Protein Degradation Pathways Research Articles

The identification of high-performing antibodies for Sequestosome-1 for use in Western blot, immunoprecipitation and immunofluorescence.

Sequestosome-1, encoded by the gene SQSTM1, functions as a bridge between ubiquitinated proteins and the proteasome or autophagosome, thereby regulating protein degradation pathways. Loss of Sequestosome-1 is hypothesized to enhance neurodegeneration progression in several diseases, including amyotrophic lateral sclerosis (ALS) and frontotemporal disorders (FTD). Sequestosome-1 reproducible research would be facilitated with the availability of well-characterized anti-Sequestosome-1 antibodies. In this study, we characterized seventeen Sequestosome-1 commercial antibodies for Western blot, immunoprecipitation, and immunofluorescence using a standardized experimental protocol based on comparing read-outs in knockout cell lines and isogenic parental controls. We identified many high-performing antibodies and encourage readers to use this report as a guide to select the most appropriate antibody for their specific needs.

Research Progress in the Regulation of the ABA Signaling Pathway by E3 Ubiquitin Ligases in Plants.

E3 ubiquitin ligases (UBLs), as enzymes capable of specifically recognizing target proteins in the process of protein ubiquitination, play crucial roles in regulating responses to abiotic stresses such as drought, salt, and temperature. Abscisic acid (ABA), a plant endogenous hormone, is essential to regulating plant growth, development, disease resistance, and defense against abiotic stresses, and acts through a complex ABA signaling pathway. Hormone signaling transduction relies on protein regulation, and E3 ubiquitin ligases play important parts in regulating the ABA pathway. Therefore, this paper reviews the ubiquitin-proteasome-mediated protein degradation pathway, ABA-related signaling pathways, and the regulation of ABA-signaling-pathway-related genes by E3 ubiquitin ligases, aiming to provide references for further exploration of the relevant research on how plant E3 ubiquitin ligases regulate the ABA pathway.

OIP5-AS1 enhances the malignant characteristics and resistance to chemotherapy of pancreatic cancer cells by targeting miR-30d-5p/MARCH8

MARCH8, an E3 ubiquitin ligase, plays a crucial role in regulating various cellular processes such as protein degradation and signaling pathways and is implicated in the development and spread of pancreatic cancer. Analysis of pancreatic cancer tissues compared to adjacent normal tissues showed a decrease in miRNA-30d-5p levels and an increase in OIP5-AS1 and MARCH8 levels, as confirmed by qRT-PCR and Western blot analysis. The dual-luciferase reporter assay demonstrated a binding relationship between OIP5-AS1 and miRNA-30d-5p, as well as between miRNA-30d-5p and MARCH8 in PACN-1 cells, derived from a human pancreatic carcinoma specimen. Further investigations utilizing various assays revealed that OIP5-AS1 inhibited apoptosis and promoted cell proliferation, invasion, and migration in PACN-1 cells via the miRNA-30d-5p/MARCH8 axis in vitro. Tumor experiments in nude mice confirmed that OIP5-AS1 enhanced PACN-1 cell growth in vivo through the miRNA-30d-5p/MARCH8 axis. Additionally, OIP5-AS1 was found to activate downstream genes of the JAK-STAT pathway, namely IFNAR2, SOCS3, and JAK1, in PACN-1 cells. Furthermore, OIP5-AS1 increased the IC50 values for doxorubicin, gemcitabine, and cisplatin in PACN-1 cells, as determined by the Cell Counting Kit-8 assay. Overall, OIP5-AS1 was shown to promote aggressive traits and resistance to chemotherapy in PACN-1 cells through the miRNA-30d-5p/MARCH8 axis.

Lead toxicity regulation via protein degradation and tetrapyrrole biosynthesis pathways in Brassica species: A comparative quantitative analysis of proteomic study

Understanding the heavy metals (HMs) tolerance mechanism is crucial for improving plant growth in metal-contaminated soil. In order to evaluate the lead (Pb) tolerance mechanism in Brassica species, a comparative proteomic study was used. Thirteen-day-old seedlings of B. juncea and B. napus were treated with different Pb(NO3)2 concentrations at 0, 3, 30, and 300 mg/L. Under 300 mg/L Pb(NO3)2 concentration, B. napus growth was significantly decreased, while B. juncea maintained normal growth similar to the control. The Pb accumulation was also higher in B. napus root and shoot compared to B. juncea. Gel-free proteomic analysis of roots revealed a total of 68 and 37 differentially abundant proteins (DAPs) in B. juncea and B. napus-specifically, after 300 mg/L Pb exposure. The majority of these proteins are associated with protein degradation, cellular respiration, and enzyme classification. The upregulated RPT2 and tetrapyrrole biosynthesis pathway-associated proteins maintain the cellular homeostasis and photosynthetic rate in B. juncea. Among the 55 common DAPs, S-adenosyl methionine and TCA cycle proteins were upregulated in B. juncea and down-regulated in B. napus after Pb exposure. Furthermore, higher oxidative stress also reduced the antioxidant enzyme activity in B. napus. The current finding suggests that B. juncea is more Pb tolerant than B. napus, possibly due to the upregulation of proteins involved in protein recycling, degradation, and tetrapyrrole biosynthesis pathway.

StaufenC facilitates utilization of the ERAD pathway to transport dsRNA through the endoplasmic reticulum to the cytosol

RNA interference (RNAi) is more efficient in coleopteran insects than other insects. StaufenC (StauC), a coleopteran-specific double-stranded RNA (dsRNA)-binding protein, is required for efficient RNAi in coleopterans. We investigated the function of StauC in the intracellular transport of dsRNA into the cytosol, where dsRNA is digested by Dicer enzymes and recruited by Argonauts to RNA-induced silencing complexes. Confocal microscopy and cellular organelle fractionation studies have shown that dsRNA is trafficked through the endoplasmic reticulum (ER) in coleopteran Colorado potato beetle (CPB) cells. StauC is localized to the ER in CPB cells, and StauC-knockdown caused the accumulation of dsRNA in the ER and a decrease in the cytosol, suggesting that StauC plays a key role in the intracellular transport of dsRNA through the ER. Using immunoprecipitation, we showed that StauC is required for dsRNA interaction with ER proteins in the ER-associated protein degradation (ERAD) pathway, and these interactions are required for RNAi in CPB cells. These results suggest that StauC works with the ERAD pathway to transport dsRNA through the ER to the cytosol. This information could be used to develop dsRNA delivery methods aimed at improving RNAi.

Integration of transcriptomics, metabolomics, and hormone analysis revealed the formation of lesion spots inhibited by GA and CTK was related to cell death and disease resistance in bread wheat (Triticum aestivum L.)

BackgroundWheat is one of the important grain crops in the world. The formation of lesion spots related to cell death is involved in disease resistance, whereas the regulatory pathway of lesion spot production and resistance mechanism to pathogens in wheat is largely unknown.ResultsIn this study, a pair of NILs (NIL-Lm5W and NIL-Lm5M) was constructed from the BC1F4 population by the wheat lesion mimic mutant MC21 and its wild genotype Chuannong 16. The formation of lesion spots in NIL-Lm5M significantly increased its resistance to stripe rust, and NIL-Lm5M showed superiour agronomic traits than NIL-Lm5W under stripe rust infection.Whereafter, the NILs were subjected to transcriptomic (stage N: no spots; stage S, only a few spots; and stage M, numerous spots), metabolomic (stage N and S), and hormone analysis (stage S), with samples taken from normal plants in the field. Transcriptomic analysis showed that the differentially expressed genes were enriched in plant-pathogen interaction, and defense-related genes were significantly upregulated following the formation of lesion spots. Metabolomic analysis showed that the differentially accumulated metabolites were enriched in energy metabolism, including amino acid metabolism, carbohydrate metabolism, and lipid metabolism. Correlation network diagrams of transcriptomic and metabolomic showed that they were both enriched in energy metabolism. Additionally, the contents of gibberellin A7, cis-Zeatin, and abscisic acid were decreased in leaves upon lesion spot formation, whereas the lesion spots in NIL-Lm5M leaves were restrained by spaying GA and cytokinin (CTK, trans-zeatin) in the field.ConclusionThe formation of lesion spots can result in cell death and enhance strip rust resistance by protein degradation pathway and defense-related genes overexpression in wheat. Besides, the formation of lesion spots was significantly affected by GA and CTK. Altogether, these results may contribute to the understanding of lesion spot formation in wheat and laid a foundation for regulating the resistance mechanism to stripe rust.

Toll-like receptor signaling in multiple myeloma cells promotes the expression of pro-survival genes B-cell lymphoma 2 and MYC and modulates the expression of B-cell maturation antigen.

Infections are common in plasma cell cancer multiple myeloma (MM) due to disease-related immune deficiencies and cancer treatment. Myeloma cells express Toll-like receptors (TLRs), and TLR activation has been shown to induce proliferative and pro-survival signals in cancer cells. MM is a complex and heterogeneous disease, and expression levels of TLRs as well as downstream signaling components are likely to differ between patients. Here, we show that in a large cohort of patients, TLR1, TLR4, TLR6, TLR9, and TLR10 are the most highly expressed in primary CD138+ cells. Using an MM cell line expressing TLR4 and TLR9 as a model, we demonstrate that TLR4 and TLR9 activation promoted the expression of well-established pro-survival and oncogenes in MM such as MYC, IRF4, NFKB, and BCL2. TLR4 and TLR9 activation inhibited the efficacy of proteasome inhibitors bortezomib and carfilzomib, drugs used in the treatment of MM. Inhibiting the autophagosome-lysosome protein degradation pathway by hydroxychloroquine (HCQ) diminished the protective effect of TLR activation on proteasome inhibitor-induced cytotoxicity. We also found that TLR signaling downregulated the expression of TNFRSF17, the gene encoding for B-cell maturation antigen (BCMA). MYC, BCL2, and BCL2L1 were upregulated in approximately 50% of primary cells, while the response to TLR signaling in terms of TNFRSF17 expression was dichotomous, as an equal fraction of patients showed upregulation and downregulation of the gene. While proteasome inhibitors are part of first-line MM treatment, several of the new anti-MM immune therapeutic drugs target BCMA. Thus, TLR activation may render MM cells less responsive to commonly used anti-myeloma drugs.

Direct and acute effects of advanced glycation end products on proteostasis in isolated mouse skeletal muscle.

Advanced glycation end products (AGEs) have been implicated in several skeletal muscle dysfunctions. However, whether the adverse effects of AGEs on skeletal muscle are because of their direct action on the skeletal muscle tissue is unclear. Therefore, this study aimed to investigate the direct and acute effects of AGEs on skeletal muscle using an isolated mouse skeletal muscle to eliminate several confounders derived from other organs. The results showed that the incubation of isolated mouse skeletal muscle with AGEs (1 mg/mL) for 2-6 h suppressed protein synthesis and the mechanistic target of rapamycin signaling pathway. Furthermore, AGEs showed potential inhibitory effects on protein degradation pathways, including autophagy and the ubiquitin-proteasome system. Additionally, AGEs stimulated endoplasmic reticulum (ER) stress by modulating the activating transcription factor 6, PKR-like ER kinase, C/EBP homologous protein, and altered inflammatory cytokine expression. AGEs also stimulated receptor for AGEs (RAGE)-associated signaling molecules, including mitogen-activated protein kinases. These findings suggest that AGEs have direct and acute effect on skeletal muscle and disturb proteostasis by modulating intracellular pathways such as RAGE signaling, protein synthesis, proteolysis, ER stress, and inflammatory cytokines.

New insights into the dominance of mixed fermentation of Staphylococcus cohnii and Staphylococcus saprophyticus in Chinese bacon: Complete genomic and comparative genomic perspectives

Previous studies have demonstrated that Staphylococcus cohnii WX_M8 and S. saprophyticus MY_A10 significantly enhanced the flavor of Chinese bacon in a mixed fermentation. However, due to the complexity of the processing, the contribution of the bacteria is deceptive when investigating only the phenotypic changes at the time of fermentation. In order to clarify the metabolic mechanisms of mixed fermentation, a technological characterization, whole genome and comparative genomics analysis, and metabolites were approached in this study. Results showed that differences in tolerance characteristics existed between WX_M8 and MY_A10. And the genomes of both the two strains consisted of one chromosome and four circular plasmids. Their genome sizes were 2.74 Mp and 2.62 Mp, the GC contents were 32.45% and 33.18%, and the predicted coding genes (CDS) were 2564 and 2541, respectively. Based on the annotation of gene functions and assessment of metabolic pathways in the KEGG database, WX_M8 and MY_A10 strains were found to harbor complete protein degradation and amino acid metabolic pathways, pyruvate and butanol metabolic pathways, and isoleucine metabolic pathways, and their diverse enzyme-encoding genes superimposed the metabolic functions, whereas the alcohol dehydrogenase genes, adh and frmA, achieved complementary functions in the production of esters. Comparative genomics analysis revealed a diversity of encoding genes of aminotransferases and a greater metabolism for sulfur-containing amino acids, aromatic amino acids, and branched-chain amino acids in the mixed fermentation of strains WX_M8 and MY_A10. Metabolites analysis showed that MY_A10 focused on the production of soluble peptides and free amino acids (FAAs), while WX_M8 focused on volatile organic compounds (VOCs), resulting in a significant enhancement of the flavor of Chinese bacon when the two were mixed fermented. This result may provide direction for strains WX_M8 and MY_A10 to be used as starter cultures and targeted to regulate flavor.

Skeletal muscle preservation in arctic ground squirrels during hibernation season

Reduced skeletal loading leads to muscle atrophy in humans and most mammals. By contrast, hibernating mammals demonstrate limited loss of skeletal muscle mass and strength by the end of winter after being physically inactive for several months. The present study objective was to establish potential underlying processes for the lack of muscle loss during the hibernation season of arctic ground squirrels (AGS). Our hypothesis was that early in the the hibernation season, protective mechanisms of AGS are not yet mobilized and muscles show signs of atrophy, while compensatory processes for preventing muscle loss appear at a later time point. Quadriceps muscles of juvenile male AGS were collected shortly before hibernation, and on 2, 6, 10-12 and 16-22 weeks during hibernation. Pre-hibernating animals were used as controls. Fiber cross-sectional area (CSA) and fiber type composition were determined with immunohistochemistry. Amount of total RNA, ribosomal RNA content and expression levels of genes involved in 2 pathways of protein degradation, ubiquitination and autophagy, were analyzed by real-time PCR, to evaluate molecular mechanisms of muscle maintenance. One-Way ANOVA was used to statistically analyze the group differences. We found that CSA was not different between the groups (P > 0.05). CSA frequency distribution curves did not reveal any shift to a smaller or larger size range. No difference was detected in myofiber composition between the hibernation groups compared to the control. Total RNA and ribosomal RNA content were not significantly different between the groups during hibernation. Muscle atrophy marker FBXO32 (Atrogin-1, MAFBX) and autophagy related genes MAP1LC3A and BECN1 did not show different level of expression between the groups. Only another muscle atrophy marker TRIM63 (MURF-1, Iris Ring Finger Protein) was significantly over-expressed at the time point of 2 weeks of hibernation. These results indicate that throughout the hibernation season, AGS preserve muscle fiber CSA showing limited signs of muscle atrophy only during the first weeks of hibernation, and yet-to-be determined processes exist to suppress protein degradation in AGS muscles during hibernation. The work was supported by Center of Biomedical Research Excellence under grant number [P20GM130443]. This is the full abstract presented at the American Physiology Summit 2024 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.

Multiple strategies to improve the secretory expression of prolyl endopeptidase in Aspergillus niger

Prolyl Endopeptidase (PEP) has the unique ability to hydrolyze the carboxyl terminal peptide bonds of Proline. This enzyme holds great value in various fields including medical research, disease treatment, human diet, and food production. Aspergillus niger is a significant food-grade protein expression system renowned for its high safety. It is considered an ideal system for producing Aspergillus-derived PEP due to its ability to secrete a large amount of endogenous proteins. Our research group successfully constructed the Aspergillus niger PEP engineering strain in the early stages by eliminating the background protein AsaA, thereby increasing secretion protein production. In this article, we explore new solutions to further enhance production. Experimental investigations were conducted to regulate secretory protein production at different levels by modifying the 5′UTR and reconstructing the secretion pathways. The results demonstrate that the strain modified by the 5′UTR and Kozak sequence (glaA5'UTR-GCCACC, glaA::protA) exhibited a 1.88-fold increase in gene transcription and a 105% increase in extracellular PEP activity compared to the non-modified strain. Co-expression of the ERdjs gene AnScj1 further increased extracellular PEP activity by 168%. However, deletion of derA or vps10, key genes in the Aspergillus niger secretory protein degradation pathway, resulted in reduced levels of gene transcription and secretory expression. This article holds practical significance in enhancing the secretion and expression of PEP, obtaining higher-yield strains, and exploring optimization strategies for highly expressed proteins.

The Influence of Stress and Binge-Patterned Alcohol Drinking on Mouse Skeletal Muscle Protein Synthesis and Degradation Pathways.

Adverse experiences (e.g., acute stress) and alcohol misuse can both impair skeletal muscle homeostasis, resulting in reduced protein synthesis and greater protein breakdown. Exposure to acute stress is a significant risk factor for engaging in alcohol misuse. However, little is known about how these factors together might further affect skeletal muscle health. To that end, this study investigated the effects of acute stress exposure followed by a period of binge-patterned alcohol drinking on signaling factors along mouse skeletal muscle protein synthesis (MPS) and degradation (MPD) pathways. Young adult male C57BL/6J mice participated in the Drinking in the Dark paradigm, where they received 2-4 h of access to 20% ethanol (alcohol group) or water (control group) for four days to establish baseline drinking levels. Three days later, half of the mice in each group were either exposed to a single episode of uncontrollable tail shocks (acute stress) or remained undisturbed in their home cages (no stress). Three days after stress exposure, mice received 4 h of access to 20% ethanol (alcohol) to model binge-patterned alcohol drinking or water for ten consecutive days. Immediately following the final episode of alcohol access, mouse gastrocnemius muscle was extracted to measure changes in relative protein levels along the Akt-mTOR MPS, as well as the ubiquitin-proteasome pathway (UPP) and autophagy MPD pathways via Western blotting. A single exposure to acute stress impaired Akt singling and reduced rates of MPS, independent of alcohol access. This observation was concurrent with a potent increase in heat shock protein seventy expression in the muscle of stressed mice. Alcohol drinking did not exacerbate stress-induced alterations in the MPS and MPD signaling pathways. Instead, changes in the MPS and MPD signaling factors due to alcohol access were primarily observed in non-stressed mice. Taken together, these data suggest that exposure to a stressor of sufficient intensity may cause prolonged disruptions to signaling factors that impact skeletal muscle health and function beyond what could be further induced by periods of alcohol misuse.

TurboID-Based IRE1 Interactome Reveals Participants of the Endoplasmic Reticulum-Associated Protein Degradation Machinery in the Human Mast Cell Leukemia Cell Line HMC-1.2.

The unfolded protein response is an intricate system of sensor proteins in the endoplasmic reticulum (ER) that recognizes misfolded proteins and transmits information via transcription factors to either regain proteostasis or, depending on the severity, to induce apoptosis. The main transmembrane sensor is IRE1α, which contains cytoplasmic kinase and RNase domains relevant for its activation and the mRNA splicing of the transcription factor XBP1. Mast cell leukemia (MCL) is a severe form of systemic mastocytosis. The inhibition of IRE1α in the MCL cell line HMC-1.2 has anti-proliferative and pro-apoptotic effects, motivating us to elucidate the IRE1α interactors/regulators in HMC-1.2 cells. Therefore, the TurboID proximity labeling technique combined with MS analysis was applied. Gene Ontology and pathway enrichment analyses revealed that the majority of the enriched proteins are involved in vesicle-mediated transport, protein stabilization, and ubiquitin-dependent ER-associated protein degradation pathways. In particular, the AAA ATPase VCP and the oncoprotein MTDH as IRE1α-interacting proteins caught our interest for further analyses. The pharmacological inhibition of VCP activity resulted in the increased stability of IRE1α and MTDH as well as the activation of IRE1α. The interaction of VCP with both IRE1α and MTDH was dependent on ubiquitination. Moreover, MTDH stability was reduced in IRE1α-knockout cells. Hence, pharmacological manipulation of IRE1α-MTDH-VCP complex(es) might enable the treatment of MCL.

TCEB3 initiates ovarian cancer apoptosis by mediating ubiquitination and degradation of MCL-1.

Platinum resistance remains a major contributor to the poor prognosis of ovarian cancer. Anti-apoptotic protein myeloid cell leukemia-1 (MCL-1) has emerged as a promising target for overcoming drug resistance, but different cancer cells utilize distinct protein degradation pathways to alter MCL-1 level. We systematically investigated E3 ligases to identify novel candidates that mediate platinum resistance in ovarian cancer. Transcription Elongation Factor B (TCEB3) has been identified as a novel E3 ligase recognition subunit that targets MCL-1 in the cytoplasm during platinum treatment other than its traditional function of targeting the Pol II in the nuclear compartment. TCEB3 expression is downregulated in platinum-resistant cell lines and this low expression is associated with poor prognosis. The ubiquitination of MCL-1 induced by TCEB3 leads to cell death in ovarian cancer. Moreover, platinum treatment increased the cytoplasm proportion of TCEB3, and the cytoplasm localization of TCEB3 is important for its targeting of MCL-1. This study emphasizes the dual function of TCEB3 in homeostasis maintenance and in cell fate determination under different conditions, and provides a new insight into drug resistance in ovarian cancer.

Targeting Molecular Mechanisms of Obesity- and Type 2 Diabetes Mellitus-Induced Skeletal Muscle Atrophy with Nerve Growth Factor.

Skeletal muscle plays a critical role in metabolic diseases, such as obesity and type 2 diabetes mellitus (T2DM). Muscle atrophy, characterized by a decrease in muscle mass and function, occurs due to an imbalance between the rates of muscle protein synthesis and degradation. This study aimed to investigate the molecular mechanisms that lead to muscle atrophy in obese and T2DM mouse models. Additionally, the effect of nerve growth factor (NGF) on the protein synthesis and degradation pathways was examined. Male mice were divided into three groups: a control group that was fed a standard chow diet, and two experimental groups that were fed a Western diet. After 8 weeks, the diabetic group was injected with streptozotocin to induce T2DM. Each group was then further divided into NGF-treated or non-treated control group. In the gastrocnemius muscles of the Western diet group, increased expressions of myostatin, autophagy markers, and ubiquitin ligases were observed. Skeletal muscle tissue morphology indicated signs of muscle atrophy in both obese and diabetic mice. The NGF-treated group showed a prominent decrease in the protein levels of myostatin and autophagy markers. Furthermore, the NGF-treated group showed an increased Cyclin D1 level. Western diet-induced obesity and T2DM may be linked to muscle atrophy through upregulation of myostatin and subsequent increase in the ubiquitin and autophagy systems. Moreover, NGF treatment may improve muscle protein synthesis and cell cycling.

Ferroptosis and the ubiquitin-proteasome system: exploring treatment targets in cancer.

Ferroptosis is an emerging mode of programmed cell death fueled by iron buildup and lipid peroxidation. Recent evidence points to the function of ferroptosis in the aetiology and development of cancer and other disorders. Consequently, harnessing iron death for disease treatment has diverted the interest of the researchers in the field of basic and clinical research. The ubiquitin-proteasome system (UPS) represents a primary protein degradation pathway in eukaryotes. It involves labelling proteins to be degraded by ubiquitin (Ub), followed by recognition and degradation by the proteasome. Dysfunction of the UPS can contribute to diverse pathological processes, emphasizing the importance of maintaining organismal homeostasis. The regulation of protein stability is a critical component of the intricate molecular mechanism underlying iron death. Moreover, the intricate involvement of the UPS in regulating iron death-related molecules and signaling pathways, providing valuable insights for targeted treatment strategies. Besides, it highlights the potential of ferroptosis as a promising target for cancer therapy, emphasizing the combination between ferroptosis and the UPS. The molecular mechanisms underlying ferroptosis, including key regulators such as glutathione peroxidase 4 (GPX4), cysteine/glutamate transporter (system XC-), and iron metabolism, are thoroughly examined, alongside the role of the UPS in modulating the abundance and activity of crucial proteins for ferroptotic cell death, such as GPX4, and nuclear factor erythroid 2-related factor 2 (NRF2). As a pivotal regulatory system for macromolecular homeostasis, the UPS substantially impacts ferroptosis by directly or indirectly modulating iron death-related molecules or associated signaling pathways. This review explores the involvement of the UPS in regulating iron death-related molecules and signaling pathways, providing valuable insights for the targeted treatment of diseases associated with ferroptosis.

Lactobacillus gasseri BNR17 Ameliorates Dexamethasone-Induced Muscle Loss in BALB/c Mice and C2C12 Myotubes.

This study aimed to investigate the effects and mechanism of Lactobacillus gasseri BNR17, a probiotic strain isolated from human breast milk, on dexamethasone-induced muscle loss in mice and cultured myotubes. BALB/c mice were intraperitoneally injected with dexamethasone, and orally administered L. gasseri BNR17 for 21 days. L. gasseri BNR17 treatment ameliorated dexamethasone-induced decline in muscle function, as evidenced by an increase in forelimb grip strength, treadmill running time, and rotarod retention time in both female and male mice. In addition, L. gasseri BNR17 treatment significantly increased the mass of the gastrocnemius and quadriceps muscles. Dual-energy X-ray absorptiometry showed a significant increase in lean body mass and a decrease in fat mass in both whole body and hind limb after treatment with L. gasseri BNR17. It was found that L. gasseri BNR17 treatment downregulated serum myostatin level and the protein degradation pathway composed of muscle-specific ubiquitin E3 ligases, MuRF1 and MAFbx, and their transcription factor FoxO3. In contrast, L. gasseri BNR17 treatment upregulated serum insulin-like growth factor-1 level and Akt-mTOR-p70S6K signaling pathway involved in protein synthesis in muscle. As a result, L. gasseri BNR17 treatment significantly increased the levels of major muscular proteins such as myosin heavy chain and myoblast determination protein 1. Consistent with in vivo results, L. gasseri BNR17 culture supernatant significantly ameliorated dexamethasone-induced C2C12 myotube atrophy in vitro. In conclusion, L. gasseri BNR17 ameliorates muscle loss by downregulating the protein degradation pathway and upregulating the protein synthesis pathway.

Coculture with Colon-26 cancer cells decreases the protein synthesis rate and shifts energy metabolism toward glycolysis dominance in C2C12 myotubes.

Cancer cachexia is the result of complex interorgan interactions initiated by cancer cells and changes in patient behavior such as decreased physical activity and energy intake. Therefore, it is crucial to distinguish between the direct and indirect effects of cancer cells on muscle mass regulation and bioenergetics to identify novel therapeutic targets. In this study, we investigated the direct effects of Colon-26 cancer cells on the molecular regulating machinery of muscle mass and its bioenergetics using a coculture system with C2C12 myotubes. Our results demonstrated that coculture with Colon-26 cells induced myotube atrophy and reduced skeletal muscle protein synthesis and its regulating mechanistic target of rapamycin complex 1 signal transduction. However, we did not observe any activating effects on protein degradation pathways including ubiquitin-proteasome and autophagy-lysosome systems. From a bioenergetic perspective, coculture with Colon-26 cells decreased the complex I-driven, but not complex II-driven, mitochondrial ATP production capacity, while increasing glycolytic enzyme activity and glycolytic metabolites, suggesting a shift in energy metabolism toward glycolysis dominance. Gene expression profiling by RNA sequencing showed that the increased activity of glycolytic enzymes was consistent with changes in gene expression. However, the decreased ATP production capacity of mitochondria was not in line with the gene expression. The potential direct interaction between cancer cells and skeletal muscle cells revealed in this study may contribute to a better fundamental understanding of the complex pathophysiology of cancer cachexia.NEW & NOTEWORTHY We explored the potential direct interplay between colon cancer cells (Colon-26) and skeletal muscle cells (C2C12 myotubes) employing a noncontact coculture experimental model. Our findings reveal that coculturing with Colon-26 cells substantially impairs the protein synthesis rate, concurrently instigating a metabolic shift toward glycolytic dominance in C2C12 myotubes. This research unveils critical insights into the intricate cellular cross talk underpinning the complex pathophysiology of cancer cachexia.

Derlin-1 promotes diet-induced non-alcoholic fatty liver disease via increasing RIPK3-mediated necroptosis

Background & aimsUnrestricted endoplasmic reticulum (ER) stress and the continuous activation of ER associated protein degradation (ERAD) pathway might lead to the aggravation of non-alcoholic steatohepatitis (NASH). Derlin-1 has been considered to be an integral part of the ERAD pathway, which is involved in the regulation of the transport and excretion of protein degradation products within ER. However, the regulatory role and mechanism of Derlin-1 in NASH remains unclear. MethodsThe expression of Derlin-1 was firstly detected in the liver of normal and NASH animal model and patient. Then, western diet (WD)-induced NASH mice were administrated with the lentivirus-mediated Derlin-1 knockdown or overexpression. Finally, RIPK3 knockout mice were used to explore the mechanism. The liver injury, hepatic steatosis, inflammation, and fibrosis as well as ER stress signal pathway were evaluated. ResultsThe levels of Derlin-1 were significantly elevated in the liver of WD-fed mice and NASH patients when compared to the control group. Furthermore, Derlin-1 knockdown attenuated WD-induced liver injury, lipid accumulation, inflammatory response, and fibrosis. Conversely, overexpression of Derlin-1 presented the completely opposite results. Mechanistically, Derlin-1 enhanced ER stress pathways and led to necroptosis, and RIPK3 knockout dramatically reduced Derlin-1 expression and reversed the progression of NASH aggravated by Derlin-1. ConclusionsNotably, Derlin-1 is a critical modulator in NASH. It may accelerate the progression of NASH by regulating the activation of the ERAD pathway and further aggravating the ER stress, which might be involved in RIPK3-mediated necroptosis. Therefore, targeting Derlin-1 as a novel intervention point holds the potential to delay or even reverse NASH.

Abstract 4316: Investigation the role of DDI2 in autophagy

Abstract Background: The ubiquitin-proteasome system (UPS) and autophagy-lysosomal pathway (ALP) are critical intracellular protein degradation pathways. In response to proteasome dysfunction, cells initiate compensatory autophagy to manage proteotoxic stress, which impacts cancer therapy efficacy. Despite current insights, the exact molecular mechanisms underlying the induction of autophagy due to impaired proteasome function remain unclear. The transcription factor nuclear factor erythroid-2-related factor 1 (NRF1) is the master regulator of proteasome subunit genes, which is activated by the protease DNA damage inducible 1 homolog 2 (DDI2) upon proteasome inhibition. DDI2 also acts as an ubiquitin shuttling factor, and its depletion causes the accumulation of highly ubiquitinated proteins, resulting in increased sensitivity to proteasome inhibition in cancer cells. However, cancer cells might trigger compensatory autophagy in response to DDI2 depletion to degrade ubiquitinated protein aggregates. Here, we demonstrated that DDI2 depletion plays a role in autophagy induction. Methods: A panel of cell lines of different origins (EW16 and ES1 human Ewing sarcoma, MIA PaCa-2 human pancreatic cancer, and NIH-3T3 murine fibroblasts) either control or DDI2-deficient were examined for autophagy activity following treatment with the autophagy inhibitor chloroquine (CQ). Through total proteome analysis, we identified an increase in cellular communication network factor 1 (CCN1) protein levels in DDI2-depleted cells, which plays a role in autophagy induction. To determine whether CCN1 is required and sufficient to induce autophagy, we applied small interfering RNA (siRNA), and viral constructs to knockdown and overexpress CCN1. Results: The autophagic flux measurements in four different DDI2-deficient cell lines revealed elevated LC3B-II protein levels, a known autophagy marker, following CQ treatment compared to their control. Western blotting analysis confirmed increased CCN1 protein levels in the same cell lines. The autophagy induction observed in DDI2-deficient cells was diminished by CCN1-siRNA, demonstrating that CCN1 may be required for autophagy induction. Furthermore, overexpressing CCN1 in wild-type cells resulted in increased LC3B-II protein levels, indicating that CCN1 alone is sufficient to induce autophagy. Conclusion: Targeting autophagy is a promising therapeutic strategy to improve the effects of anti-cancer therapies. We demonstrated that upon depletion of ubiquitin shuttle factor DDI2, secretory protein CCN1 accumulates leading to the subsequent induction of autophagy. These findings improve our understanding of the molecular basis governing DDI2’s role in crosstalk between protein degradation pathways. Targeting DDI2 in combination with autophagy inhibition could further potentiate these drugs as anti-cancer therapies. Citation Format: Nayyerehalsadat Hosseini, Holly A. Byers, Senthil K. Radhakrishnan. Investigation the role of DDI2 in autophagy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4316.