Protein Degradation Research Articles

The MON1-CCZ1 complex plays dual roles in autophagic degradation and vacuolar protein transport in rice.

Autophagy is a highly conserved cellular program in eukaryotic cells which mediates the degradation of cytoplasmic components through the lysosome, also named the vacuole in plants. However, the molecular mechanisms underlying the fusion of autophagosomes with the vacuole remain unclear. Here, we report the functional characterization of a rice (Oryza sativa) mutant with defects in storage protein transport in endosperm cells and accumulation of numerous autophagosomes in root cells. Cytological and immunocytochemical experiments showed that this mutant exhibits a defect in the fusion between autophagosomes and vacuoles. The mutant harbors a loss-of-function mutation in the rice homolog of Arabidopsis thaliana MONENSIN SENSITIVITY1 (MON1). Biochemical and genetic evidence revealed a synergistic interaction between rice MON1 and AUTOPHAGY-RELATED 8a in maintaining normal growth and development. In addition, the rice mon1 mutant disrupted storage protein sorting to protein storage vacuoles. Furthermore, quantitative proteomics verified that the loss of MON1 function influenced diverse biological pathways including autophagy and vacuolar transport, thus decreasing the transport of autophagic and vacuolar cargoes to vacuoles. Together, our findings establish a molecular link between autophagy and vacuolar protein transport, and offer insights into the dual functions of the MON1-CCZ1 (CAFFEINE ZINC SENSITIVITY1) complex in plants.

Protein Degradation in Focus

Protein Degradation in Focus

Effect of synchronicity of amino acid supply on the synthesis of protein in C2C12 myotubes cultured in vitro

Previous studies inferred that the synthesis rate/efficiency of protein in body tissue is probably affected by synchronicity of different amino acid (AA) supply in its metabolic pool. In order to further observe the influence of synchronicity of AA supply on the synthesis of protein in cell level, a cell culture experiment in vitro was conducted with C2C12 myotubes. C2C12 myotubes were cultured for 24 h, meanwhile the culture medium was replaced for each 8 h. Those myotubes were subjected to 3 treatments (1 for controlled and 2 for tested), control myotubes were cultured with same normal complete medium within the whole 24 h, and the 2 tested myotubes were cultured with asynchronous amino acid supply medium in which the levels of different AAs (Lysine, threonine, methionine, leucine, valine and glutamic acid) either increased and then decreased or decreased and then increased, at different replaced medium time point (at 0, 8, and 16 h). However, during the whole experiment period all the 3 treated myotubes received same amount of each AA. The sample of the myotubes were used for myotube morphology, protein, AA, and proteomic analysis. The results showed that asynchronous AA nutrition affect the synthesis and degradation of myotube proteins, and the AAAS in the medium increase, thus decreasing the synthesis rate of myotube proteins (p < 0.05) and decreasing the diameter of myotubes (p < 0.05). The process of reduced protein synthesis affects the PI3K-AKT and FoxO signaling pathway by downregulating the levels of IRS1 and EGFR, and the degradation amplitude is greater than the synthesis amplitude. Therefore, this study further revealed the effect of the asynchronous supply of amino acids on myotube protein synthesis and the underlying mechanism and provided a theoretical reference for the precision of nutrition to animals.

Involvement of E3 Ubiquitin Ligases in Viral Infections of the Human Host.

Viral infections are one of the principal causes of global primary health crises, with increased rate of infection and mortality demonstrated by the newer progeny of viruses. Viral invasion of the host involves utilization of various cellular machinery. Ubiquitination is one of a few central regulatory systems used by viruses for establishment of the infections in the host. Members of the ubiquitination system are involved in carrying out proteasomal degradation or functional modification of proteins in numerous cellular processes. E3 ubiquitin ligases play a major role in this system through recognition and recruitment of protein substrates and catalyzing the transfer of ubiquitin to these substrates. The versatility of ubiquitin ligases frequently makes them useful tools for the viruses, for either utilizing or degrading other cellular machineries, for carrying out their multiplication or inactivating the defensive strategies of the host. Therefore, these ligases are important targets for aiming at major pathways causing viral protein degradation or functional modification of the infection process. In this review, we have discussed the role and mechanism of different types of ubiquitin ligases in the context of infections of mainly human viruses, highlighting the viral proteins directly interacting with the ligases. Knowledge about these direct interactions is central in understanding the ubiquitin-dependent processes. This comprehensive account may also be beneficial for pharmaceutical exploration of E3 ligase-based broad-spectrum antiviral treatment.

ATPase Valosin-Containing Protein (VCP) Is Involved During the Replication and Egress of Sialodacryoadenitis Virus (SDAV) in Neurons

Sialodacryoadenitis virus (SDAV) has been identified as the etiological agent responsible for the respiratory system and salivary gland infections in rats. The existing literature on SDAV infections is insufficient to address the topic adequately, particularly in relation to the central nervous system. In order to ascertain how SDAV gains access to neuronal cells and subsequently exits, our attention was focused on the small molecule valosin-containing protein (VCP), which is an ATPase. VCP is acknowledged for its function in the ubiquitin-mediated proteasomal degradation of proteins, including those of viral origin. To ascertain the potential influence of VCP on SDAV replication and egress, high-content screening was employed to determine the viral titer and protein content. Western blot analysis was employed to ascertain the relative expression of VCP. Real-time imaging of SDAV-infected cells and confocal imaging for qualitative morphological analysis were conducted. The Eeyarestatin I (EerI) inhibitor was employed to disrupt VCP involvement in the endoplasmic reticulum-associated protein degradation pathway (ERAD) in both pre- and post-incubation systems, with concentrations of 5 μM/mL and 25 μM/mL, respectively. We demonstrated for the first time that SDAV productively replicates in cultured primary neurons. VCP expression is markedly elevated during SDAV infection. The application of 5 μM/mL EerI in the post-treatment system yielded a statistically significant inhibition of the SDAV yield. It is likely that this modulates the efficacy of virion assembly by arresting viral proteins in the submembrane area.

Novel TBC1D8B variant causes neonatal nephrotic syndrome combined with acute kidney injury

BackgroundNephrotic syndrome (NPHS), characterized by proteinuria, hypoalbuminemia, and edema, can be caused by genetic variations. TBC1D8B was recently discovered as a novel disease-causing gene for X-linked NPHS. With only a few reported cases, the clinical manifestations associated with variants of this gene need to be further examined.MethodsWe recruited a newborn with NPHS complicated by acute kidney injury (AKI) and his parents and tested the potential genetic cause of the disease through trio-whole exome sequencing and Sanger sequencing. Western blotting (WB) was performed using a mutant plasmid to evaluate mutant protein expression levels. Since the TBC1D8B protein interacts with RAB proteins to catalyze the GTPase hydrolysis process, immunofluorescence (IF) can be used to verify the interaction between the TBC1D8B mutant protein and RAB11A/RAB11B, and thus to confirm its effect on the endocytosis and vesicle recycling functions of RAB proteins within the cell.ResultsThe child, at 1 month, showed severe edema and proteinuria and unexplained coma with epilepsy. Ultrasound examination revealed multiple organ enlargement, and MRI showed nonspecific high diffusion-weighted imaging signal characteristics in the splenium of the corpus callosum. Hematoxylin and eosin staining showed diffuse inflammatory cell infiltration in the renal interstitium and multifocal renal tubule lumen expansion. Diffuse fusion of podocyte foot processes was observed under electron microscopy, indicating glomerular podocyte lesions. Genetic testing revealed a maternally inherited novel hemizygous variant, NM_017752: c.628 A > T, p.Lys210Ter, in TBC1D8B. In vitro functional experiments showed that this variant may lead to TBC1D8B protein degradation. IF results showed disrupted interaction with RAB11A/RAB11B, that then affects the biological function of RAB proteins in the process of cell intimal vesicle formation and intracellular transport.ConclusionThis study will enrich the mutational and phenotypic spectra of TBC1D8B and demonstrate the potential of this gene variants to cause early-onset NPHS leading to severe kidney disease.

High-level secretory expression and characterization of an acid protease in Komagataella phaffii and its application in soybean meal protein degradation

Acid proteases play a crucial role in the industrial enzyme market, but low yield limits their widespread application. In this study, we focused on enhancing the secretory expression level of an acid protease (AopepA) from Aspergillus oryzae in Komagataella phaffii through stepwise genetic modification strategies. These included the co-expression of endoplasmic reticulum secretion-associated factors, overexpression of eukaryotic translation initiation factors, knockout of the β-1,3-glucanosyltransferase gene, disruption of the hypoxic heme-dependent repressor gene, and co-expression of the hemoglobin gene. After these modifications, protease activity increased by 4.2-fold, reaching 536.6 U/mL in a shaking flask. The engineered strain produced protease activity of up to 17,392.0 U/mL with a protein concentration of 44.6 g/L in a 5 L fermenter, representing the highest secretory expression level of acid proteases in K. phaffii ever reported. The optimal conditions of AopepA were pH 3.0 and 50 °C. AopepA demonstrated broad hydrolysis activity towards various protein substrates. It efficiently degraded soybean meal proteins into low molecular weight (Mw < 1 kDa, accounting for 82 %) oligopeptides to enhance protein utilization. This study provides valuable insights into improving the secretory expression of acid proteases in K. phaffii and identifies a suitable acid protease for enhancing soybean meal protein utilization.

Pathogenic mechanism and therapeutic intervention of impaired N7-methylguanosine (m7G) tRNA modification

Mutations modification enzymes including the tRNA N7-methylguanosine (m7G) methyltransferase complex component WDR4 were frequently found in patients with neural disorders, while the pathogenic mechanism and therapeutic intervention strategies are poorly explored. In this study, we revealed that patient-derived WDR4 mutation leads to temporal and cell-type-specific neural degeneration, and directly causes neural developmental disorders in mice. Mechanistically, WDR4 point mutation disrupts the interaction between WDR4 and METTL1 and accelerates METTL1 protein degradation. We further uncovered that impaired tRNA m7G modification caused by Wdr4 mutation decreases the mRNA translation of genes involved in mTOR pathway, leading to elevated endoplasmic reticulum stress markers, and increases neural cell apoptosis. Importantly, treatment with stress-attenuating drug Tauroursodeoxycholate (TUDCA) significantly decreases neural cell death and improves neural functions of the Wdr4 mutated mice. Moreover, adeno-associated virus mediated transduction of wild-type WDR4 restores METTL1 protein level and tRNA m7G modification in the mouse brain, and achieves long-lasting therapeutic effect in Wdr4 mutated mice. Most importantly, we further demonstrated that both TUDCA treatment and WDR4 restoration significantly improve the survival and functions of human iPSCs-derived neuron stem cells that harbor the patient's WDR4 mutation. Overall, our study uncovers molecular insights underlying WDR4 mutation in the pathogenesis of neural diseases and develops two promising therapeutic strategies for treatment of neural diseases caused by impaired tRNA modifications.

Protein degradation of antizyme depends on the N-terminal degrons.

Antizyme (AZ) is a regulatory protein that plays a crucial role in modulating the activity of ornithine decarboxylase (ODC), which is the initial and rate-limiting enzyme in the complex pathway of polyamine biosynthesis. AZ facilitates the swift degradation of ODC, thereby modulating the levels of cellular polyamines. This study unveils a new ubiquitin-independent mechanism for AZ degradation, emphasizing the essential role of N-terminal degrons. Contrary to traditional ubiquitin-dependent degradation, our findings reveal that AZ degradation is significantly influenced by its N-terminal region. By conducting a series of experiments, including in vitro degradation assays, cycloheximide chase experiments, differential scanning calorimetry, and measurement of cellular concentrations of polyamines, we demonstrate that N-terminal truncation significantly enhances AZ's stability and facilitates the reduction of polyamine levels by accelerating ODC degradation. The removal of the N-terminal portion of AZ results in a reduced degradation rate and enhanced thermal stability of the protein, leading to a more efficient inhibition of polyamine synthesis. These findings are corroborated by the analysis of AZ isoforms, AZ1, AZ2, and AZ3, which display differential degradation patterns based on the specific N-terminal segments. This substantiates a degradation mechanism driven by an intrinsically disordered N-terminal region acting as a degron, independent of lysine ubiquitination. These results underscore the significant regulatory function of the N-terminal domain in the activity of AZ and the maintenance of polyamine homeostasis.

Targeted protein degradation via cellular trafficking of nanoparticles.

Strategies that selectively bind proteins of interest and target them to the intracellular protein recycling machinery for targeted protein degradation have recently emerged as powerful tools for undruggable targets in biomedical research and the pharmaceutical industry. However, targeting any new protein of interest with current degradation tools requires a laborious case-by-case design for different diseases and cell types, especially for extracellular targets. Here we observe that nanoparticles can mediate specific receptor-independent internalization of a bound protein and further develop a general strategy for degradation of extracellular proteins of interest by making full use of clinically approved components. This extremely flexible strategy aids in targeted protein degradation tool development and provides knowledge for targeted drug therapies and nanomedicine design.

Compound heterozygous RYR1-RM mouse model reveals disease pathomechanisms and muscle adaptations to promote postnatal survival.

Pathogenic variants in the type I ryanodine receptor (RYR1) result in a wide range of muscle disorders referred to as RYR1-related myopathies (RYR1-RM). We developed the first RYR1-RM mouse model resulting from co-inheritance of two different RYR1 missense alleles (Ryr1TM/SC-ΔL mice). Ryr1TM/SC-ΔL mice exhibit a severe, early onset myopathy characterized by decreased body/muscle mass, muscle weakness, hypotrophy, reduced RYR1 expression, and unexpectedly, incomplete postnatal lethality with a plateau survival of ~50% at 12 weeks of age. Ryr1TM/SC-ΔL mice display reduced respiratory function, locomotor activity, and invivo muscle strength. Extensor digitorum longus muscles from Ryr1TM/SC-ΔL mice exhibit decreased cross-sectional area of type IIb and type IIx fibers, as well as a reduction in number of type IIb fibers. Exvivo functional analyses revealed reduced Ca2+ release and specific force production during electrically-evoked twitch stimulation. In spite of a ~threefold reduction in RYR1 expression in single muscle fibers from Ryr1TM/SC-ΔL mice at 4 weeks and 12 weeks of age, RYR1 Ca2+ leak was not different from that of fibers from control mice at either age. Proteomic analyses revealed alterations in protein synthesis, folding, and degradation pathways in the muscle of 4- and 12-week-old Ryr1TM/SC-ΔL mice, while proteins involved in the extracellular matrix, dystrophin-associated glycoprotein complex, and fatty acid metabolism were upregulated in Ryr1TM/SC-ΔL mice that survive to 12 weeks of age. These findings suggest that adaptations that optimize RYR1 expression/Ca2+ leak balance, sarcolemmal stability, and fatty acid biosynthesis provide Ryr1TM/SC-ΔL mice with an increased survival advantage during postnatal development.

Moyamoya disease: insights into the clinical implications of the RNF213 gene

Moyamoya disease (MMD) is a rare cerebrovascular disorder characterized by progressive stenosis of the terminal internal carotid arteries and the formation of compensatory collateral vessels, which appear as a “puff of smoke” on cerebral angiography. It is a significant cause of stroke in East Asia, with an incidence of 0.5 to 1.5 cases per 100,000 people annually. The etiology of MMD remains unclear; however, the identification of the RNF213 gene, particularly the R4810K variant, as a major susceptibility factor among the East Asian population, has provided crucial insights into the disease's pathophysiology and clinical manifestations. MMD typically presents with transient ischemic attacks, ischemic and hemorrhagic strokes, seizures, headaches, and cognitive deficits. Diagnostic criteria have evolved to emphasize advanced imaging techniques. Pathological features include fibrocellular intimal thickening, irregular undulation of the elastic lamina, and the formation of moyamoya vessels. The mutation in the RNF213 gene impairs the degradation of proteins involved in vessel development, leading to abnormal angiogenesis. Genotype-phenotype studies indicate that the RNF213 variant is associated with an earlier onset, transient ischemic attacks, infarctions, and involvement of the posterior cerebral artery, although its effects vary between regions. Additionally, the homozygous RNF213 variant consistently correlates with an earlier age of onset and a higher risk of cerebral infarction. However, further research is necessary to fully understand its long-term impacts and its relationship with revascularization outcomes. Ongoing research is crucial to fully comprehend the pathophysiology and genetics of MMD, improve prognostic predictions, and develop novel therapies.

Muscle RING-finger proteins (MuRF) regulate protein kinase A activity via retrograde vesicular transport of PKA RI-alpha

Abstract Background Muscle RING finger (MuRF) proteins are muscle-restricted E3 ubiquitin ligases that control protein degradation in striated muscles. MuRF proteins have coordinate functions for maintenance of striated muscle structure and function that depends on their target proteins. MuRF1 is a key enzyme in muscle atrophy. MuRF2 and MuRF3 bind to and stabilize microtubules which affects striated muscle differentiation and contraction. Although some MuRF-interaction partners have been described only few have been proven to be targeted for proteasomal degradation. To better understand the function of MuRF proteins, we set out to identify and functionally characterize novel MuRF target proteins. Methods and Results Stable isotope labeling of amino acids in cell culture followed by affinity purification and quantitative mass spectrometry analysis (SILAC-AP-MS) was used to identify the interactome of MuRF proteins. We identified the mammalian retromer subunit sorting nexin 5 (SNX5) that is involved in subcellular trafficking as a novel MuRF2 and MuRF3 interaction partner. Coimmunoprecipitation experiments and immunocytochemistry confirmed physical interaction and colocalization between SNX5 and MuRF2 or MuRF3. The interaction domains were mapped. MuRF2, MuRF3 and SNX5 were colocalized to early endosomes at microtubules in myocytes. MuRF2 mediated the ubiquitin proteasome system-dependent degradation of SNX5 in a RING-finger dependent manner, whereas MuRF3 stabilized SNX5. Using mass spectrometry, we identified the regulatory subunit of protein kinase A (PKA-RI-α) as a cargo of SNX5 coated early endosomes in myocytes. CRISPR-Cas9 and siRNA experiments showed that SNX5 regulates PKA activity by stabilizing PKA-RI-α in myocytes. Deletion of SNX5 resulted in PKA activation and inhibition of the HDAC5-MEF2 axis that disturbed myogenic differentiation. Conclusion MuRF2 and MuRF3 play opposing roles in SNX5-mediated retrograde transport of early endosomes along microtubules in myocytes. This mechanism is involved in regulation of PKA catalytic activity via stabilization of PKA-RI-α and controls differentiation of striated muscles.

Skeletal myocyte-specific knockout of Ptpn1 and Ptpn2 enhances inflammatory response in muscle and increases mortality in polymicrobial sepsis

Abstract Background Critically ill patients often suffer from heart and skeletal muscle atrophy and failure (intensive care unit acquired weakness; ICUAW). Inflammation and sepsis are major risk factors for ICUAW. Critically ill patients with sepsis frequently develop insulin resistance that decreases protein synthesis and increases protein degradation in muscle eventually leading to ICUAW. The link between inflammation and insulin resistance is not well understood, but protein tyrosine phosphatases (PTP), that inhibit insulin signaling via insulin receptor (INSR) dephosphorylation as well as inflammation-associated pathways, are implicated. Hypothesis: We hypothesized that PTP1B and TC-PTP mediate inflammation-induced insulin resistance and muscle atrophy in heart and skeletal muscle. Methods and Results qRT-PCR analyses showed that the proinflammatory cytokines tumor necrosis factor (TNF), interleukin (IL)-1β, and IL-6 caused a ~2-fold increase in Ptpn1/PTP1B and Ptpn2/TC-PTP mRNA expression in C2C12 myocytes as well as in neonatal rat ventricular cardiomyocytes. Inhibition of the IL-6/GP130 pathway by siRNA and inhibitors in myocytes in vitro and skeletal muscle specific gp130 knockout mice in vivo attenuated IL-6-induced Ptpn1/PTP1B and Ptpn2/TC-PTP mRNA expression. CLP-induced polymicrobial sepsis was associated with a significant increase in gene expression and protein content of pro-inflammatory cytokines (Tnf, Il1b, Ifng) and leukocyte markers (CD68, CD11c, F4/80) in muscle of skeletal myocyte specific double knockout (Ptpn1+Ptpn2loxP/loxP, MCK cre, dKO) compared to wildtype (Ptpn1+Ptpn2loxP/loxP) mice in sepsis. This also resulted in a lower survival rate of dKO mice in sepsis (42% WT vs. 23% dKO). Immunohistological analyses revealed centralized nuclei, macrophage/monocyte infiltration and enhanced regeneration (Myh3, Pax7, Ki67) in tibialis anterior muscle of septic dKO mice. A strong activation of the NLRP3-inflammasome, as indicated by cleavage of caspase 1, GSDMD, GSDME and caspase 8, indicative for a pronounced inflammation and activated cell death pathways was observed in muscle of septic dKO mice. Conclusion The proinflammatory cytokine IL-6 increases the expression of Ptpn1 and Ptpn2 and enhances overall PTP activity. Selective inhibition of the IL-6R/GP130 signaling pathway by JAK2- and STAT3-inhibition as well as Gp130 deletion in myocytes in vivo attenuates those effects. Muscle-specific deletion of Ptpn1 and Ptpn2 in skeletal muscle leads to increased mortality, enhanced pro-inflammatory response and induced inflammatory, programmed cell death pathways in sepsis. Our data show that Ptpn1 and Ptpn2 play a key anti-inflammatory role in skeletal muscle.

Cardioprotective effects of nicorandil on mitochondrial apoptosis signaling in the failing heart of cardiac-specific Bcl-2-associated athanogene (BAG) 3 knockout mice

Abstract Background Bcl-2-associated athanogene (BAG) 3 is a known regulator of mitochondrial apoptotic cell death and autophagic protein degradation in the heart. Disruption of the BAG3 gene in a cardiac-specific manner can lead to cardiac failure in mice. However, therapeutic approaches for cardiac failure induced by BAG3 gene disruption remain poorly understood. Method and results We characterized mice with the ablated BAG3 gene using the Cre-recombinase-loxp site system in a cardiac-specific manner (BAG3f/f crossbred with cardiac specific Cre transgenic mice; BAG3 cKO) and examined the therapeutic effect of nicorandil, an antianginal drug, in BAG3 cKO mice. Cardiac BAG3 levels were markedly reduced in the cytoplasmic and mitochondrial fractions from the hearts of BAG3 cKO mice compared to BAG3f/f mice. BAG3cKO mice exhibited cardiac disease such as a reduction in fractional shortening detected by echocardiography, cardiac fibrosis at 6 months of age, and approximately 40% premature death by 6 months of age. Electron microscopy analysis confirmed cardiomyocyte loss and mitochondrial abnormalities in some areas of the heart section from BAG3cKO mice at 4.5 months of age. A marked decrease in the level of cytochrome C in the mitochondrial fraction and a slight increase in the cytoplasmic fraction, along with apoptotic cell death, were observed in hearts from BAG3 cKO mice at 4.5 months. Mitochondrial levels of anti-apoptotic factors, such as Bcl-2 and Bcl-xl, were reduced, while there was a marked increase in Bak1, an apoptotic inducible factor, in hearts from BAG3 cKO mice at 4.5 months of age. These results suggest that cardiac mitochondrial impairment and subsequent apoptotic cell death occurred in BAG3cKO mice. Nicorandil treatment (81 mg/L drinking water) initiated at 2 months of age prevented the reduction of fractional shortening, cardiac fibrosis, ultrastructural cardiac abnormalities, and premature death. Furthermore, nicorandil treatment prevented the decrease in mitochondrial cytochrome C and the increase in cytoplasmic cytochrome C in hearts from BAG3 cKO mice at 4.5 months of age. Nicorandil treatment also inhibited the elevation of mitochondrial Bak1 levels, reduction in Bcl-2 levels, and occurrence of apoptotic cell death in hearts from BAG3 cKO mice at 4.5 months of age. Conclusion Long-term treatment with nicorandil can inhibit cardiac disease induced by cardiac- specific BAG3 ablation through mitochondrial protection via the prevention of activation of mitochondrial apoptosis signaling.

The Role and Interactive Mechanism of Endoplasmic Reticulum Stress and Ferroptosis in Musculoskeletal Disorders

Endoplasmic reticulum (ER) stress is a cellular phenomenon that arises in response to the accumulation of misfolded proteins within the ER. This process triggers the activation of a signalling pathway known as the unfolded protein response (UPR), which aims to restore ER homeostasis by reducing protein synthesis, increasing protein degradation, and promoting proper protein folding. However, excessive ER stress can perturb regular cellular function and contribute to the development of diverse pathological conditions. As is well known, ferroptosis is a kind of programmed cell death characterized by the accumulation of lipid peroxides and iron-dependent reactive oxygen species (ROS), resulting in oxidative harm to cellular structures. In recent years, there has been increasing evidence indicating that ferroptosis occurs in musculoskeletal disorders (MSDs), with emerging recognition of the complex relationship between ER stress and ferroptosis. This review presents a summary of ER stress and the ferroptosis pathway. Most importantly, it delves into the significance of ER stress in the ferroptosis process within diverse skeletal or muscle cell types. Furthermore, we highlight the potential benefits of targeting the correlation between ER stress and ferroptosis in treating degenerative MSDs.

Amelioration of Cancer Cachexia by Dalbergia odorifera Extract Through AKT Signaling Pathway Regulation

Background/Objectives: Cancer cachexia is a multifactorial syndrome characterized by the progressive loss of skeletal muscle mass and adipose tissue. Dalbergia odorifer is widely used in traditional medicine in Korea and China to treat various diseases. However, its exact role and underlying mechanism in regulating cancer cachexia have not been elucidated yet. This research was conducted to investigate the effect of D. odorifer extract (DOE) in preventing the development of cancer-induced cachexia symptoms and figure out the relevant mechanisms. Methods: A cancer cachexia model was established in Balb/c mice using the CT26 colon carcinoma cell line. To evaluate the anti-cachexia effect of Dalbergia odorifer extract (DOE), CT26-bearing mice were orally administered with DOE at concentrations of 50 and 100 mg/kg BW for 14 days. C2C12 myotubes and 3T3L1 adipocytes were treated with 80% CT26 conditioned medium, DOE, and wortmannin, a particular AKT inhibitor to determine the influence of DOE in the AKT signaling pathway. Mice body weight, food intake, myofiber cross-sectional area, adipocyte size, myotube diameter, lipid accumulation, and relevant gene expression were analyzed. Results: The oral administration of DOE at doses of 50 and 100 mg/kg body weight to CT26 tumor-bearing mice resulted in a significant reduction in body weight loss, an increase in food intake, and a decrease in serum glycerol levels. Furthermore, DOE treatment led to an increase in muscle mass, larger muscle fiber diameter, and elevated expression levels of MyH2 and Igf1, while simultaneously reducing the expression of Atrogin1 and MuRF1. DOE also attenuated adipose tissue wasting, as evidenced by increased epididymal fat mass, enlarged adipocyte size, and upregulated Pparγ expression, alongside a reduction in Ucp1 and IL6 levels. In cachectic C2C12 myotubes and 3T3-L1 adipocytes induced by the CT26 conditioned medium, DOE significantly inhibited muscle wasting and lipolysis by activating the AKT signaling pathway. The treatment of wortmannin, a specific AKT inhibitor, effectively neutralized DOE’s impact on the AKT pathway, myotube diameter, and lipid accumulation. Conclusions: DOE ameliorates cancer cachexia through the expression of genes involved in protein synthesis and lipogenesis, while suppressing those related to protein degradation, suggesting its potential as a plant-derived therapeutic agent in combating cancer cachexia.

Baicalein inhibits cell proliferation and induces apoptosis in brain glioma cells by downregulating the LGR4-EGFR pathway.

Patients diagnosed with brain glioma have a poor prognosis and limited therapeutic options. LGR4 is overexpressed in brain glioma and involved in the tumorigenesis of many tumors. Baicalein (BAI) is a kind of flavonoid that has exhibited anti-tumor effects in various tumors. Nevertheless, the functions and associations of BAI and LGR4 in brain glioma remain unclear. In this study, Gene Expression Profiling Interactive Analysis and Human Protein Atlas databases were used to perform expression and survival analysis of LGR4 in brain glioma patients. Subsequently, the significance of LGR4-EGFR in brain glioma cells (HS683 and KNS89) and brain glioma animal models was explored by RNA interference and subcutaneous transplantation. Additionally, brain glioma cells were treated with BAI to explore the roles and mechanisms of BAI in brain glioma. The results showed that LGR4 was highly expressed in brain glioma and was related to a poor prognosis. LGR4 knockdown repressed the proliferation and EGFR phosphorylation but induced apoptosis in brain glioma cells. However, these effects were reversed by EGFR overexpression and CBL knockdown. In contrast, both in vitro and in vivo experiments revealed that LGR4 overexpression facilitated brain glioma cell malignant behavior and promoted tumor development, but these effects were rescued by BAI and an EGFR inhibitor. Furthermore, si-LGR4 accelerated EGFR protein degradation, while oe-LGR4 exhibited the opposite effect. Without affecting normal cellular viability, BAI inhibited malignant behavior, interacted with LGR4, and blocked the LGR4-EGFR pathway for brain glioma cells. In conclusion, our data suggested that BAI inhibited brain glioma cell proliferation and induced apoptosis by downregulating the LGR4-EGFR pathway, which provides a novel strategy and potential therapeutic targets to treat brain glioma.

The Dsc ubiquitin ligase complex identifies transmembrane degrons to degrade orphaned proteins at the Golgi

The Golgi apparatus is essential for protein sorting, yet its quality control mechanisms are poorly understood. Here we show that the Dsc ubiquitin ligase complex uses its rhomboid pseudo-protease subunit, Dsc2, to assess the hydrophobic length of α-helical transmembrane domains (TMDs) at the Golgi. Thereby the Dsc complex likely interacts with orphaned ER and Golgi proteins that have shorter TMDs and ubiquitinates them for targeted degradation. Some Dsc substrates will be extracted by Cdc48 for endosome and Golgi associated proteasomal degradation (EGAD), while others will undergo ESCRT dependent vacuolar degradation. Some substrates are degraded by both, EGAD- or ESCRT pathways. The accumulation of Dsc substrates entails a specific increase in glycerophospholipids with shorter and asymmetric fatty acyl chains. Hence, the Dsc complex mediates the selective degradation of orphaned proteins at the sorting center of cells, which prevents their spreading across other organelles and thereby preserves cellular membrane protein and lipid composition.

The quest for the best target genes for RNAi-mediated pest control.

RNA interference (RNAi) has emerged as an eco-friendly alternative to classic pesticides for pest control. This review highlights the importance of identifying the best target genes for RNAi-mediated pest control. We argue that the knowledge-based approach to predicting effective targets is limited by our current gaps of knowledge, making unbiased screening a superior method for discovering the best target processes and genes. We emphasize the recent evidence that suggests targeting conserved basic cellular processes, such as protein degradation and translation, is more effective than targeting the classic pesticide target processes. We support these claims by comparing the efficacy of previously reported RNAi target genes and classic insecticide targets with data from our genome-wide RNAi screen in the red flour beetle, Tribolium castaneum. Finally, we provide practical advice for identifying excellent target genes in other pests, where large-scale RNAi screenings are typically challenging.