Investigation of the effects of nanoplastic polyethylene terephthalate on environmental toxicology using model Drosophila melanogaster.

Plastic pollution has become a major environmental concern, and various plastic polymers are used daily. A study was conducted to examine the toxic effects of polyethylene terephthalate (PET) nanoplastics (NPLs) on Drosophila melanogaster. We have successfully synthesized PET NPLs and characterized using DLS, Zeta potential, TEM, HRTEM, SAED, XRD, FTIR, and Raman spectroscopy to gain crucial insights into the structure and properties. We fed PET NPLs to Drosophila to assess toxicity. ROS was quantified using DCFH-DA and NBT, and the nuclear degradation was checked by DAPI staining. Quantification of protein and activity of antioxidant enzymes like SOD, catalase depicted the adverse consequences of PET NPLs exposure. The dorsal side of the abdomens, eyes, and wings were also defective when phenotypically analyzed. These results substantiate the genotoxic and cytotoxic impact of nanoplastics. Notably, behavioral observations encompassing larval crawling and climbing of adults exhibit normal patterns, excluding the presence of neurotoxicity. Adult Drosophila showed decreased survivability, and fat accumulation enhanced body weight. These findings contribute to unraveling the intricate mechanisms underlying nanoplastic toxicity and emphasize its potential repercussions for organismal health and ecological equilibrium.

Mechanisms and targeting of proteosome-dependent androgen receptor degradation in prostate cancer.

The androgen receptor (AR) remains to be a key target for the treatment of prostate cancer, including the majority of castration-resistant prostate cancer (CRPC). AR is stabilized in CRPC and the ubiquitin-proteasome system (UPS) plays a major role in AR degradation. Targeting AR for degradation provides a potential approach to overcome the resistance of CRPC to current AR antagonists, including the next generation AR signaling inhibitors. Different types of AR degraders have been developed, including the proteolysis-targeting chimeras (PROTACs), selective AR degraders (SARDs), and novel AR degraders, with several AR PROTACs currently in clinical trials. The present mini-review discusses the regulation of AR degradation by the UPS, the potential role of a novel nuclear degradation signal in AR, and different types of AR degraders.

Atg5 Regulates Selective Autophagy of the Parental Macronucleus during Tetrahymena Sexual Reproduction.

Nuclear autophagy is an important selective autophagy process. The selective autophagy of sexual development micronuclei (MICs) and the programmed nuclear degradation of parental macronucleus (paMAC) occur during sexual reproduction in Tetrahymena thermophila. The molecular regulatory mechanism of nuclear selective autophagy is unclear. In this study, the autophagy-related protein Atg5 was identified from T. thermophila. Atg5 was localized in the cytoplasm in the early sexual-development stage and was localized in the paMAC in the late sexual-development stage. During this stage, the degradation of meiotic products of MIC was delayed in atg5i mutants. Furthermore, paMAC was abnormally enlarged and delayed or failed to degrade. The expression level and lipidation of Atg8.2 significantly decreased in the mutants. All these results indicated that Atg5 was involved in the regulation of the selective autophagy of paMAC by regulating Atg8.2 in Tetrahymena.

Establishment of conidial fusion in the asexual fungus Verticillium dahliae as a useful system for the study of non-sexual genetic interactions.

Cell-to-cell fusion is a fundamental biological process across the tree of life. In filamentous fungi, somatic fusion (or anastomosis) is required for the normal development of their syncytial hyphal networks, and it can initiate non-sexual genetic exchange processes, such as horizontal genetic transfer and the parasexual cycle. Although these could be important drivers of the evolution of asexual fungi, this remains a largely unexplored possibility due to the lack of suitable resources for their study in these puzzling organisms. We thus aimed at the characterization of cell fusion in the important asexual fungus Verticillium dahliae via Conidial Anastomosis Tubes (CATs), which can be useful for the analysis of parasexuality. We optimized appropriate procedures for their highly reproducible quantification and live-cell imaging, which were used to characterize their physiology and cell biology, and to start elucidating their underlying genetic machinery. Formation of CATs was shown to depend on growth conditions and require functional Fus3 and Slt2 MAP kinases, as well as the NADPH oxidase NoxA, whereas the GPCR Ste2 and the mating-type protein MAT1-2-1 were dispensable. We show that nuclei and other organelles can migrate through CATs, which often leads to the formation of transient dikaryons. Their nuclei have possible windows of opportunity for genetic interaction before degradation of one by a presumably homeostatic mechanism. We establish here CAT-mediated fusion in V. dahliae as an experimentally convenient system for the cytological analysis of fungal non-sexual genetic interactions. We expect that it will facilitate the dissection of sexual alternatives in asexual fungi.

Out or decay: fate determination of nuclear RNAs.

In eukaryotes, RNAs newly synthesized by RNA polymerase II (RNAPII) undergo several processing steps prior to transport to the cytoplasm. It has long been known that RNAs with defects in processing or export are removed in the nucleus. Recent studies revealed that RNAs without apparent defects are also subjected to nuclear degradation, indicating that nuclear RNA fate is determined in a more complex and dynamic way than previously thought. Nuclear RNA sorting directly determines the quality and quantity of RNA pools for future translation and thus is of significant importance. In this essay, we will summarize recent studies on this topic, mainly focusing on findings in mammalian system, and discuss about important remaining questions and possible biological relevance for nuclear RNA fate determination.

A Nuclear Export Block Triggers the Decay of Newly Synthesized Polyadenylated RNA.

Genomes are promiscuously transcribed, necessitating mechanisms that facilitate the sorting of RNA for function or destruction. The polyA (pA) tail is one such distinguishing feature, which in the Saccharomyces cerevisiae nucleus is bound by the Nab2p protein, yielding transcript protection. As Nab2p also contacts the main nuclear export factor Mex67p, we asked whether transport kinetics contributes to RNA sorting. Indeed, 3' end sequencing of newly transcribed pA+ RNAs demonstrates that nuclear depletion of Mex67p elicits their instant and global decay. A similar phenotype is evident upon inactivation of other export factors and proportional to the amount of nuclear pA+ RNA. As RNA expression is partially rescued by Nab2p overexpression, we propose that an export block out-titrates Nab2p onto nuclear-retained pA+ RNA, reducing the pool of Nab2p available to protect new transcripts. More generally, we suggest that nuclear RNA decay, negotiated by Nab2p availability, aids in balancing cellular transcript supply with demand.

Tau Positive Neurons Show Marked Mitochondrial Loss and Nuclear Degradation in Alzheimer's Disease.

BACKGROUND: Alzheimer's disease (AD) pathology consists of intraneuronal neurofibrillary tangles, made of hyperphosphorylated tau and extracellular accumulation of beta amyloid (Aß) in Aß plaques. There is an extensive debate as to which pathology initiates and is responsible for cellular loss in AD. METHODS: Using confocal and light microscopy, post mortem brains from control and AD cases, an antibody to SOD2 as a marker for mitochondria and an antibody to all forms of tau, we analyzed mitochondrial density in tau positive neurons along with nuclear degradation by calculating the raw integrative density. RESULTS: Our findings showed an extensive staining of aggregated tau in cell bodies, dystrophic neurites and neurofilaments in AD with minimal staining in control tissue, along with a marked decrease in mitochondria in tau positive (tau+) neurons. The control or tau negative (tau-) neurons in AD contained an even distribution of mitochondria, which was greatly diminished in tau+ neurons by 40%. There were no significant differences between control and tau- neurons in AD. Tau+ neurons showed marked nuclear degradation which appeared to progress with the extent of tau aggregation. The aggregated tau infiltrated and appeared to break the nuclear envelope with progressively more DNA exiting the nucleus and associating with the aggregated intracellular tau. CONCLUSION: We report that the mitochondrial decrease is likely due to a decrease in the protein synthesis rather than a redistribution of mitochondria because of the decreased axonal transport. We suggest that the decrease in mitochondria and nuclear degradation are key mechanisms for the neuronal loss seen in AD.

PML-Nuclear Bodies Regulate the Stability of the Fusion Protein Dendra2-Nrf2 in the Nucleus.

BACKGROUND/AIMS: Nuclear factor erythroid 2-related factor 2 (Nrf2) is a basic leucine-zipper transcription factor essential for cellular responses to oxidative stress. Degradation of Nrf2 in the cytoplasm, mediated by Keap1-Cullin3/RING box1 (Cul3-Rbx1) E3 ubiquitin ligase and the proteasome, is considered the primary pathway controlling the cellular abundance of Nrf2. Although the nucleus has been implicated in the degradation of Nrf2, little information is available on how this compartment participates in degrading Nrf2. METHODS: Here, we fused the photoconvertible fluorescent protein Dendra2 to Nrf2 and capitalized on the irreversible change in color (green to red) that occurs when Dendra2 undergoes photoconversion to study degradation of Dendra2-Nrf2 in single live cells. RESULTS: Using this approach, we show that the half-life (t1/2) of Dendra2-Nrf2 in the whole cell, under homeostatic conditions, is 35 min. Inhibition of the proteasome with MG-132 or induction of oxidative stress with tert-butylhydroquinone (tBHQ) extended the half-life of Dendra2-Nrf2 by 6- and 28-fold, respectively. By inhibiting nuclear export using Leptomycin B, we provide direct evidence that degradation of Nrf2 also occurs in the nucleus and involves PML-NBs (Promyelocytic Leukemia-nuclear bodies). We further demonstrate that co-expression of Dendra2-Nrf2 and Crimson-PML-I lacking two PML-I sumoylation sites (K65R and K490R) changed the decay rate of Dendra2-Nrf2 in the nucleus and stabilized the nuclear derived Nrf2 levels in whole cells. CONCLUSION: Altogether, our findings provide direct evidence for degradation of Nrf2 in the nucleus and suggest that modification of Nrf2 in PML nuclear bodies contributes to its degradation in intact cells.

Arkadia (RING Finger Protein 111) Mediates Sumoylation-Dependent Stabilization of Nrf2 Through K48-Linked Ubiquitination.

BACKGROUND/AIMS: The transcription factor Nrf2 is a master regulator of the antioxidant defense system, protecting cells from oxidative damage. We previously reported that the SUMO-targeted E3 ubiquitin ligase (STUbL), RING finger protein 4 (RNF4) accelerated the degradation rate of Nrf2 in promyelocytic leukemia-nuclear body (PML-NB)-enriched fractions and decreased Nrf2-mediated gene transcription. The mechanisms that regulate Nrf2 nuclear levels are poorly understood. In this study, we aim to explore the role of the second mammalian STUbL, Arkadia/RNF111 on Nrf2. METHODS: Arkadia mediated ubiquitination was detected using co-immunoprecipitation assays in which whole cell lysates were immunoprecipated with anti-Nrf2 antibody and Western blotted with anti-hemagglutinin (HA) antibody or anti-Lys-48 ubiquitin-specific antibody. The half-life of Nrf2 was detected in whole cell lysates and promyelocytic leukemia-nuclear body enriched fractions by cycloheximide-chase. Reporter gene assays were performed using the antioxidant response element (ARE)-containing promoter Heme oxygenase-1 (HO-1). RESULTS: We show that Arkadia/RNF111 is able to ubiquitinate Nrf2 resulting in the stabilization of Nrf2. This stabilization was mediated through Lys-48 ubiquitin chains, contrary to traditionally degradative role of Lys-48 ubiquitination, suggesting that Lys-48 ubiquitination of Nrf2 protects Nrf2 from degradation thereby allowing Nrf2-dependent gene transcription. CONCLUSION: Collectively, these findings highlight a novel mechanism to positively regulate nuclear Nrf2 levels in response to oxidative stress through Arkadia-mediated K48-linked ubiquitination of Nrf2.

Uncovering mechanisms of nuclear degradation in keratinocytes: A paradigm for nuclear degradation in other tissues.

Eukaryotic nuclei are essential organelles, storing the majority of the cellular DNA, comprising the site of most DNA and RNA synthesis, controlling gene expression and therefore regulating cellular function. The majority of mammalian cells retain their nucleus throughout their lifetime, however, in three mammalian tissues the nucleus is entirely removed and its removal is essential for cell function. Lens fibre cells, erythroblasts and epidermal keratinocytes all lose their nucleus in the terminal differentiation pathways of these cell types. However, relatively little is known about the pathways that lead to complete nuclear removal and about how these pathways are regulated. In this review, we aim to discuss the current understanding of nuclear removal mechanisms in these three cell types and expand upon how recent studies into nuclear degradation in keratinocytes, an easily accessible experimental model, could contribute to a wider understanding of these molecular mechanisms in both health and pathology.

A novel mitochondrial nuclease-associated protein: a major executor of the programmed nuclear death in Tetrahymena thermophila.

BACKGROUND INFORMATION: Programmed nuclear death (PND) in the ciliate Tetrahymena is an apoptosis-like phenomenon that occurs in a restricted space of cytoplasm during conjugation. In the process, only the parental macronucleus is selectively eliminated from the progeny cytoplasm, in conjunction with differentiation of new macronuclei for the next generation. For the last decade, mitochondria have been elucidated to be a crucial executioner like apoptosis: apoptosis-inducing factor and yet-unidentified nucleases localised in mitochondria are major factors for PND. RESULTS: To identify such nucleases, we performed a DNase assay in a PAGE (SDS-DNA-PAGE) using total mitochondrial proteins. Some proteins showed DNase activity, but particularly a 17 kDa protein exhibited the highest and predominant activity. Mass spectrometric analysis revealed a novel mitochondrial nuclease, named TMN1, whose homologue has been discovered only in the ciliate Paramecium tetraurelia, but not in other eukaryotes. Gene disruption of TMN1 led to a drastic reduction of mitochondrial nuclease activity and blocked nuclear degradation during conjugation, but did not affect accumulation of autophagic and lysosomal machinery around the parental macronucleus. CONCLUSIONS: These observations strongly suggest that the mitochondrial nuclease-associated protein plays a key role in PND as a major executor. Taking the novel protein specific to ciliates in consideration, Tetrahymena would have diverted a different protein from common apoptotic factors shared in eukaryotes to PND in the course of ciliate evolution.