peritoneal mesothelioma.

BACKGROUND: Serum high mobility group box 1 (HMGB1) serves as a diagnostic biomarker for malignant peritoneal mesothelioma (MPM) patients, yet its diagnostic significance within MPM tumor tissues remains uncertain. This study aims to elucidate the roles of HMGB1 in MPM. METHODS: HMGB1 expression analysis was conducted in both tumor and adjacent non-cancerous tissues collected from MPM patients. The two-year follow-up of MPM patients commenced from the diagnosis date. Inflammatory cytokine analysis was performed on these tissues, and Pearson correlation coefficient analysis was applied to examine variable relationships. In vitro assays included constructing an HMGB1 knockdown cell line, assessing cell viability, apoptosis, and inflammatory cytokine levels to delineate HMGB1's roles in MPM. RESULTS: HMGB1 overexpression was observed in MPM tumor tissues, particularly in stages III-IV. Diagnostic implications of HMGB1 for MPM were evident, augmenting its diagnostic value. HMGB1 overexpression correlated with diminished survival rates. Positive correlations existed between inflammatory cytokines and HMGB1 in MPM tumor tissues and cell lines. Suppression of HMGB1 regulated cell growth and apoptosis in MPM cell lines. CONCLUSION: HMGB1 exhibits diagnostic potential for MPM and modulates inflammatory responses within the disease context.

Angiotensin II type-2 receptor signaling facilitates liver injury repair and regeneration via inactivation of Hippo pathway.

The angiotensin II type 2 receptor (AT2R) is a well-established component of the renin-angiotensin system and is known to counteract classical activation of this system and protect against organ damage. Pharmacological activation of the AT2R has significant therapeutic benefits, including vasodilation, natriuresis, anti-inflammatory activity, and improved insulin sensitivity. However, the precise biological functions of the AT2R in maintaining homeostasis in liver tissue remain largely unexplored. In this study, we found that the AT2R facilitates liver repair and regeneration following acute injury by deactivating Hippo signaling and that interleukin-6 transcriptionally upregulates expression of the AT2R in hepatocytes through STAT3 acting as a transcription activator binding to promoter regions of the AT2R. Subsequently, elevated AT2R levels activate downstream signaling via heterotrimeric G protein Gα12/13-coupled signals to induce Yap activity, thereby contributing to repair and regeneration processes in the liver. Conversely, a deficiency in the AT2R attenuates regeneration of the liver while increasing susceptibility to acetaminophen-induced liver injury. Administration of an AT2R agonist significantly enhances the repair and regeneration capacity of injured liver tissue. Our findings suggest that the AT2R acts as an upstream regulator in the Hippo pathway and is a potential target in the treatment of liver damage.

Appendicitis combined with Meckel's diverticulum obstruction, perforation, and inflammation in children: Three case reports.

BACKGROUND: Meckel's diverticulum is a common congenital malformation of the small intestine, with the three most common complications being obstruction, perforation, and inflammation. To date, only a few cases have been reported worldwide. In children, the clinical symptoms are similar to appendicitis. As most of the imaging features are nonspecific, the preoperative diagnosis is not precise. In addition, the clinical characteristics are highly similar to pediatric acute appendicitis, thus special attention is necessary to distinguish Meckel's diverticulum from pediatric appendicitis. Patients with poor disease control should undergo laparoscopic exploration to avoid serious complications, including intestinal necrosis, intestinal perforation and gastrointestinal bleeding. CASE SUMMARY: This report presents three cases of appendicitis in children combined with intestinal obstruction, which was caused by fibrous bands (ligaments) arising from the top part of Meckel's diverticulum, diverticular perforation, and diverticular inflammation. All three patients, aged 11-12 years, had acute appendicitis as their initial clinical presentation. All were treated by laparoscopic surgery with a favorable outcome. A complete dataset including clinical presentation, diagnostic imaging, surgical information, and histopathologic findings was also provided. CONCLUSION: Preoperative diagnosis of Meckel's diverticulum and its complications is challenging because its clinical signs and complications are similar to those of appendicitis in children. Laparoscopy combined with laparotomy is useful for diagnosis and treatment.

Laser-driven hierarchical "gas-needles" for programmable and high-precision proximity transfer printing of microchips.

Micro-transfer printing (µTP) techniques are essential for advanced electronics. However, current contact/noncontact µTP techniques fail to simultaneously achieve high selectivity and transfer accuracy. Here, a laser projection proximity transfer (LaserPPT) technique is presented, which assembles the microchips in an approach-and-release manner, combining high-precision parallelism with individual chip control. An embedded carbon layer with a thin gas layer is generated by an ultraviolet laser, followed by absorbing heat from the infrared laser, to enable the sequential expansion of hierarchical "gas-needles." The level 1 large gas-needle with a substantially growing height can reduce the gap between the microchip and the receiver. Then, the level 2 small gas-needles enable the gentle release of a chip. Therefore, the LaserPPT can obtain a strong adhesion modulation (~1000 times), excellent size scalability (<100 micrometers), and high transfer accuracy of ~4 micrometers. Last, the assembly of a micro-light-emitting diode display demonstrates the capabilities for deterministic assembly of microarrays.

ETV2 Enhances CXCL5 Secretion from Endothelial Cells, Leading to the Promotion of Vascular Smooth Muscle Cell Migration.

Abnormal communication between endothelial cells (ECs) and vascular smooth muscle cells (VSMCs) promotes vascular diseases, including atherogenesis. ETS variant transcription factor 2 (ETV2) plays a substantial role in pathological angiogenesis and the reprogramming of ECs; however, the role of ETV2 in the communication between ECs and VSMCs has not been revealed. To investigate the interactive role of ETV2 in the EC to VSMC phenotype, we first showed that treatment with a conditioned medium from ETV2-overexpressed ECs (Ad-ETV2 CM) significantly increased VSMC migration. The cytokine array showed altered levels of several cytokines in Ad-ETV2 CM compared with those in normal CM. We found that C-X-C motif chemokine 5 (CXCL5) promoted VSMC migration using the Boyden chamber and wound healing assays. In addition, an inhibitor of C-X-C motif chemokine receptor 2 (CXCR2) (the receptor for CXCL5) significantly inhibited this process. Gelatin zymography showed that the activities of matrix metalloproteinase (MMP)-2 and MMP-9 increased in the media of VSMCs treated with Ad-ETV2 CM. Western blotting revealed a positive correlation between Akt/p38/c-Jun phosphorylation and CXCL5 concentration. The inhibition of Akt and p38-c-Jun effectively blocked CXCL5-induced VSMC migration. In conclusion, CXCL5 from ECs induced by ETV2 promotes VSMC migration via MMP upregulation and the activation of Akt and p38/c-Jun.

Noninvasive prediction of node-positive breast cancer response to presurgical neoadjuvant chemotherapy therapy based on machine learning of axillary lymph node ultrasound.

OBJECTIVES: To explore an optimal model to predict the response of patients with axillary lymph node (ALN) positive breast cancer to neoadjuvant chemotherapy (NAC) with machine learning using clinical and ultrasound-based radiomic features. METHODS: In this study, 1014 patients with ALN-positive breast cancer confirmed by histological examination and received preoperative NAC in the Affiliated Hospital of Qingdao University (QUH) and Qingdao Municipal Hospital (QMH) were included. Finally, 444 participants from QUH were divided into the training cohort (n = 310) and validation cohort (n = 134) based on the date of ultrasound examination. 81 participants from QMH were used to evaluate the external generalizability of our prediction models. A total of 1032 radiomic features of each ALN ultrasound image were extracted and used to establish the prediction models. The clinical model, radiomics model, and radiomics nomogram with clinical factors (RNWCF) were built. The performance of the models was assessed with respect to discrimination and clinical usefulness. RESULTS: Although the radiomics model did not show better predictive efficacy than the clinical model, the RNWCF showed favorable predictive efficacy in the training cohort (AUC, 0.855; 95% CI 0.817-0.893), the validation cohort (AUC, 0.882; 95% CI 0.834-0.928), and the external test cohort (AUC, 0.858; 95% CI 0.782-0.921) compared with the clinical factor model and radiomics model. CONCLUSIONS: The RNWCF, a noninvasive, preoperative prediction tool that incorporates a combination of clinical and radiomics features, showed favorable predictive efficacy for the response of node-positive breast cancer to NAC. Therefore, the RNWCF could serve as a potential noninvasive approach to assist personalized treatment strategies, guide ALN management, avoiding unnecessary ALND.

Expanding the Promiscuity of a Copper-Dependent Oxidase for Enantioselective Cross-Coupling of Indoles.

Herein, we disclose the highly enantioselective oxidative cross-coupling of 3-hydroxyindole esters with various nucleophilic partners as catalyzed by copper efflux oxidase. The biocatalytic transformation delivers functionalized 2,2-disubstituted indolin-3-ones with excellent optical purity (90-99 % ee), which exhibited anticancer activity against MCF-7 cell lines, as shown by preliminary biological evaluation. Mechanistic studies and molecular docking results suggest the formation of a phenoxyl radical and enantiocontrol facilitated by a suited enzyme chiral pocket. This study is significant with regard to expanding the catalytic repertoire of natural multicopper oxidases as well as enlarging the synthetic toolbox for sustainable asymmetric oxidative coupling.

Analysis of the Curative Effect of Temporomandibular Joint Disc Release and Fixation Combined with Chitosan Injection in the Treatment of Temporomandibular Joint Osteoarthrosis.

OBJECTIVE: Temporomandibular joint osteoarthritis (TMJ-OA) is common in clinic. The purpose of this study was to evaluate the efficacy of disc release, fixation and chitosan injection in the treatment of TMJ-OA. METHODS: From March 2021 to March 2022, 32 patients who underwent the unilateral reduction and fixation of temporomandibular joint disc release were retrospectively studied. All patients were diagnosed with TMJ-OA and were treated with chitosan injection. This group of patients was analyzed by the visual analog scale (VAS) for pain and improvement of maximum comfortable mouth opening before treatment and 6 months after treatment. A paired t-test was used to evaluate the treatment effect, and p < 0.05 indicated that the difference was statistically significant. RESULTS: All 32 patients were successfully treated by surgery and chitosan injection in the second week after operation. The duration of disease in this group ranged from 1 to 10 months, with an average of 5.7 months. After 6 months of follow up, 30 patients were satisfied with the treatment and two were unsatisfied. The difference in the treatment effect was found to be statistically significant (p < 0.05). CONCLUSIONS: Temporomandibular joint disc release and fixation combined with chitosan injection is effective in the treatment of TMJ-OA.

Correlation of serum-soluble mesothelin-related protein, HMGB1 and CA125 in diffuse malignant peritoneal mesothelioma and their combined measurement for prognosis.

Diagnosis of diffuse malignant peritoneal mesothelioma (DMPM) is challenging due to the lack of efficient biomarkers for early-stage DMPM. This study was designed to characterize three serum-soluble mesothelium-related proteins, including soluble mesothelin-related protein (SMRP), high mobility group box 1 (HMGB1), and cancer antigen 125 (CA125) in diagnosing DMPM. Serum samples of DMPM patients and healthy controls were collected and an enzyme-linked immunosorbent assay (ELISA) was used to measure the levels of HMGB1, CA125, and SMRP. Correlations between these three serum proteins were examined and the diagnostic values of the biomarkers were assessed by receiver operating curve (ROC) analysis. The combined expression levels of the three markers were also analyzed in terms of predicting patient survival. Higher levels of CA125, SMRP, and HMGB1 was found to be a prominent characteristic of DMPM patients (with > two-fold higher for all levels in DMPM patients compared to control patients, all p < .001), particularly for those with higher-stage DMPM (stage III-IV) compared with lower-stage DMPM (stage I-II) (all p < .05). HMGB1, CA125, and SMRP were all significantly inter-correlated with each other (all p < .05), the combination of the three serum markers had high sensitivity and specificity for diagnosing DMPM. Combined values of the three markers demonstrated a high AUC of 0.85, sensitivity of 78.95%, specificity of 82.75% for identifying DMPM. The combined level of the three markers also demonstrated a significant positive correlation with poor survival of DMPM patients (p = .022). CA125, SMRP, and HMGB1 are potentially valuable diagnostic biomarkers to facilitate the diagnosis of DMPM.

PKCßII activation requires nuclear trafficking for phosphorylation and Mdm2-mediated ubiquitination.

PKCßII, a conventional PKC family member, plays critical roles in the regulation of a variety of cellular functions. Here, we employed loss-of-function approaches and mutants of PKCßII with altered phosphorylation and protein interaction behaviors to identify the cellular mechanisms underlying the activation of PKCßII. Our results show that 3-phosphoinositide-dependent protein kinase-1 (PDK1)-mediated constitutive phosphorylation of PKCßII at the activation loop (T500) is required for phorbol ester-induced nuclear entry and subsequent Mdm2-mediated ubiquitination of PKCßII, whereas ubiquitination of PKCßII is required for the PDK1-mediated inducible phosphorylation of PKCßII at T500 in the nucleus. After moving out of the nucleus, PKCßII interacts with actin, undergoes inducible mTORC2-mediated phosphorylation at the turn motif (T641), interacts with clathrin, and then translocates to the plasma membrane. This overall cascade of cellular events intertwined with the phosphorylation at critical residues and Mdm2-mediated ubiquitination in the nucleus and along with interactions with actin and clathrin plays roles that encompass the core processes of PKC activation.

Efficacy analysis of splint combined with platelet-rich plasma in the treatment of temporomandibular joint osteoarthritis.

Objective: To evaluate the efficacy of splints combined with PRP for the treatment of temporomandibular joint osteoarthritis. Methods: Thirty-one patients with temporomandibular joint osteoarthritis who were treated with splints combined with platelet-rich plasma (PRP) from January 2021 to June 2021 at the Department of Oral and Maxillofacial Surgery, School of Stomatology, China Medical University (Shenyang, China) were retrospectively reviewed. The VAS scores of all the patients were recorded before and 6 months after treatment, and the maximum comfortable mouth opening was recorded. All data were analyzed by the paired t-test using SPSS software, and a p-value < 0.05 indicated statistically significant differences. Results: Splint + PRP treatment was successful in 31 patients. The mean pretreatment VAS score was 6.1, and the mean VAS score 6 months posttreatment was 4.1. The posttreatment VAS score was significantly lower than the preoperative VAS score (p < 0.05). The mean pretreatment maximum comfortable mouth opening (MCMO) was 27.6 mm, and the mean MCMO 6 months posttreatment was 34.8 mm. The MCMO was significantly increased (p < 0.05). Conclusion: Splint + PRP is an effective treatment for temporomandibular joint osteoarthritis.

Magnetic thermo-responsive branched polymer for fast extraction and enrichment of phenolic acids in olive oil with tunable and enhanced performance.

Magnetic thermo-responsive branched polymer (Fe3O4@poly(glycidyl methacrylate)@poly(N-isopropylacrylamide)) was fabricated for the first time and applied for microwave-assisted magnetic solid phase extraction of phenolic acids in olive oil samples followed by ultra-high performance liquid chromatography-tandem mass spectrometry analysis in multiple reaction monitoring mode. Owing to the controllable molecular weight of poly(glycidyl methacrylate) synthesized by atom transfer radical polymerization and the thermo-responsive characteristic of poly(N-isopropylacrylamide), extraction performance could be efficiently tuned and enhanced. The whole sample pretreatment process was accomplished within 1 min with the help of the microwave. The nanocomposites were characterized by transmission electron microscope, scanning electron microscope, Fourier transform infrared spectroscopy, thermogravimetric analysis, vibrating sample magnetometer, water contact angles and dynamic light scattering. The adsorption experimental data fitted well with the Freundlich isotherm model and followed the pseudo-second-order kinetic model. The factors affecting the extraction process including adsorbent amount, adsorption time, sample volume, desorption conditions and interferents were investigated and optimized. Under the most favorable conditions, the developed method showed good linearity (R2 ≥ 97.98%) in the range of 0.2-30 µg L-1, low limits of detection (0.005-0.030 µg L-1) and limits of quantification (0.016-0.098 µg L-1) as well as satisfactory precision (RSDs≤4.85%). Our proposed method was successfully used for determination of phenolic acids in olive oil samples and satisfactory recoveries at three spiked concentration levels were in the range of 84.6-108.1% with RSDs less than 9.20%. Coupled with principal component analysis, our developed method proved promising for fast and convenient differentiation between extra virgin olive oils and refined olive oils.

Accurate transfer of bimaxillary orthognathic surgical plans using computer-aided intraoperative navigation.

OBJECTIVE: To examine the accuracy of computer-aided intraoperative navigation (Ci-Navi) in bimaxillary orthognathic surgery by comparing preoperative planning and postoperative outcome. METHODS: The study comprised 45 patients with congenital dentomaxillofacial deformities who were scheduled to undergo bimaxillary orthognathic surgery. Virtual bimaxillary orthognathic surgery was simulated using Mimics software. Intraoperatively, a Le Fort I osteotomy of the maxilla was performed using osteotomy guide plates. After the Le Fort I osteotomy and bilateral sagittal split ramus osteotomy of the mandible, the mobilized maxilla and the distal mandibular segment were fixed using an occlusal splint, forming the maxillomandibular complex (MMC). Realtime Ci-Navi was used to lead the MMC in the designated direction. Osteoplasty of the inferior border of the mandible was performed using Ci-Navi when facial symmetry and skeletal harmony were of concern. Linear and angular distinctions between preoperative planning and postoperative outcomes were calculated. RESULTS: The mean linear difference was 0.79 mm (maxilla: 0.62 mm, mandible: 0.88 mm) and the overall mean angular difference was 1.20°. The observed difference in the upper incisor point to the Frankfort horizontal plane, midfacial sagittal plane, and coronal plane was < 1 mm in 40 cases. CONCLUSIONS: This study demonstrates the role of Ci-Navi in the accurate positioning of bone segments during bimaxillary orthognathic surgery. Ci-Navi was found to be a reliable method for the accurate transfer of the surgical plan during an operation.

Randomized Controlled Clinical Trial to Assess the Utility of Computer-Aided Intraoperative Navigation in Bimaxillary Orthognathic Surgery.

ABSTRACT: Accurate application of the preoperative surgical plan in actual surgical settings is of paramount importance in orthognathic surgery. This randomized controlled clinical trial aimed to evaluate the accuracy of computer-aided intraoperative navigation (Ci-Navi) compared with that of conventional navigation methods in bimaxillary orthognathic surgery. Fifty-two patients were randomly divided into 2 groups. Group A (n = 26) patients underwent surgery assisted with Ci-Navi and group B (n = 26) patients underwent surgery assisted with conventional intraoperative navigation methods. During the operation, after LeFort I osteotomy, the mobile maxilla was repositioned to the designated position either using assistance from real-time Ci-Navi (group A) or using an intermediate splint (group B). Intra- and intergroup linear and angular differences between preoperative planning and postoperative outcomes were calculated. In group A, the overall mean linear difference was 0.79 mm (0.62 mm for the maxilla and 0.88 mm for the mandible) and the overall mean angular difference was 1.20°. In 23 cases, the difference from the upper incisor point to the Frankfort horizontal plane, midfacial sagittal plane, and coronal plane was less than 1 mm. In group B, the overall mean linear difference was 1.98 mm (1.76 mm for the maxilla and 2.02 mm for the mandible) and the overall mean angular difference was 2.08°. The difference from the upper incisor point to the Frankfort horizontal plane, midfacial sagittal plane, and coronal plane was less than 1 mm in 15 cases. This study demonstrates the utility of Ci-Navi is superior to the conventional methods in aiding the accurate repositioning of bony segments in bimaxillary orthognathic surgery.

Cytoplasmic recruitment of Mdm2 as a common characteristic of G protein-coupled receptors that undergo desensitization.

Desensitization of G protein-coupled receptors (GPCRs) represents a gradual attenuation of receptor responsiveness by continuous or repeated exposure to agonists. The most widely accepted molecular mechanism responsible for desensitization is that of GRK2-mediated receptor phosphorylation followed by association with ß-arrestins. However, in most cases, this mechanism cannot explain the desensitization of GPCRs. In this study, we investigated whether there exists a direct correlation between desensitization and certain cellular events that commonly observed with desensitizing receptors. Our study showed that constitutive ubiquitination of ß-arrestin, accompanied by nuclear to cytoplasmic translocation of Mdm2, was observed in cells expressing desensitizing GPCRs (dopamine D3 receptor, K149C-dopamine D2 receptor, ß2 adrenoceptor, and lysophosphatidic acid receptor 1). In contrast, Mdm2 was observed in the nucleus in cells expressing non-desensitizing GPCRs (dopamine D2 receptor, C147K-dopamine D3 receptor, and dopamine D4 receptor). Molecular manipulation to convert the characteristics of the dopamine D4 receptor from non-desensitizing to desensitizing changed the status of subcellular localization of Mdm2 from nuclear to cytoplasmic. With repeated agonist treatments of desensitizing receptors, Mdm2 translocated from cytoplasm to nucleus, resulting in the deubiquitination of ß-arrestins. This study suggests that the property of a receptor that causes a change in subcellular localization of Mdm2, from the nuclear to cytoplasmic, could be used as a biomarker to predict the desensitization of a receptor.

A novel molecular mechanism responsible for phosphorylation-independent desensitization of G protein-coupled receptors exemplified by the dopamine D3 receptor.

GRK-mediated receptor phosphorylation followed by association with ß-arrestins has been proposed to be the molecular mechanism involved in the desensitization of G protein-coupled receptors (GPCRs). However, this mechanism does not explain the desensitization of some GPCRs, such as dopamine D3 receptor (D3R), which does not undergo GRK-mediated phosphorylation. Loss-of-function approaches and mutants of dopamine D2 receptor and D3R, which exhibit different desensitization properties, were used to identify the cellular components and processes responsible for desensitization. D3R mediated the recruitment of Mdm2 to the cytosol, which resulted in the constitutive ubiquitination of ß-arrestin2 in the resting state. Under desensitization conditions, cytosolic Mdm2 returned to the nucleus, resulting in the deubiquitination of cytosolic ß-arrestins. Deubiquitinated ß-arrestins formed a tight complex with Gßγ, thereby sequestering it, causing interference in D3R signaling. In conclusion, this study shows that ß-arrestins, depending on their ubiquitination status, control the G protein cycling by regulating their interactions with Gßγ. This is a novel mechanism proposed to explain how certain GPCRs can undergo desensitization without receptor phosphorylation.

Highly Efficient Peptide-Based Click Chemistry for Proteomic Profiling of Nascent Proteins.

Copper-catalyzed azide-alkyne cycloaddition (CuAAC) has been widely used in a variety of scientific research, including dynamic proteomics. The current well-established protocols of CuAAC for proteomics analysis introduce labeling tags (azide- or alkyne-containing reagents) at the protein level, followed by downstream analysis by mass spectrometry. In the present study, a new method for proteomic profiling of nascent proteins relying on highly efficient peptide-based click chemistry is proposed, in which the CuAAC reaction was performed at the peptide level, leading to a significant increase in efficiency of the click conjugation reaction. A remarkable improvement in identification rate for spectrum, distinct peptide, and protein was achieved when proteins to be analyzed were proteolytically cleaved into peptides prior to the click conjugation reaction, which would be beneficial to downstream proteomics analysis, especially for the detection of AHA-tagged proteins in very low amounts.

Mdm2-mediated ubiquitination of ß-arrestin2 in the nucleus occurs in a Gßγ- and clathrin-dependent manner.

The fate and activity of ß-arrestin2, a key player in the regulation of desensitization and endocytosis of G protein-coupled receptors (GPCRs), are regulated by mouse double minute 2 homolog (Mdm2)-mediated ubiquitination. However, details of the molecular mechanisms of ß-arrestin2 ubiquitination remain unclear. Studies on ß-arrestin2 and Mdm2 mutants with modified nucleocytoplasmic shuttling properties have revealed that ß-arrestin2 ubiquitination occurs in the nucleus in a Gßγ- and clathrin-dependent manner. The nuclear entry of both ß-arrestin2 and Mdm2 commonly relies on the presence of importin complex but can occur independently of each other. Gßγ and clathrin regulated the nuclear entry of ß-arrestin2 by mediating the interaction between ß-arrestin2 and importin ß1. In contrast, Akt-mediated phosphorylation of two serine residues of Mdm2 partly regulated the nuclear entry of Mdm2. Ubiquitinated ß-arrestin2 along with Mdm2 translocated to the cytoplasm where they play various functional roles including receptor endocytosis and ubiquitination of other cytoplasmic proteins. The nuclear export of Mdm2 required nuclear entry and interaction of ß-arrestin2 with Mdm2. Ubiquitination was required for the translocation of ß-arrestin2 toward activated receptors on the plasma membrane and for its endocytic activity. The current study revealed the cellular components and processes involved in the ubiquitination of ß-arrestin2, and these findings could be quintessential for providing directions and detailed strategies for the manipulation of GPCR functions and development of GPCR-related therapeutics.