Proteolytic Pathway Research Articles

In-Depth Characterization of Apoptosis N-Terminome Reveals a Link Between Caspase-3 Cleavage and Posttranslational N-Terminal Acetylation

The N termini of proteins contain information about their biochemical properties and functions. These N termini can be processed by proteases and can undergo other co- or posttranslational modifications. We have developed LATE (LysN Amino Terminal Enrichment), a method that uses selective chemical derivatization of α-amines to isolate the N-terminal peptides, in order to improve N-terminome identification in conjunction with other enrichment strategies. We applied LATE alongside another N-terminomic method to study caspase-3-mediated proteolysis both in vitro and during apoptosis in cells. This has enabled us to identify many unreported caspase-3 cleavages, some of which cannot be identified by other methods. Moreover, we have found direct evidence that neo-N-termini generated by caspase-3 cleavage can be further modified by Nt-acetylation. Some of these neo-Nt-acetylation events occur in the early phase of the apoptotic process and may have a role in translation inhibition. This has provided a comprehensive overview of the caspase-3 degradome and has uncovered previously unrecognized cross talk between posttranslational Nt-acetylation and caspase proteolytic pathways.

LncRNA H19 Regulates Breast Cancer DNA Damage Response and Sensitivity to PARP Inhibitors via Binding to ILF2.

Although DNA damage repair plays a critical role in cancer chemotherapy, the function of lncRNAs in this process remains largely unclear. In this study, in silico screening identified H19 as an lncRNA that potentially plays a role in DNA damage response and sensitivity to PARP inhibitors. Increased expression of H19 is correlated with disease progression and with a poor prognosis in breast cancer. In breast cancer cells, forced expression of H19 promotes DNA damage repair and resistance to PARP inhibition, whereas H19 depletion diminishes DNA damage repair and increases sensitivity to PARP inhibitors. H19 exerted its functional roles via direct interaction with ILF2 in the cell nucleus. H19 and ILF2 increased BRCA1 stability via the ubiquitin-proteasome proteolytic pathway via the H19- and ILF2-regulated BRCA1 ubiquitin ligases HUWE1 and UBE2T. In summary, this study has identified a novel mechanism to promote BRCA1-deficiency in breast cancer cells. Therefore, targeting the H19/ILF2/BRCA1 axis might modulate therapeutic approaches in breast cancer.

Antagonizing the irreversible thrombomodulin-initiated proteolytic signaling alleviates age-related liver fibrosis via senescent cell killing.

Cellular senescence is a stress-induced, stable cell cycle arrest phenotype which generates a pro-inflammatory microenvironment, leading to chronic inflammation and age-associated diseases. Determining the fundamental molecular pathways driving senescence instead of apoptosis could enable the identification of senolytic agents to restore tissue homeostasis. Here, we identify thrombomodulin (THBD) signaling as a key molecular determinant of the senescent cell fate. Although normally restricted to endothelial cells, THBD is rapidly upregulated and maintained throughout all phases of the senescence program in aged mammalian tissues and in senescent cell models. Mechanistically, THBD activates a proteolytic feed-forward signaling pathway by stabilizing a multi-protein complex in early endosomes, thus forming a molecular basis for the irreversibility of the senescence program and ensuring senescent cell viability. Therapeutically, THBD signaling depletion or inhibition using vorapaxar, an FDA-approved drug, effectively ablates senescent cells and restores tissue homeostasis in liver fibrosis models. Collectively, these results uncover proteolytic THBD signaling as a conserved pro-survival pathway essential for senescent cell viability, thus providing a pharmacologically exploitable senolytic target for senescence-associated diseases.

The effects of sucrose and arsenic on muscular insulin signaling pathways differ between the gastrocnemius and quadriceps muscles.

Insulin resistance in muscle can originate from a sedentary lifestyle, hypercaloric diets, or exposure to endocrine-disrupting pollutants such as arsenic. In skeletal muscle, insulin stimulates glucose uptake by translocating GLUT4 to the sarcolemma. This study aimed to evaluate the alterations induced by sucrose and arsenic exposure in vivo on the pathways involved in insulinstimulated GLUT4 translocation in the quadriceps and gastrocnemius muscles. Male Wistar rats were treated with 20% sucrose (S), 50 ppm sodium arsenite (A), or both (A+S) in drinking water for 8 weeks. We conducted an intraperitoneal insulin tolerance (ITT) test on the seventh week of treatment. The quadriceps and gastrocnemius muscles were obtained after overnight fasting or 30 min after intraperitoneal insulin injection. We assessed changes in GLUT4 translocation to the sarcolemma by cell fractionation and abundance of the proteins involved in GLUT4 translocation by Western blot. Male rats consuming S and A+S gained more weight than control and Atreated animals. Rats consuming S, A, and A+S developed insulin resistance assessed through ITT. Neither treatments nor insulin stimulation in the quadriceps produced changes in GLUT4 levels in the sarcolemma and Akt phosphorylation. Conversely, A and A+S decreased protein expression of Tether containing UBX domain for GLUT4 (TUG), and A alone increased calpain-10 expression. All treatments reduced this muscle's protein levels of VAMP2. Conversely, S and A treatment increased basal GLUT4 levels in the sarcolemma of the gastrocnemius, while all treatments inhibited insulin-induced GLUT4 translocation. These effects correlated with lower basal levels of TUG and impaired insulin-stimulated TUG proteolysis. Moreover, animals treated with S had reduced calpain-10 protein levels in this muscle, while A and A+S inhibited insulin-induced Akt phosphorylation. Arsenic and sucrose induce systemic insulin resistance due to defects in GLUT4 translocation induced by insulin. These defects depend on which muscle is being analyzed, in the quadriceps there were defects in GLUT4 retention and docking while in the gastrocnemius the Akt pathway was impacted by arsenic and the proteolytic pathway was impaired by arsenic and sucrose.

Insights into Polyprotein Processing and RNA-Protein Interactions in Foot-and-Mouth Disease Virus Genome Replication.

Foot-and-mouth disease virus (FMDV) is a picornavirus, which infects cloven-hoofed animals to cause foot-and-mouth disease (FMD). The positive-sense RNA genome contains a single open reading frame, which is translated as a polyprotein that is cleaved by viral proteases to produce the viral structural and nonstructural proteins. Initial processing occurs at three main junctions to generate four primary precursors; Lpro and P1, P2, and P3 (also termed 1ABCD, 2BC, and 3AB1,2,3CD). The 2BC and 3AB1,2,3CD precursors undergo subsequent proteolysis to generate the proteins required for viral replication, including the enzymes 2C, 3Cpro, and 3Dpol. These precursors can be processed through both cis and trans (i.e., intra- and intermolecular proteolysis) pathways, which are thought to be important for controlling virus replication. Our previous studies suggested that a single residue in the 3B3-3C junction has an important role in controlling 3AB1,2,3CD processing. Here, we use in vitro based assays to show that a single amino acid substitution at the 3B3-3C boundary increases the rate of proteolysis to generate a novel 2C-containing precursor. Complementation assays showed that while this amino acid substitution enhanced production of some nonenzymatic nonstructural proteins, those with enzymatic functions were inhibited. Interestingly, replication could only be supported by complementation with mutations in cis acting RNA elements, providing genetic evidence for a functional interaction between replication enzymes and RNA elements. IMPORTANCE Foot-and-mouth disease virus (FMDV) is responsible for foot-and-mouth disease (FMD), an important disease of farmed animals, which is endemic in many parts of the world and can results in major economic losses. Replication of the virus occurs within membrane-associated compartments in infected cells and requires highly coordinated processing events to produce an array of nonstructural proteins. These are initially produced as a polyprotein that undergoes proteolysis likely through both cis and trans alternative pathways (i.e., intra- and intermolecular proteolysis). The role of alternative processing pathways may help coordination of viral replication by providing temporal control of protein production and here we analyze the consequences of amino acid substitutions that change these pathways in FMDV. Our data suggest that correct processing is required to produce key enzymes for replication in an environment in which they can interact with essential viral RNA elements. These data further the understanding of RNA genome replication.

FOXO1 cooperates with C/EBPΔ and ATF4 to regulate skeletal muscle atrophy transcriptional program during fasting

Catabolic conditions, such as starvation, inactivity, and cancer cachexia, induce Forkhead box O (FOXO) transcription factor(s) expression and severe muscle atrophy via the induction of ubiquitin–proteasome system-mediated muscle proteolysis, resulting in frailty and poor quality of life. Although FOXOs are clearly essential for the induction of muscle atrophy, it is unclear whether there are other factors involved in the FOXO-mediated transcriptional regulation. As such, we identified FOXO–CCAAT/enhancer binding protein Δ (C/EBPΔ) signaling pathway as a novel proteolytic pathway. By comparing the gene expression profiles of FOXO1-transgenic (gain-of-function model) and FOXO1,3a,4–/–(loss-of-function model) mice, we identified several novel FOXO1-target genes in skeletal muscle including Redd1, Sestrin1, Castor2, Chac1, Depp1, Lat3, as well as C/EBPΔ. During starvation, C/EBPΔ abundance was increased in a FOXOs-dependent manner. Notably, knockdown of C/EBPΔ prevented the induction of the ubiquitin–proteasome system and decrease of myofibers in FOXO1-activated myotubes. Conversely, C/EBPΔ overexpression in primary myotubes induced myotube atrophy. Furthermore, we demonstrated that FOXO1 enhances the promoter activity of target genes in cooperation with C/EBPΔ and ATF4. This research comprehensively identifies novel FOXO1 target genes in skeletal muscle and clarifies the pathophysiological role of FOXO1, a master regulator of skeletal muscle atrophy. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Platinum-based anticancer drugs-induced downregulation of myosin heavy chain isoforms in skeletal muscle of mouse

Cisplatin, a platinum-based anticancer drug used frequently in cancer treatment, causes skeletal muscle atrophy. It was predicted that the proteolytic pathway is enhanced as the mechanism of this atrophy. Therefore, we investigated whether a platinum-based anticancer drug affects the expression of the major proteins of skeletal muscle, myosin heavy chain (MyHC). Mice were injected with cisplatin or oxaliplatin for four consecutive days. C2C12 myotubes were treated using cisplatin and oxaliplatin. Administration of platinum-based anticancer drug reduced quadriceps mass and muscle strength compared to the control group. Protein levels of all MyHC isoforms were reduced in the platinum-based anticancer drug groups. However, only Myh2 (MyHC-IIa) gene expression in skeletal muscle of mice treated with platinum-based anticancer drugs was found to be reduced. Treatment of C2C12 myotubes with platinum-based anticancer drugs reduced the protein levels of all MyHCs, and treatment with the proteasome inhibitor MG-132 restored this reduction. The expression of Mef2c, which was predicted to act upstream of Myh2, was reduced in the skeletal muscle of mice treated systemically with platinum-based anticancer drug. Degradation of skeletal muscle MyHCs by proteasomes may be a factor that plays an important role in muscle mass loss in platinum-based anticancer drug-induced muscle atrophy.

Effect of Sodium Hypochlorite 0.05% on MMP-9 Extracellular Release in Chronic Wounds.

In chronic wounds, high concentrations of matrix metalloproteinases (MMPs) can cause excessive proteolysis and slow wound healing. Consequently, restoring a proper MMP balance can help reduce the risk of a chronic wound. An antiseptic solution containing 0.05% sodium hypochlorite (Amukine Med 0.05%, Angelini S.p.A.; hereafter termed NaClO solution) is available on the market. The NaClO solution was proven effective and safe in managing infected skin wounds. To further characterize its activity, this study evaluated the in vitro activity of the NaClO solution on the monocyte release of MMPs. Human monocytic THP-1 (ATCC® TIB-202™) cell lines were differentiated into macrophages and treated with different concentrations of NaClO (from 0.05% to 5 × 10-7%). In addition, the THP-1 cell line was stimulated with wound fluid (WF) from patients with active venous leg ulcers in the inflammatory phase. The effect of NaClO (0.025-0.0062%) was also evaluated on healthy human peripheral blood serum samples. The effects of treatments on the gelatinolytic activity of MMP-9 were evaluated by gelatin zymography. The effects on MMPs release were evaluated through the Pro™ Human MMP 9-plex Assay. An exploratory scratch wound healing assay was also performed. The NaClO solution reduced the gelatinolytic activity of MMP-9 and its activated form. The downregulation of MMP-9 gelatinolytic activity was also observed in peripheral blood serum. The MMPs profile showed a reduction in MMP-1 release (p < 0.05) and a slight reduction of the release of MMP-9 and MMP-12 after the treatment with LPS and the NaClO solution. A slight improvement in wound healing was observed after macrophage activation and treatment with the NaClO solution. The results obtained suggest a possible ability of the NaClO solution to modulate the proteolytic pathways in the wound microenvironment, further characterizing its activity and use in clinical practice during wound care.

Identification and dimorphic expression of sex-related genes in Pacific abalone (Haliotis discus hannai)

Sex determination is a hot topic in evolutionary biology. The mechanism of sex determination is complex and diverse in shellfish, while no sex chromosomes have been found in mollusks. The Pacific abalone (Haliotis discus hannai) is one of the most economically important cultured shellfish species in China, and its sex determination mechanism is still unclear. Here, we identified the sex-determination region (SDR) of Pacific abalone and developed five stable sex-specific markers through genome-wide association studies (GWAS) based on re-sequencing data from 803 abalones. These markers can be used for sex discrimination in polyploid and sexually immature individuals in abalone breeding projects. In addition, haplotype analysis and quantitative expression of candidate sex-related genes showed that RhoGAPp190, Skp1, Cul2, and Otulin were potentially involved in the sex determination of Pacific abalone by the ubiquitin-mediated proteolysis pathway. Our study could help to better understand the molecular mechanisms that underpin sex determination and reproductive regulation in mollusks.

Immunopeptidomics reveals determinants of Mycobacterium tuberculosis antigen presentation on MHC class I.

CD8+ T cell recognition of Mycobacterium tuberculosis (Mtb)-specific peptides presented on major histocompatibility complex class I (MHC-I) contributes to immunity to tuberculosis (TB), but the principles that govern presentation of Mtb antigens on MHC-I are incompletely understood. In this study, mass spectrometry (MS) analysis of the MHC-I repertoire of Mtb-infected primary human macrophages reveals that substrates of Mtb's type VII secretion systems (T7SS) are overrepresented among Mtb-derived peptides presented on MHC-I. Quantitative, targeted MS shows that ESX-1 activity is required for presentation of Mtb peptides derived from both ESX-1 substrates and ESX-5 substrates on MHC-I, consistent with a model in which proteins secreted by multiple T7SSs access a cytosolic antigen processing pathway via ESX-1-mediated phagosome permeabilization. Chemical inhibition of proteasome activity, lysosomal acidification, or cysteine cathepsin activity did not block presentation of Mtb antigens on MHC-I, suggesting involvement of other proteolytic pathways or redundancy among multiple pathways. Our study identifies Mtb antigens presented on MHC-I that could serve as targets for TB vaccines, and reveals how the activity of multiple T7SSs interacts to contribute to presentation of Mtb antigens on MHC-I.

Abstract 3433: Global proteomic analysis of urine using a label-free LC-MS/MS approach identifies potential biomarkers of metastatic breast cancer

Abstract Introduction: Breast cancer (BC) is a complex heterogenous disease and is a leading cause of death in women. Following primary BC therapy ~40% of patients will develop metastatic BC (MBC) and 60-70% of the recurrent disease will occur at distant sites. Median survival of MBC patients with distant recurrence is ~2-3 yr. While the early detection and monitoring of BC progression are essential to improve prognosis and reduce BC-related mortality, there is a lack of surveillance strategies for monitoring patients for distant recurrence of MBC. The aim of our study was to identify urinary biomarkers for detection and monitoring of MBC. Method: We have conducted a comparative label-free LC-MS/MS analysis of the urinary proteome of patients with MBC and healthy age-matched controls (HC). A hybrid quadrupole time of flight (Q-Tof™) mass spectrometer was used for urine analysis via liquid chromatography (LC) with tandem mass spectrometry (MS/MS). Three different database search algorithms were used for peptide identification including MASCOT, Protein Lynx Global Server and Ion Accounting software. Only peptides detected in at least two out of three replicated LC-MS experiments were counted towards protein identification. Identified candidate biomarkers were validated via ELISA assays. Results: Using this approach, we identified ~400 urinary proteins of which ~104 and 89 were unique to the MBC and HC groups, respectively. Upregulated proteins in the MBC cohort were associated with angiogenesis, apoptosis, proteolysis, ECM regulation, and cell adhesion pathways. Using bioinformatic and in silico approaches, we identified a specific metastasis signature comprised of CALB1, S100A8, ZAG, VTN and TN. Diagnostic accuracies of these candidate markers were validated using independent training and validation sets according to the REMARK criteria. Urinary VTN (uVTN) and uTN levels correlated significantly with disease status in MBC patients when compared to patients with locally invasive BC and HC. Interestingly, a small subset of patients with bone metastasis also had significantly higher uVTN levels. A multiplexed marker panel of uVTN and uTN could discriminate between HC and MBC groups (AUC=0.831, P&amp;lt;0.001). Blinded testing indicated that uVTN levels (cutoff&amp;gt;1018 ng/ml) could discriminate (AUC=0.782, P&amp;lt;0.006) between MBC and HC. Longitudinal analysis of samples from MBC patients indicated a strong correlation between uVTN levels and disease status. Conclusions: Our findings suggest that uVTN and uTN have the potential to serve as biomarkers for the detection of MBC. While validation in larger cohorts is necessary, these results may be useful in the development of noninvasive diagnostic tests for monitoring BC progression and recurrence. [Supported by: the Advanced Medical Research Foundation, the Jo Ann Webb Fund for Angiogenesis Research and the S. Elizabeth O’Brien Trust] Citation Format: Roopali Roy, Elisa Schunkert, Petra Olivova, Martin Gilar, Scott Geromanos, Guo-Zhong Li, John Gebler, Adelle Dagher, Andrew El-Hayek, Rama Aldakhlallah, David Zurakowski, Susan Pories, Marsha A. Moses. Global proteomic analysis of urine using a label-free LC-MS/MS approach identifies potential biomarkers of metastatic breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3433.

Transcriptome profiling provides insight into root development at low temperature by chimeric metacaspase for directing Pep1 formation

Plant elicitor peptides (Peps) and low temperatures have both emerged as important regulators of root development. The intracellular formation of Pep1 by metacaspase 4 at low temperature is expectedly linked to root-growth regulation. Here, we engineered a chimeric metacaspase 4 (cMC4) protein with altered structure by substituting linker of MC4 with linker of MC7 to efficiently direct Pep1 generation at low temperature (15 °C). Arabidopsis seedlings exhibited significantly root length inhibition at low temperature by incubating with in vitro Pep1 producing solution from cMC4-mediated cleavage, which was simulated as intracellular Pep1 of damaged root cell. Further investigation of root transcriptomic profiling for these treated seedlings indicated that low-temperature-responsive genes involving in cellular ion/cation homeostasis, response to karrikin, plant-type vacuole and cell wall, along with metabolic networks of protein processing in endoplasmic reticulum and phenylpropanoid biosynthesis potentially contributed to low-temperature resistance of Arabidopsis root. Root-developmental-response genes of pollen tube growth, meristem growth, root hair cell development, and cell projection, along with ubiquitin mediated proteolysis pathways and defense-related genes were positively correlated with Pep1 generation pattern for triggering defense response. To the best of our knowledge, this is the first time to engineer chimeric protein to strengthen Pep1-triggered immunity for root development at low temperature, which could provide a potential clue for controlling root growth against stress.

VspC from Vibrio splendidus is responsible for collagen degradation in Apostichopus japonicus

Vibrio splendidus is a marine opportunistic pathogen that can infect diverse aquaculture animals. In this study, the catalytic domain of the vspC gene, encoding an M9 family metalloproteinase, was cloned from V. splendidus AJ01, and the recombinant catalytic domain rVspC was purified using Ni-NTA agarose. The rVspC can directly degrade collagen collected from body wall as well as the collagen in the coelomocytes of Apostichopus japonicus. Transcriptomics analysis of coelomocytes collected from A. japonicus injected with rVspC showed that there were 52 upregulated genes and 163 downregulated genes, of which the levels of MMP-14 were upregulated while laminin was downregulated in the extracellular matrix. Immune-related pathways of apoptosis, lysosome, phagosome, ubiquitin-mediated proteolysis and antigen processing and presentation were downregulated, while endocytosis was upregulated. Furthermore, one regulatory pathway of “Hfq-SmcR-VspC” at high cell density of V. splendidus was determined. The protein level of SmcR was reduced in Δhfq at high cell density, suggesting that Hfq positively regulates SmcR, and SmcR can bind to the promoters of vspC gene and positively regulates the expression of the vspC gene. In addition, comparative analysis of the sequences of the catalytic domain of VspC revealed that collagenase homologous to VspC are widely present in bacteria, and they may play an important role in colonization, environmental adaptation or virulence in different habitats.

Investigation of targets and anticancer mechanisms of covalently acting natural products by functional proteomics.

Eriocalyxin B (EB), 17-hydroxy-jolkinolide B (HJB), parthenolide (PN), xanthatin (XT) and andrographolide (AG) are terpenoid natural products with a variety of promising antitumor activities, which commonly bear electrophilic groups (α,β-unsaturated carbonyl groups and/or epoxides) capable of covalently modifying protein cysteine residues. However, their direct targets and underlying molecular mechanisms are still largely unclear, which limits the development of these compounds. In this study, we integrated activity-based protein profiling (ABPP) and quantitative proteomics approach to systematically characterize the covalent targets of these natural products and their involved cellular pathways. We first demonstrated the anti-proliferation activities of these five compounds in triple-negative breast cancer cell MDA-MB-231. Tandem mass tag (TMT)-based quantitative proteomics showed all five compounds commonly affected the ubiquitin mediated proteolysis pathways. ABPP platform identified the preferentially modified targets of EB and PN, two natural products with high anti-proliferation activity. Biochemical experiments showed that PN inhibited the cell proliferation through targeting ubiquitin carboxyl-terminal hydrolase 10 (USP10). Together, this study uncovered the covalently modified targets of these natural products and potential molecular mechanisms of their antitumor activities.

Abstract P2-03-20: Clinical profiling and comprehensive analysis of candidate genes related to breast cancer estrogen receptor intratumour heterogeneity

Abstract 1. Background and Aim Breast cancer has the highest incidence among the world’s population. One third of patients diagnosed with early breast cancer progressed into metastatic outcomes due to the inherent heterogeneity and evolutionary features of tumors. The main clinical manifestation is the discordance of receptor expression in metastases. ESR1 the estrogen receptor (ER) encoding gene, has been proved affected ER expression. Nevertheless, the correlation between ER transformation and the somatic mutation profiles in breast cancer metastatic lesions remains unclear. Availability of advanced high-throughput technologies, and the development of bioinformatics tools has greatly accelerated our cognition of the molecular basis of cancer. In this study, we aimed to evaluate the frequency, clinical characteristics and prognostic value of ER receptor conversion between primary and first metastatic lesions in 115 patients whose primary ER status are positive. We also compared the results of NGS of 42 patients and explored latent genes related to ER discordance in expression. 2. Method Qualified metastatic tumor tissue sequencing information was available for 42 patients. Samples were accessed using the FoundationOne CDx assay (Foundation Medicine, Cambridge, MA, USA) panel including 733 tumor related genes. High frequency mutant genes were defined as the genes count more than 4 times in each group. All data were analyzed using SPSS 25.0 software (Chicago, IL, USA). Pearson’s chi-squared (χ2) test, Fisher’s exact test, were used to test the associations between different variables. The Cox proportional hazard models was used to evaluate the relationship between KMT2C mutation and Disease free survival (DFS) in the patients cohorts. The results with p &amp;lt; 0.05 were considered statistically significant. 3. Results and Conclusions All 115 patients (primary ER status is positive) were divided into two groups, 70(60.87%) patients ER status remained positive while 45(39.14%) patients ER status transformed into negative. The histological type (p=0.008) and DFS (p=0.004) were significantly different in two subgroups. There were no significant correlations between the age at the time of diagnosis, BMI classification, menopausal status, surgery, histological grade, tumor size, lymph node status, metastasis, Ki67, adjuvant radiotherapy, neoadjuvant, metastasis site before second biopsy, first treatment after recurrent and first PFS after rebiopsy. Univariable analysis of DFS proved metastasis to be distinct risk factor for poor survival (hazard ratio [HR] &amp;gt; 1, p = 0.001). By contrast, neoadjuvant proved to be protective factor for better survival (hazard ratio [HR] &amp;lt; 1, p &amp;lt; 0.001). The surgery, histological type, histological grade, tumorsize, metastasis, neoadjuvant, first treatment after recurrent, first PFS after rebiopsy were subsequently analyzed with the multivariable Cox analysis. Metastasis (HR&amp;gt;1, p=0.007) and neoadjuvant (HR&amp;lt;1, p&amp;lt;0.001) remained independent prognostic factors of DFS. We compared the differentially mutated genes in the ER differential expression BC patient groups. Mutations in TP53 (n=24) and PIK3CA (n=19) occurred most frequently in both groups and account for 57.14% and 49.23% separately. Notably, mutations in KMT2C (n=6) was detected in ER+ to – subgroup and accounted for 37.5%. According to the results of NGS, the distribution of KMT2C mutations was different among the two cohorts. We identified the mutation in codon 321 was the hot spot of KMT2C in our patient cohort. Next, we used the Kyoto Encyclopedia of Genes and Genomes (KEGG) database to analyze the enriched pathways of KMT2C. KEGG pathway analysis revealed that patients with KMT2C mutations harbored significantly more mutations in genes involved in the Ubiquitin mediated proteolysis and Lysine degradation signaling pathway. Table 1. Patients’ basic clinicopathological characteristics among two groups Table 2. Univariate and Multivariate Analysis Between Clinicopathological Characteristics and DFS of 115 BC patients Table 3. Mutated Genes Rank and patients number Citation Format: Xi Wang, Yongmei Yin, Ziyi Fu. Clinical profiling and comprehensive analysis of candidate genes related to breast cancer estrogen receptor intratumour heterogeneity [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P2-03-20.

Combined enteral infusion of glutamine, carbohydrates, and antioxidants modulates gut protein metabolism in humans

Background:Available data suggest that nutrients can affect intestinal protein metabolism, which contributes to the regulation of gut barrier function. Objective:We aimed to assess whether an oral nutritional supplement (ONS) containing glutamine (as the dipeptide Ala-Gln), carbohydrates, and antioxidants would modulate duodenal protein metabolism in healthy humans. Design:Thirty healthy control subjects were included and, over a period of 5 h, received by nasogastric tube either saline or ONS providing 11.7 kcal/kg as 0.877 g Ala-Gln/kg, 3.9 g carbohydrates/kg, and antioxidants (29.25 mg vitamin C/kg, 9.75 mg vitamin E/kg, 195 μg β-carotene/kg, 5.85 mg Se/kg, and 390 μg Zn/kg) or glutamine (0.585 g/kg, 2.34 kcal/kg). Simultaneously, a continuous intravenous infusion of l-[1-13C]-leucine was done until endoscopy. Leucine enrichment was assessed by using gas chromatography–mass spectrometric analysis, and mucosal fractional synthesis rate was calculated by using intracellular amino acid enrichment as precursor. Mucosal proteolytic pathways were also evaluated. Results:ONS infusion resulted in a doubling increase (P < 0.01) of duodenal fractional synthesis rate and a significant (P < 0.05) decrease in cathepsin D–mediated proteolysis compared with saline, whereas proteasome and Ca2+-dependent activities were unaffected. ONS infusion significantly (P < 0.01) decreased duodenal glutathione but not glutathione disulfide concentrations or the ratio of glutathione to glutathione disulfide. Insulinemia increased after ONS infusion, whereas plasma essential amino acids decreased. Infusion of glutamine alone did not reproduce ONS effects. Conclusions:ONS infusion improves duodenal protein balance in healthy humans. Further investigations are needed to study the origin of these effects and to evaluate ONS supply in stressed persons.

Multiple ubiquitin E3 ligase genes antagonistically regulate chloroplast-associated protein degradation

The chloroplast is the most prominent member of a diverse group of plant organelles called the plastids, and it is characterized by its vital role in photosynthesis.1,2,3 Most of the ∼3,000 different proteins in chloroplasts are synthesized in the cytosol in precursor (preprotein) form, each with a cleavable transit peptide.4,5,6,7,8 Preproteins are imported via translocons in the outer and inner envelope membranes of the chloroplast, termed TOC and TIC, respectively.9,10,11,12,13 Discovery of the chloroplast-localized ubiquitin E3 ligase SUPPRESSOR OF PPI1 LOCUS1 (SP1) demonstrated that the nucleocytosolic ubiquitin-proteasome system (UPS) targets the TOC apparatus to dynamically control protein import and chloroplast biogenesis in response to developmental and environmental cues. The relevant UPS pathway is termed chloroplast-associated protein degradation (CHLORAD).14,15,16 Two homologs of SP1 exist, SP1-like1 (SPL1) and SPL2, but their roles have remained obscure. Here, we show that SP1 is ubiquitous in the Viridiplantae and that SPL2 and SPL1 appeared early during the evolution of the Viridiplantae and land plants, respectively. Through genetic and biochemical analysis, we reveal that SPL1 functions as a negative regulator of SP1, potentially by interfering with its ability to catalyze ubiquitination. In contrast, SPL2, the more distantly related SP1 homolog, displays partial functional redundancy with SP1. Both SPL1 and SPL2 modify the extent of leaf senescence, like SP1, but do so in diametrically opposite ways. Thus, SPL1 and SPL2 are bona fide CHLORAD system components with negative and positive regulatory functions that allow for nuanced control of this vital proteolytic pathway.

Stability of Dengue 2 Nonstructural Glycoprotein 1 (NS1) Is Affected by the Nature of Basic Residue at Position NS1-324.

Dengue is the most prevalent mosquito-borne viral disease. It is caused by the infection of any of the four dengue virus (DENV) serotypes DENV-1 to DENV-4. The DENV non-structural glycoprotein 1 (NS1) plays an important role in virus replication and the immunopathogenesis of virus infection. The NS1 protein has been identified as both a cell-associated homodimer and a soluble secreted lipoprotein nanoparticle. The nature of the residues at positions NS1-272 and NS1-324 in the β-ladder domain may have an effect on the biological behaviors of DENV-2 NS1 protein in human hepatoma Huh7 cells. The stability of the NS1 protein from the Reunion 2018 DENV-2 strain was affected by the presence of lysine residues at positions 272 and 324. In the present study, we evaluated the impact of mutations into lysine at positions 272 and 324 on recombinant NS1 protein from the DES-14 DENV-2 strain bearing arginine residue on these two positions. The DES-14 NS1 protein mutant bearing a lysine at position 324 was deficient in protein stability and secretion compared to wild-type protein. The defect in the DES-14 NS1 protein mutant was associated to oxidative stress and pro-inflammatory cytokine activation in Huh7 cells. The ubiquitin-proteasome proteolytic pathway might play a key role in the stability of DENV-2 protein bearing a lysine residue at position 324.

Immortalized Alzheimer’s Disease Astrocytes: Characterization of Their Proteolytic Systems

Alzheimer’s disease (AD) is a progressive neurodegeneration with dysfunctions in both the ubiquitin–proteasome system (UPS) and autophagy. Astroglia participation in AD is an attractive topic of research, but molecular patterns are partially defined and available in vitro models have technical limitations. Immortalized astrocytes from the hippocampus of 3xTg-AD and wild-type mice (3Tg-iAstro and WT-iAstro, respectively) have been obtained as an attempt to overcome primary cell line limitations and this study aims at characterizing their proteolytic systems, focusing on UPS and autophagy. Both 26S and 20S proteasomal activities were downregulated in 3Tg-iAstro, in which a shift in catalytic subunits from constitutive 20S proteasome to immunoproteasome occurred, with consequences on immune functions. In fact, immunoproteasome is the specific complex in charge of clearing damaged proteins under inflammatory conditions. Parallelly, augmented expression and activity of the lysosomal cathepsin B, enhanced levels of lysosomal-associated membrane protein 1, beclin1, and LC3-II, together with an increased uptake of monodansylcadaverine in autophagic vacuoles, suggested autophagy activation in 3Tg-iAstro. The two proteolytic pathways were linked by p62 that accumulated in 3Tg-iAstro due to both increased synthesis and decreased degradation in the UPS defective astrocytes. Treatment with 4-phenylbutyric acid, a neuroprotective small chemical chaperone, partially restored proteasome and autophagy-mediated proteolysis in 3Tg-iAstro. Our data shed light on the impaired proteostasis in 3Tg-iAstro with proteasome inhibition and autophagic compensatory activation, providing additional validation of this AD in vitro model, and propose a new mechanism of action of 4-phenylbutyric acid in neurodegenerative disorders.

FMRP, FXR1 protein and Dlg4 mRNA, which are associated with fragile X syndrome, are involved in the ubiquitin–proteasome system

The ubiquitin–proteasome system (UPS) is a proteolytic pathway that is essential for life maintenance and vital functions, and its disruption causes serious impairments, e.g., disease development. Thus, the UPS is properly regulated. Here we show novel UPS-related factors: the fragile X mental retardation 1 (FMR1) and Fmr1 autosomal homolog 1 (FXR1) proteins and discs large MAGUK scaffold protein 4 (Dlg4) mRNA, which are associated with Fragile X syndrome, are involved in UPS activity. Fmr1-, Fxr1- and Dlg4-knockdown and Fmr1- and Fxr1-knockdown resulted in increased ubiquitination and proteasome activity, respectively. FXR1 protein was further confirmed to be associated with proteasomes, and Dlg4 mRNA itself was found to be involved in the UPS. Knockdown of these genes also affected neurite outgrowth. These findings provide new insights into the regulatory mechanism of the UPS and into the interpretation of the pathogenesis of diseases in which these genes are involved as UPS-related factors.