Cellular Degradation Pathway Research Articles

Zebrafish reveal new roles for Fam83f in hatching and the DNA damage-mediated autophagic response.

The FAM83 (Family with sequence similarity 83) family is highly conserved in vertebrates, but little is known of the functions of these proteins beyond their association with oncogenesis. Of the family, FAM83F is of particular interest because it is the only membrane-targeted FAM83 protein. When overexpressed, FAM83F activates the canonical Wnt signalling pathway and binds to and stabilizes p53; it therefore interacts with two pathways often dysregulated in disease. Insights into gene function can often be gained by studying the roles they play during development, and here we report the generation of fam83f knock-out (KO) zebrafish, which we have used to study the role of Fam83f in vivo. We show that endogenous fam83f is most strongly expressed in the hatching gland of developing zebrafish embryos, and that fam83f KO embryos hatch earlier than their wild-type (WT) counterparts, despite developing at a comparable rate. We also demonstrate that fam83f KO embryos are more sensitive to ionizing radiation than WT embryos-an unexpected finding, bearing in mind the previously reported ability of FAM83F to stabilize p53. Transcriptomic analysis shows that loss of fam83f leads to downregulation of phosphatidylinositol-3-phosphate (PI(3)P) binding proteins and impairment of cellular degradation pathways, particularly autophagy, a crucial component of the DNA damage response. Finally, we show that Fam83f protein is itself targeted to the lysosome when overexpressed in HEK293T cells, and that this localization is dependent upon a C' terminal signal sequence. The zebrafish lines we have generated suggest that Fam83f plays an important role in autophagic/lysosomal processes, resulting in dysregulated hatching and increased sensitivity to genotoxic stress in vivo.

HaloTag as a substrate-based macroautophagy reporter

Macroautophagy is a conserved cellular degradation pathway that, upon upregulation, confers resilience toward various stress conditions, including protection against proteotoxicity associated with neurodegenerative diseases, leading to cell survival. Monitoring autophagy regulation in living cells is important to understand its role in physiology and pathology, which remains challenging. Here, we report that when HaloTag is expressed within a cell of interest and reacts with tetramethylrhodamine (TMR; its ligand attached to a fluorophore), the rate of fluorescent TMR-HaloTag conjugate accumulation in autophagosomes and lysosomes, observed by fluorescence microscopy, reflects the rate of autophagy. Notably, we found that TMR-HaloTag conjugates were mainly degraded by the proteasome (~95%) under basal conditions, while lysosomal degradation (~10% upon pharmacological autophagy activation) was slow and incomplete, forming a degraded product that remained fluorescent within a SDS-PAGE gel, in agreement with previous reports that HaloTag is resistant to lysosomal degradation when fused to proteins of interest. Autophagy activation is distinguished from autophagy inhibition by the increased production of the degraded TMR-HaloTag band relative to the full-length TMR-HaloTag band as assessed by SDS-PAGE and by a faster rate of TMR-HaloTag conjugate lysosomal puncta accumulation as observed by fluorescence microscopy. Pharmacological proteasome inhibition leads to accumulation of TMR-HaloTag in lysosomes, indicating possible cross talk between autophagy and proteasomal degradation.

Crosstalk between degradation and bioenergetics: how autophagy and endolysosomal processes regulate energy production.

Cells undergo metabolic reprogramming to adapt to changes in nutrient availability, cellular activity, and transitions in cell states. The balance between glycolysis and mitochondrial respiration is crucial for energy production, and metabolic reprogramming stipulates a shift in such balance to optimize both bioenergetic efficiency and anabolic requirements. Failure in switching bioenergetic dependence can lead to maladaptation and pathogenesis. While cellular degradation is known to recycle precursor molecules for anabolism, its potential role in regulating energy production remains less explored. The bioenergetic switch between glycolysis and mitochondrial respiration involves transcription factors and organelle homeostasis, which are both regulated by the cellular degradation pathways. A growing body of studies has demonstrated that both stem cells and differentiated cells exhibit bioenergetic switch upon perturbations of autophagic activity or endolysosomal processes. Here, we highlighted the current understanding of the interplay between degradation processes, specifically autophagy and endolysosomes, transcription factors, endolysosomal signaling, and mitochondrial homeostasis in shaping cellular bioenergetics. This review aims to summarize the relationship between degradation processes and bioenergetics, providing a foundation for future research to unveil deeper mechanistic insights into bioenergetic regulation.

In the fed state, autophagy plays a crucial role in assisting the insect vector Rhodnius prolixus mobilize TAG reserves under forced flight activity.

Autophagy is a cellular degradation pathway mediated by highly conserved autophagy-related genes (Atgs). In our previous work, we showed that inhibiting autophagy under starvation conditions leads to significant physiological changes in the insect vector of Chagas disease Rhodnius prolixus; these changes include triacylglycerol (TAG) retention in the fat body, reduced survival and impaired locomotion and flight capabilities. Herein, because it is known that autophagy can be modulated in response to various stimuli, we further investigated the role of autophagy in the fed state, following blood feeding. Interestingly, the primary indicator for the presence of autophagosomes, the lipidated form of Atg8 (Atg8-II), displayed 20%-50% higher autophagic activation in the first 2weeks after feeding compared to the third week when digestion was complete. Despite the elevated detection of autophagosomes, RNAi-mediated suppression of RpAtg6 and RpAtg8 did not cause substantial changes in TAG or protein levels in the fat body or the flight muscle during blood digestion. We also found that knockdown of RpAtg6 and RpAtg8 led to modest modulations in the gene expression of essential enzymes involved in lipid metabolism and did not significantly stimulate the expression of the chaperones BiP and PDI, which are the main effectors of the unfolded protein response. These findings indicate that impaired autophagy leads to slight disturbances in lipid metabolism and general cell proteostasis. However, the ability of insects to fly during forced flight until exhaustion was reduced by 60% after knockdown of RpAtg6 and RpAtg8. This change was accompanied by TAG and protein increases as well as decreased ATP levels in the fat body and flight muscle, indicating that autophagy during digestion, i.e., under fed conditions, is necessary to sustain high-performance activity.

Abstract 1866: Discovery of mutation-independent EGFR degrading bispecific antibodies that suppress tumor growth in preclinical tumor models

Abstract Extracellular targeted protein degradation (eTPD) has emerged as a promising new drug modality focused on targeted elimination of extracellular and transmembrane proteins. In contrast to intracellular protein degraders, such as proteolysis targeting chimeras (PROTACs) and molecular glues which require ubiquitin-proteosome cellular degradation pathways, extracellular protein degraders can additionally harness endosomal-lysosomal protein degradation. Two recently published examples of extracellular protein degraders include AbTACs (antibody-based PROTAC) which co-engage a protein of interest (POI) and transmembrane E3 ligases, and KineTACs (cytokine receptor-targeting chimeras) which utilize endogenous cytokine receptors to degrade extracellular POIs. These bispecific antibody degrader platforms not only have advantageous pharmacological and drug-like manufacturing properties, but can also be engineered for tissue-specificity and to address multiple complementary targets, with the goal of increased efficacy and decreased toxicity. To that end, we have greatly expanded the extracellular degrader repertoire beyond AbTACs and KineTACs, with a novel bispecific antibody degrader platform called TrainTACs (tissue receptor antigen internalization targeting chimeras). To demonstrate the potential of this novel extracellular degrading platform, we developed degraders for the canonical receptor tyrosine kinase epidermal growth factor receptor (EGFR). EGFR is an oncogenic driver, that has been clinically validated in lung, colorectal and head and neck cancers, but patient benefit has been limited by treatment-related acquired resistance mutations and on-target/off-tumor dose-limiting toxicities. The potential of our TrainTACs, AbTACs and KineTACs to overcome the limitations of current therapeutic modalities was assessed. Gene expression profiles from tumor and normal tissues were evaluated to identify potential degraders co-expressed with EGFR in tumors. More than 70 unique bispecific antibody constructs spanning 20 receptors were generated and screened in tumor cell-based assays to evaluate EGFR antagonism, internalization, and degradation. TrainTACs, AbTACs and KineTACs degraded EGFR in multiple tumor cell lines covering a range of EGFR mutations. Degradation of EGFR led to deep inhibition of EGFR signaling, robust inhibitory effects on tumor spheroids, and in xenograft mouse tumor models. In conclusion, eTPD represents a promising new drug modality, and TrainTACs, AbTACs and KineTACs have expanded the toolbox of extracellular targeted protein degraders that can be utilized in a target-, tissue- and disease-specific manner. Citation Format: Jonathan Sitrin, Lisa Marshall, Hai Tran, Kenneth Ng, Kimberly Hoi, Josef Gramespacher, Zhong Huang, Andy Goodrich, Filomena Housley, May Dayao, Man-Tzu Wang, Katarina Pance, Aleysha Chen, Kevin Carlin, Lichao Zhang, James Lee, Rami Hannoush, Ken Flanagan, Maia Vinogradova, Isaac Rondon, Shyra Gardai. Discovery of mutation-independent EGFR degrading bispecific antibodies that suppress tumor growth in preclinical tumor models [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 1866.

Down-Regulation of TRIM11 in Lysosomal Regulation: Investigating its Impact on Cellular Degradation Pathways

Alzheimer’s disease is characterized by the accumulation of Aβ plaques and tau protein tangles, with TRIM11 playing a crucial role in tau regulation [1]. However, TRIM11’s degradation in Alzheimer’s remains unclear. This study investigates whether lysosomes, responsible for cellular degradation, are involved in TRIM11 degradation [2]. Lysosome inhibitors were applied, and TRIM11 levels were examined. Immunostaining for TRIM11 and lysosome inhibitors was conducted to assess co-localization, indicating potential interaction. The findings suggest a connection between TRIM11 and lysosomes in Alzheimer’s pathology, warranting further research for potential therapeutic implications [3].

Expression of RNautophagy/DNautophagy-related genes is regulated under control of an innate immune receptor

ABSTRACT Double-stranded RNA (dsRNA) is a molecular pattern uniquely produced in cells infected with various viruses as a product or byproduct of replication. Cells detect such molecules, which indicate non-self invasion, and induce diverse immune responses to eliminate them. The degradation of virus-derived molecules can also play a role in the removal of pathogens and suppression of their replication. RNautophagy and DNautophagy are cellular degradative pathways in which RNA and DNA are directly imported into a hydrolytic organelle, the lysosome. Two lysosomal membrane proteins, SIDT2 and LAMP2C, mediate nucleic acid uptake via this pathway. Here, we showed that the expression of both SIDT2 and LAMP2C is selectively upregulated during the intracellular detection of poly(I:C), a synthetic analog of dsRNA that mimics viral infection. The upregulation of these two gene products upon poly(I:C) introduction was transient and synchronized. We also observed that the induction of SIDT2 and LAMP2C expression by poly(I:C) was dependent on MDA5, a cytoplasmic innate immune receptor that directly recognizes poly(I:C) and induces various antiviral responses. Finally, we showed that lysosomes can target viral RNA for degradation via RNautophagy and may suppress viral replication. Our results revealed a novel degradative pathway in cells as a downstream component of the innate immune response and provided evidence suggesting that the degradation of viral nucleic acids via RNautophagy/DNautophagy contributes to the suppression of viral replication.

Autophagy confers aluminum tolerance by enhancing the antioxidant capacity and promoting oxidized protein degradation in wheat (Triticum aestivum.) roots

Aluminum (Al) usually disturbs redox homeostasis and causes irreversible oxidative damage to biomolecules in plants, efficiently removing damaged and toxic macromolecules is essential to Al tolerance. Here, we investigated the role of autophagy, a conserved cellular degradation pathway, in plant Al tolerance using two wheat genotypes with different Al sensitivity. Compared to the sensitive genotype, Al significantly upregulated the expression of autophagy-related genes (ATGs) and enhanced the formation of autophagosomes. Reinforcing autophagy by rapamycin substantially alleviated the inhibition of root growth by Al stress, accompanied by low levels of reactive oxygen species (ROS) accumulation and oxidative damage in wheat roots; whereas inhibition of autophagy by 3-methyladenine (3-MA) aggravated the adverse impacts of Al toxicity. Further experiments showed that Al significantly triggered the accumulation of oxidized proteins, particularly in the sensitive genotype. The content of Al-induced oxidized proteins was reduced by rapamycin but further increased by 3-MA in wheat roots. Wheat plants treated with the inhibitor of vacuole H+-ATPase, concanamycin A, were more sensitive to Al stress and accumulated higher levels of oxidatively-damaged proteins compared to Al treatment. These results reveal that autophagy-mediated redox homeostasis and the clearance of oxidatively damaged proteins play a crucial role in plant's tolerance to Al stress.

Degradation of p0071 and p120-catenin during adherens junction disassembly by Leptospira interrogans.

Leptospira interrogans disseminates hematogenously to reach the target organs by disrupting epithelial adherens junctions (AJs), thus causing leptospirosis, which is a globally neglected zoonotic disease. L. interrogans induces E-cadherin (E-cad) endocytosis and cytoskeletal rearrangement during AJ disassembly, but the detailed mechanism remains unknown. Elucidation of AJ disassembly mechanisms will guide new approaches to developing vaccines and diagnostic methods. In this study, we combine proteomic and imaging analysis with chemical inhibition studies to demonstrate that disrupting the AJs of renal proximal tubule epithelial cells involves the degradation of two armadillo repeat-containing proteins, p0071 and p120-catenin, that stabilize E-cad at the plasma membrane. Combining proteasomal and lysosomal inhibitors substantially prevented p120-catenin degradation, and monolayer integrity destruction without preventing p0071 proteolysis. In contrast, the pan-caspase inhibitor Z-VAD-FMK inhibited p0071 proteolysis and displacement of both armadillo repeat-containing proteins from the cell-cell junctions. Our results show that L. interrogans induces p120-catenin and p0071 degradation, which mutually regulates E-cad stability by co-opting multiple cellular degradation pathways. This strategy may allow L. interrogans to disassemble AJs and disseminate through the body efficiently.

Autophagy ablation in skeletal muscles worsens sepsis-induced muscle wasting, impairs whole-body metabolism, and decreases survival

Septic patients frequently develop skeletal muscle wasting and weakness, resulting in severe clinical consequences and adverse outcomes. Sepsis triggers sustained induction of autophagy, a key cellular degradative pathway, in skeletal muscles. However, the impact of enhanced autophagy on sepsis-induced muscle dysfunction remains unclear. Using an inducible and muscle-specific Atg7 knockout mouse model (Atg7iSkM-KO), we investigated the functional importance of skeletal muscle autophagy in sepsis using the cecal ligation and puncture model. Atg7iSkM-KO mice exhibited a more severe phenotype in response to sepsis, marked by severe muscle wasting, hypoglycemia, higher ketone levels, and a decreased in survival as compared to mice with intact Atg7. Sepsis and Atg7 deletion resulted in the accumulation of mitochondrial dysfunction, although sepsis did not further worsen mitochondrial dysfunction in Atg7iSkM-KO mice. Overall, our study demonstrates that autophagy inactivation in skeletal muscles triggers significant worsening of sepsis-induced muscle and metabolic dysfunctions and negatively impacts survival.

Nucleotide Exchange Factors for Hsp70 Molecular Chaperones: GrpE, Hsp110/Grp170, HspBP1/Sil1, and BAG Domain Proteins.

Molecular chaperones of the Hsp70 family are key components of the cellular protein-folding machinery. Substrate folding is accomplished by iterative cycles of ATP binding, hydrolysis, and release. The ATPase activity of Hsp70 is regulated by two main classes of cochaperones: J-domain proteins stimulate ATPase hydrolysis by Hsp70, while nucleotide exchange factors (NEFs) facilitate the conversion from the ADP-bound to the ATP-bound state, thus closing the chaperone folding cycle. NEF function can additionally be antagonized by ADP dissociation inhibitors. Beginning with the discovery of the prototypical bacterial NEF, GrpE, a large diversity of nucleotide exchange factors for Hsp70 have been identified, connecting it to a multitude of cellular processes in the eukaryotic cell. Here we review recent advances toward structure and function of nucleotide exchange factors from the Hsp110/Grp170, HspBP1/Sil1, and BAG domain protein families and discuss how these cochaperones connect protein folding with cellular quality control and degradation pathways.

The crucial role of the regulatory mechanism of the Atg1/ULK1 complex in fungi.

Autophagy, an evolutionarily conserved cellular degradation pathway in eukaryotes, is hierarchically regulated by autophagy-related genes (Atgs). The Atg1/ULK1 complex is the most upstream factor involved in autophagy initiation. Here，we summarize the recent studies on the structure and molecular mechanism of the Atg1/ULK1 complex in autophagy initiation, with a special focus on upstream regulation and downstream effectors of Atg1/ULK1. The roles of pathogenicity and autophagy aspects in Atg1/ULK1 complexes of various pathogenic hosts, including plants, insects, and humans, are also discussed in this work based on recent research findings. We establish a framework to study how the Atg1/ULK1 complex integrates the signals that induce autophagy in accordance with fungus to mammalian autophagy regulation pathways. This framework lays the foundation for studying the deeper molecular mechanisms of the Atg1 complex in pathogenic fungi.

A method for the isolation and characterization of autophagic bodies from yeast provides a key tool to investigate cargos of autophagy

Autophagy is a major cellular degradation pathway that is highly conserved among eukaryotes. The identification of cargos captured by autophagosomes is critical to our understanding of the physiological significance of autophagy in cells, but these studies can be challenging because autophagosomes disintegrate easily. In the yeast Saccharomyces cerevisiae, cells deficient in the vacuolar lipase Atg15 accumulate autophagic bodies (ABs) within the vacuole following the induction of autophagy. As ABs contain cytosolic components including proteins, RNAs, and lipids, their purification allows the identification of material targeted by autophagy for degradation. In this study, we demonstrate a method to purify intact ABs using isolated vacuoles from atg15Δ cells. Taking advantage of the size discrepancy between the vacuoles and ABs, the vacuolar membrane was disrupted by filtration to release ABs. Filtered vacuolar lysates were subjected to density gradient centrifugation to obtain AB fractions. Purified ABs retain membrane integrity and contain autophagic cargos. This technique offers a valuable tool for the identification of the cargos of autophagy, examination of autophagic cargo selectivity, and biochemical characterization of autophagosome membranes.

Autophagy Receptors in the presentation of Viral antigens by MHC-II molecules

CD4+ T lymphocytes play a major role in the establishment and maintenance of antiviral immunity. CD4+ T cells are activated by antigenic peptides, derived from pathogens, presented by MHC-II molecules. It is still widely assumed that CD4+ T cells recognize epitopes derived solely from exogenous viral particles or proteins. However, alternative sources of MHC-II-restricted antigens have been described. We showed that HIV-infected dendritic cells present MHC-II–restricted epitopes derived from newly synthesized proteins to HIV-specific CD4+ T cells. In fact, we confirmed that multiple cellular degradation pathways lead to endogenous presentation of MHC-II-restricted viral antigens, the generation of some viral epitopes being dependent and/or -independent on macroautophagy (herein referred as autophagy). We are charactering further these MHC-II-restricted endogenous presentation pathways. In particular, using RNA silencing, we asked whether autophagy receptors/adaptors that contribute to selective autophagy but also regulate endosome and autophagosome maturation might play a role. Surprisingly, although these receptors share a high structural homology and similar functions, we identified a single autophagy adaptor that strongly influences the presentation of viral antigens by MHC-II molecules. Although silencing of this autophagy adaptor does not impact the expression levels and the recycling of MHC-II molecules, it has a major influence on the ligandome of MHC-II molecules. In the absence of this adaptor, MHC-II molecules accumulate at the proximity of the nucleus in CD63+Lamp1+ acidified compartments. In addition, the lack of this autophagy adaptor has a strong influence on the invariant chain degradation and expression levels, strongly suggesting that it regulates the activation or traffic of vesicles involved in the processing of newly synthesised antigens. Interestingly, some viruses target these autophagy adaptors to favour viral replication. Our work might reveal new escape mechanisms developed by viruses to damper MHC-II restricted antigen presentation and immune responses.

Quantitative interactome proteomics identifies a proteostasis network for GABAA receptors

Gamma-aminobutyric acid type A (GABAA) receptors are the primary inhibitory neurotransmitter-gated ion channels in the mammalian central nervous system. Maintenance of GABAA receptor protein homeostasis (proteostasis) in cells utilizing its interacting proteins is essential for the function of GABAA receptors. However, how the proteostasis network orchestrates GABAA receptor biogenesis in the endoplasmic reticulum is not well understood. Here, we employed a proteomics-based approach to systematically identify the interactomes of GABAA receptors. We carried out a quantitative immunoprecipitation-tandem mass spectrometry analysis utilizing stable isotope labeling by amino acids in cell culture. Furthermore, we performed comparative proteomics by using both WT α1 subunit and a misfolding-prone α1 subunit carrying the A322D variant as the bait proteins. We identified 125 interactors for WT α1-containing receptors, 105 proteins for α1(A322D)-containing receptors, and 54 overlapping proteins within these two interactomes. Our bioinformatics analysis identified potential GABAA receptor proteostasis network components, including chaperones, folding enzymes, trafficking factors, and degradation factors, and we assembled a model of their potential involvement in the cellular folding, degradation, and trafficking pathways for GABAA receptors. In addition, we verified endogenous interactions between α1 subunits and selected interactors by using coimmunoprecipitation in mouse brain homogenates. Moreover, we showed that TRIM21 (tripartite motif containing-21), an E3 ubiquitin ligase, positively regulated the degradation of misfolding-prone α1(A322D) subunits selectively. This study paves the way for understanding the molecular mechanisms as well as fine-tuning of GABAA receptor proteostasis to ameliorate related neurological diseases such as epilepsy.

Understanding the separation of timescales in bacterial proteasome core particle assembly

The 20S proteasome core particle (CP) is a molecular machine that is a key component of cellular protein degradation pathways. Like other molecular machines, it is not synthesized in an active form but rather as a set of subunits that assemble into a functional complex. The CP is conserved across all domains of life and is composed of 28 subunits, 14 α and 14 β, arranged in four stacked seven-member rings (α7β7β7α7). While details of CP assembly vary across species, the final step in the assembly process is universally conserved: two half proteasomes (HPs; α7β7) dimerize to form the CP. In the bacterium Rhodococcus erythropolis, experiments have shown that the formation of the HP is completed within minutes, while the dimerization process takes hours. The N-terminal propeptide of the β subunit, which is autocatalytically cleaved off after CP formation, plays a key role in regulating this separation of timescales. However, the detailed molecular mechanism of how the propeptide achieves this regulation is unclear. In this work, we used molecular dynamics simulations to characterize HP conformations and found that the HP exists in two states: one where the propeptide interacts with key residues in the HP dimerization interface and likely blocks dimerization, and one where this interface is free. Furthermore, we found that a propeptide mutant that dimerizes extremely slowly is essentially always in the nondimerizable state, while the wild-type rapidly transitions between the two. Based on these simulations, we designed a propeptide mutant that favored the dimerizable state in molecular dynamics simulations. In vitro assembly experiments confirmed that this mutant dimerizes significantly faster than wild-type. Our work thus provides unprecedented insight into how this critical step in CP assembly is regulated, with implications both for efforts to inhibit proteasome assembly and for the evolution of hierarchical assembly pathways.

Regulatory roles of selective autophagy through targeting of native proteins in plant adaptive responses.

Selective autophagy functions as a regulatory mechanism by targeting native and functional proteins to ensure their proper levels and activities in plant adaptive responses. Autophagy is a cellular degradation and recycling pathway with a key role in cellular homeostasis and metabolism. Autophagy is initiated with the biogenesis of autophagosomes, which fuse with the lysosomes or vacuoles to release their contents for degradation. Under nutrient starvation or other adverse environmental conditions, autophagy usually targets unwanted or damaged proteins, organelles and other cellular components for degradation and recycling to promote cell survival. Over the past decade, however, a substantial number of studies have reported that autophagy in plants also functions as a regulatory mechanism by targeting enzymes, structural and regulatory proteins that are not necessarily damaged or dysfunctional to ensure their proper abundance and function to facilitate cellular changes required for response to endogenous and environmental conditions. During plant-pathogen interactions in particular, selective autophagy targets specific pathogen components as a defense mechanism and pathogens also utilize autophagy to target functional host factors to suppress defense mechanisms. Autophagy also targets native and functional protein regulators of plant heat stress memory, hormone signaling, and vesicle trafficking associated with plant responses to abiotic and other conditions. In this review, we discuss advances in the regulatory roles of selective autophagy through targeting of native proteins in plant adaptive responses, what questions remain and how further progress in the analysis of these special regulatory roles of autophagy can help understand biological processes important to plants.

Proteasomal and autophagy-mediated degradation of mutp53 proteins through mitochondria-targeting aggregation-induced-emission materials

Close to half of human cancers harbor point mutations in the tumor-suppressor p53 gene, giving rise to the cellular accumulation of mutant p53 (mutp53) proteins with novel neomorphic gain-of-function (GOF) properties. The destruction of mutp53 proteins through either autophagic or proteasomal degradation is a viable strategy for the targeted therapy of p53-mutated cancers. Several nanomaterials, including zinc-iron and ZIF-8 nanoparticles (NPs), have been reported to induce the proteasomal degradation of mutp53 proteins. However, how autophagy, the other major cellular degradative pathway, influences NP-induced mutp53 degradation has not been investigated. This article shows that AIE-Mit-TPP, a mitochondria-targeting material with aggregation-induced emission (AIE) characteristics, elicits ubiquitination-dependent proteasomal degradation of a broad range of mutp53 proteins. Meanwhile, AIE-Mit-TPP also induces massive mitochondrial damage and autophagy. The inhibition of autophagy further increases AIE-Mit-TPP-elicited mutp53 degradation, revealing the negative impact of autophagy on AIE-Mit-TPP-induced mutp53 degradation. As expected, the degradation of mutp53 proteins by AIE-Mit-TPP abrogated mutp53-manifested GOF, leading to reductions in cell proliferation and migration and increases in cell cycle arrest and cell death. Consequently, AIE-Mit-TPP inhibited the growth of mutp53 tumors. This paper unravels the interesting interplay between the proteasomal and autophagic degradative pathways and pinpoints the modulation of autophagy as a potential strategy for optimizing NP-induced mutp53 degradation and p53-targeted cancer therapy. Statement of significanceWe have designed three different types of AIE materials: non-targeting (AIE-Br), mitochondria-targeting (AIE-Mit-TPP), lysosome-targeting (AIE-Lyso). Our results proved that mitochondria-targeting AIE material induced degradation of mutp53 proteins via the proteasome degradation pathway and abrogated mutp53-conferred GOF phenotypes. Furthermore, we performed in vitro studies on the effect of the tested materials in mutp53-expressing cancer cells and demonstrated our findings via in vivo investigations in a mouse subcutaneous p53R175H TOV112D ovarian cancer model. Our results confirmed the link between the proteasome pathway and autophagy and thus proposed a strategy of combining AIE-Mit-TPP with autophagy inhibitors for the targeted treatment of mutp53-associated tumors. Finally, we found that AIE-Mit-TPP could induce degradation of a wide-spectrum mutp53 proteins, which makes mitochondria-targeting AIE materials an effective therapeutic strategy for p53-mutated cancers.

Autophagy-Associated Immunogenic Modulation and Its Applications in Cancer Therapy.

Autophagy, a lysosome-mediated cellular degradation pathway, recycles intracellular components to maintain metabolic balance and survival. Autophagy plays an important role in tumor immunotherapy as a “double-edged sword” that can both promote and inhibit tumor progression. Autophagy acts on innate and adaptive immunity and interacts with immune cells to modulate tumor immunotherapy. The discovery of autophagy inducers and autophagy inhibitors also provides new insights for clinical anti-tumor therapy. However, there are also difficulties in the application of autophagy-related regulators, such as low bioavailability and the lack of efficient selectivity. This review focuses on autophagy-related immunogenic regulation and its application in cancer therapy.

Targeted for destruction: degradation of singlet oxygen-damaged chloroplasts

ABSTRACT Photosynthesis is an essential process that plants must regulate to survive in dynamic environments. Thus, chloroplasts (the sites of photosynthesis in plant and algae cells) use multiple signaling mechanisms to report their health to the cell. Such signals are poorly understood but often involve reactive oxygen species (ROS) produced from the photosynthetic light reactions. One ROS, singlet oxygen (1O2), can signal to initiate chloroplast degradation, but the cellular machinery involved in identifying and degrading damaged chloroplasts (i.e., chloroplast quality control pathways) is unknown. To provide mechanistic insight into these pathways, two recent studies have investigated degrading chloroplasts in the Arabidopsis thaliana 1O2 over-producing plastid ferrochelatase two (fc2) mutant. First, a structural analysis of degrading chloroplasts was performed with electron microscopy, which demonstrated that damaged chloroplasts can protrude into the central vacuole compartment with structures reminiscent of fission-type microautophagy. 1O2-stressed chloroplasts swelled before these interactions, which may be a mechanism for their selective degradation. Second, the roles of autophagosomes and canonical autophagy (macroautophagy) were shown to be dispensable for 1O2-initiated chloroplast degradation. Instead, putative fission-type microautophagy genes were induced by chloroplast 1O2. Here, we discuss how these studies implicate this poorly understood cellular degradation pathway in the dismantling of 1O2-damaged chloroplasts.