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Protein termini are determinants of protein stability. Proteins bearing degradation signals, or degrons, at their amino‐ or carboxyl‐termini are eliminated by the N‐ or C‐degron pathways, respectively. We aimed to elucidate the function of C‐degron pathways and to unveil how normal proteomes are exempt from C‐degron pathway‐mediated destruction. Our data reveal that C‐degron pathways remove mislocalized cellular proteins and cleavage products of deubiquitinating enzymes. Furthermore, the C‐degron and N‐degron pathways cooperate in protein removal. Proteome analysis revealed a shortfall in normal proteins targeted by C‐degron pathways, but not of defective proteins, suggesting proteolysis‐based immunity as a constraint for protein evolution/selection. Our work highlights the importance of protein termini for protein quality surveillance, and the relationship between the functional proteome and protein degradation pathways. SYNOPSIS: Proteins with degradation signals (degron) at their amino‐ or carboxyl‐termini are eliminated by the N‐ or C‐degron pathways, respectively. Proteome‐wide analyses suggest functions of human C‐degrons signals in protein quality surveillance, as well as interplay with N‐degron‐dependent mechanisms. The Global Protein Stability (GPS) random peptide platform enables context‐independent characterization of degron features.The activity of C‐degrons is tuned by their surrounding sequences.Gly/C‐degron shortage is limited to functional eukaryotic proteomes accessible to C‐degron pathways.C‐degron pathways clear products of proteases and mislocalized cellular proteins.Mitochondrial localization of MIC19 is dually safeguarded by the N‐ and C‐degron pathways.Coronaviral diGly‐ending proteins evade C‐degron pathway‐mediated degradation. [ABSTRACT FROM AUTHOR]

ABSTRACT Exposure to benzidine has been known to induce human cancers, particularly bladder carcinomas. In this study, the zebrafish model was used to investigate the developmental toxicity of benzidine. Embryos at 6 h postfertilization (hpf) that were exposed to benzidine exhibited embryonic death in a dose- and time-dependent manner. Benzidine induced malformations in zebrafish, such as small brain development, shorter axes, and a slight pericardial edema. High concentrations (50, 100, and 200 µM) of benzidine triggered widespread apoptosis in the brain and dorsal neurons, as evidenced by acridine orange and terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) assays. Real-time polymerase chain reaction analysis also showed that benzidine treatment affected p53, bax, and noxa expression. Decreases in specific brain markers, such as emx1 in the telencephalon, ngn1 in differentiated neurons, and otx2 in the midbrain, were observed in benzidine-treated embryos at 24 hpf. Conversely, no overt changes to pax2.1 expression in the midbrain-hindbrain boundary were found. Moreover, the use of Tg( HuC: GFP) zebrafish showed that benzidine caused a malformation of the telencephalon region. Our findings show that benzidine exposure triggers widespread apoptosis in the zebrafish brain and dorsal neurons, resulting in the development of an abnormal telencephalon. © 2013 Wiley Periodicals, Inc. Environ Toxicol 29: 1428-1436, 2014. [ABSTRACT FROM AUTHOR]

Highlights: [•] Shikonin inhibited cell proliferation of gastric cancer cells. [•] Shikonin time-dependently induced necrosis and apoptosis in gastric cancer cells. [•] Shikonin enhanced reactive oxygen species generation in gastric cancer cells. [ABSTRACT FROM AUTHOR]

Protein termini are critical for protein functions. They are often more accessible than internal regions and thus are frequently subjected to various modifications that affect protein function. Protein termini also contribute to regulating protein lifespan. Recent studies have revealed a series of degradation signals located at protein C-termini, termed C-degrons or C-end degrons. C-degrons have been implicated as underlying a protein quality surveillance system that eliminates truncated, cleaved and mislocalized proteins. Despite the importance of C-degrons, our knowledge of them remains sparse. Here, we describe an established framework for the characterization of C-degrons by Global Protein Stability (GPS) profiling assay, a fluorescence-based reporter system for measuring protein stability in cellulo. Furthermore, we apply an approach that couples GPS with random peptide libraries for unbiased and context-independent characterization of C-degron motifs. Our methodology provides a robust and efficient platform for analyzing the degron potencies of C-terminal peptides, which can significantly accelerate our understanding of C-degrons., (Copyright © 2023 Elsevier Inc. All rights reserved.)

The proteolysis-assisted protein quality control system guards the proteome from potentially detrimental aberrant proteins. How miscellaneous defective proteins are specifically eliminated and which molecular characteristics direct them for removal are fundamental questions. We reveal a mechanism, DesCEND (destruction via C-end degrons), by which CRL2 ubiquitin ligase uses interchangeable substrate receptors to recognize the unusual C termini of abnormal proteins (i.e., C-end degrons). C-end degrons are mostly less than ten residues in length and comprise a few indispensable residues along with some rather degenerate ones. The C-terminal end position is essential for C-end degron function. Truncated selenoproteins generated by translation errors and the USP1 N-terminal fragment from post-translational cleavage are eliminated by DesCEND. DesCEND also targets full-length proteins with naturally occurring C-end degrons. The C-end degron in DesCEND echoes the N-end degron in the N-end rule pathway, highlighting the dominance of protein "ends" as indicators for protein elimination., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Bladder cancer is one of the causes of cancer‑related death and has a high mortality rate due to its metastatic ability. Naringenin, a bioactive compound predominantly found in citrus fruits, exhibits several cellular functions, including anti‑oxidant, ‑lipidemia and ‑cancer abilities. However, the effects of naringenin on bladder cancer cells are yet to be elucidated. The present study investigated the molecular mechanisms underlying the effects of naringenin on the migration of TSGH‑8301 bladder cancer cells. Treatment with naringenin at doses ranging between 0 and 300 µM over a period of 24 h was found to reduce cell viability. Furthermore, zymography and western blot analysis revealed that naringenin reduced the expression of matrix metalloproteinase (MMP)‑2 in a dose‑dependent manner, and repressed its activity. Naringenin also reduced TSGH‑8301 cell migration in a concentration‑dependent manner, as evidenced by wound healing and Transwell® assays. In addition, naringenin was found to inhibit AKT activity and block the nuclear translocation of nuclear factor κ‑light‑chain‑enhancer of activated B cells. In conclusion, the findings of the present study show that naringenin is capable of inhibiting bladder cancer cell migration through the downregulation of the AKT and MMP‑2 pathways.

In this study, we used zebrafish as an animal model to elucidate the developmental function of cdk10 in vertebrates. In situ hybridization analyses demonstrated that cdk10 is expressed throughout development with a relative enrichment in the brain in the late stages. Similar to its mammalian ortholog, cdk10 can interact with the transcription factor ETS2 and exhibit kinase activity by phosphorylating histone H1. Morpholino-based loss of cdk10 expression caused apoptosis in sox2-positive cells and decreased the expression of subsequent neuronal markers. Acetylated tubulin staining revealed a significant reduction in the number of Rohon-Beard sensory neurons in cdk10 morphants. This result is similar to that demonstrated by decreased islet2 expression in the dorsal regions. Moreover, cdk10 morphants exhibited a marked loss of huC-positive neurons in the telencephalon and throughout the spinal cord axis. The population of retinal ganglion cells was also diminished in cdk10 morphants. These phenotypes were rescued by co-injection of cdk10 mRNA. Interestingly, the knockdown of cdk10 significantly elevated raf1a mRNA expression. Meanwhile, an MEK inhibitor (U0126) recovered sox2 and ngn1 transcript levels in cdk10 morphants. Our findings provide the first functional characterization of cdk10 in vertebrate development and reveal its critical function in neurogenesis by modulation of raf1a expression.

Butein (3,4,2',4'-tetrahydroxychalcone) is a polyphenol derived from various natural plants and is capable of inducing several types of death in cancer cells. However, the molecular mechanisms underlying butein-induced breast cancer cell apoptosis remain unknown. The present study aimed to prove that butein inhibits the proliferation of MDA-MB‑231 human breast cancer cells in a dose- and time-dependent manner. Butein markedly induced the generation of reactive oxygen species (ROS), decreased the phosphorylation of extracellular signal-regulated kinase (ERK), increased p38 activity, diminished Bcl-2 expression, induced caspase 3 cleavage and was associated with poly(ADP-ribose) polymerase (PARP) cleavage. Our findings also indicate that ROS may play an important role in butein-induced apoptosis, as pre-treatment with the antioxidant, N-acetyl cysteine (NAC), prevented butein-induced apoptosis. In conclusion, our results demonstrate that butein inhibits the proliferation of breast cancer cells through the generation of ROS and the modulation of ERK and p38 activities. We also demonstrate that these effects may be abrogaged by pre-treatment with NAC. Our results suggest that butein may function as a potential therapeutic agent for the treatment of breast cancer.

Flavonoids exhibit chemopreventive and chemotherapeutic effects. Butein, a bioactive flavonoid isolated from numerous native plants, has been shown to induce apoptosis in human cancer cells. In the current study, the molecular mechanisms of butein action on cell proliferation and apoptosis of neuroblastoma cells were evaluated. Treatment with butein decreased the viability of Neuro-2A neuroblastoma cells in a dose- and time-dependent manner. The dose-dependent nature of butein-induced apoptosis was characterized by an increase in the sub-G1 phase population. Treatment with butein significantly increased intracellular reactive oxygen species (ROS)levels and reduced the Bcl-2/Bax ratio, triggering the cleavage of pro-caspase 3 and poly-(ADP-ribose) polymerase (PARP). Pre-treatment with the antioxidant agent, N-acetyl cysteine (NAC), blocks butein-induced ROS generation and cell death. NAC also recovers butein-induced apoptosis-related protein alteration. In conclusion, butein-triggered neuroblastoma cells undergo apoptosis via generation of ROS, alteration of the Bcl‑2/Bax ratio, and cleavage of pro-caspase 3 and PARP. Our results suggest that butein may serve as a potential therapeutic agent for the treatment of neuroblastoma.

The cyclin-dependent protein kinase family regulates a wide range of cellular functions such as cell cycle progression, differentiation, and apoptosis. In this study, we identified a zebrafish cyclin-dependent protein kinase-like 1 protein called zebrafish cdkl1 (zcdkl1), which shared a high degree of homology and conserved synteny with mammalian orthologs. zcdkl1 exhibited abilities for phosphorylation of myelin basic protein and histone H1. RT-PCR analysis revealed that zcdkl1 was expressed starting from fertilization and continuing thereafter. In adult tissues, zcdkl1 was predominantly detected in brain, ovary, and testis, and was expressed at low levels in other tissues. At 50% epiboly stage, zcdkl1 was widely expressed. At 12 to 48 h post-fertilization, zcdkl1 was predominantly expressed in the hypochord, the medial and lateral floor plate, and the pronephric duct. Interference of zcdkl1 expression resulted in abnormalities, such as brain and eye malformation, pericardial edema, and body axis curvature. Disruption of zcdkl1 reduced neurogenin-1 in the brain and sonic hedgehog expression in the floor plate region. These deformities were apparently rescued by co-injection of zcdkl1 mRNA. Findings of this study indicate that zcdkl1 plays an essential role in zebrafish development.

Shikonin, a major ingredient in the Chinese traditional herb Lithospermum erythrorhixon, exhibits multiple biological functions including antimicrobial, anti-inflammatory, and antitumor effects. In this study, we delineated the molecular mechanisms of shikonin in the apoptosis of 143B osteosarcoma cells. Shikonin reduced the cell viability of 143B cells in a dose- and time-dependent manner. The IC(50) at 24 h and 48 h for 143B cells was 4.55 and 2.01microM, respectively. A significantly elicited hypodiploid cell population was found in cells treated with 2, 4, and 8microM shikonin for 24 h. Moreover, treatment with shikonin induced reactive oxygen species (ROS) generation, increased extracellular signal-regulated kinase (ERK) phosphorylation, decreased B-cell lymphoma-2 (Bcl2) expression, and was accompanied by poly(ADP-ribose) polymerase (PARP) cleavage. Pretreatment with the antioxidant agent N-acetyl cysteine (NAC) not only reversed shikonin-induced ROS generation but also significantly attenuated the cytotoxic effects of shikonin in 143B cells. Furthermore, NAC attenuated shikonin-induced ERK phosphorylation. Taken together, our results reveal that shikonin increased ROS generation and ERK activation, and reduced Bcl2, which consequently caused the cells to undergo apoptosis. Therefore, shikonin may be a promising chemotherapeutic agent for osteosarcoma treatment.