use-two-dimensional-semi-denaturing-detergent-agarose-gel

Use of Two Dimensional Semi-denaturing Detergent Agarose Gel Electrophoresis to Confirm Size Heterogeneity of Amyloid or Amyloid-like Fibers

Amyloid or amyloid-like fibers have been associated with many human diseases, and are now being discovered to be important for many signaling pathways. ...

live-cell-imaging-lysosomal-membrane-permeabilization-during

Live-cell Imaging of Lysosomal Membrane Permeabilization During Necroptosis

Necroptosis, a form of regulated necrosis, culminates in cell membrane rupture. Our lab and others have discovered that lysosomal membrane ...

UT Southwestern Medical Center

University of South Florida

Mixed-lineage kinase domain-like protein (MLKL) is essential for TNF-α-induced necroptosis. How MLKL promotes cell death is still under debate. Here we report that MLKL forms SDS-resistant, disulfide bond-dependent polymers during necroptosis in both human and mouse cells. MLKL polymers are independent of receptor-interacting protein kinase 1 and 3 (RIPK1/RIPK3) fibers. Large MLKL polymers are more than 2 million Da and are resistant to proteinase K digestion. MLKL polymers are fibers 5 nm in diameter under electron microscopy. Furthermore, the recombinant N-terminal domain of MLKL forms amyloid-like fibers and binds Congo red dye. MLKL mutants that cannot form polymers also fail to induce necroptosis efficiently. Finally, the compound necrosulfonamide conjugates cysteine 86 of human MLKL and blocks MLKL polymer formation and subsequent cell death. These results demonstrate that disulfide bond-dependent, amyloid-like MLKL polymers are necessary and sufficient to induce necroptosis.

MLKL forms disulfide bond-dependent amyloid-like polymers to induce necroptosis.

Necroptosis is an immunogenic cell death program that is associated with a host of human diseases, including inflammation, infections, and cancer. Receptor-interacting protein kinase 3 (RIPK3) and its substrate mixed lineage kinase domain-like protein (MLKL) are required for necroptosis activation. Specifically, RIPK3-dependent MLKL phosphorylation promotes the assembly of disulfide bond-dependent MLKL polymers that drive the execution of necroptosis. However, how MLKL disulfide bond formation is regulated is not clear. In this study we discovered that the MLKL-modifying compound necrosulfonamide cross-links cysteine 86 of human MLKL to cysteine 32 of the thiol oxidoreductase thioredoxin-1 (Trx1). Recombinant Trx1 preferentially binds to monomeric MLKL and blocks MLKL disulfide bond formation and polymerization  Inhibition of MLKL polymer formation requires the reducing activity of Trx1. Importantly, shRNA-mediated knockdown of Trx1 promotes MLKL polymerization and sensitizes cells to necroptosis. Furthermore, pharmacological inhibition of Trx1 with compound PX-12 induces necroptosis in multiple cancer cell lines. Altogether, these findings demonstrate that Trx1 is a critical regulator of necroptosis that suppresses cell death by maintaining MLKL in a reduced inactive state. Our results further suggest new directions for targeted cancer therapy in which thioredoxin inhibitors like PX-12 could potentially be used to specifically target cancers expressing high levels of MLKL or MLKL short isoforms.

Thioredoxin-1 actively maintains the pseudokinase MLKL in a reduced state to suppress disulfide bond-dependent MLKL polymer formation and necroptosis.

Receptor-interacting protein kinase 1 (RIPK1) is a key regulator of inflammation and cell death. Many sites on RIPK1, including serine 25, are phosphorylated to inhibit its kinase activity and cell death. How these inhibitory phosphorylation sites are dephosphorylated is poorly understood. Using a sensitized CRISPR whole-genome knockout screen, we discover that protein phosphatase 1 regulatory subunit 3G (PPP1R3G) is required for RIPK1-dependent apoptosis and type I necroptosis. Mechanistically, PPP1R3G recruits its catalytic subunit protein phosphatase 1 gamma (PP1γ) to complex I to remove inhibitory phosphorylations of RIPK1. A PPP1R3G mutant which does not bind PP1γ fails to rescue RIPK1 activation and cell death. Furthermore, chemical prevention of RIPK1 inhibitory phosphorylations or mutation of serine 25 of RIPK1 to alanine largely restores cell death in PPP1R3G-knockout cells. Finally, Ppp1r3g mice are protected from tumor necrosis factor-induced systemic inflammatory response syndrome, confirming the important role of PPP1R3G in regulating apoptosis and necroptosis in vivo.

RIPK1 dephosphorylation and kinase activation by PPP1R3G/PP1γ promote apoptosis and necroptosis.

Mixed lineage kinase-like protein (MLKL) forms amyloid-like polymers to promote necroptosis; however, the mechanism through which these polymers trigger cell death is not clear. We have determined that activated MLKL translocates to the lysosomal membrane during necroptosis induction. The subsequent polymerization of MLKL induces lysosome clustering and fusion and eventual lysosomal membrane permeabilization (LMP). This LMP leads to the rapid release of lysosomal contents into the cytosol, resulting in a massive surge in cathepsin levels, with Cathepsin B (CTSB) as a significant contributor to the ensuing cell death as it cleaves many proteins essential for cell survival. Importantly, chemical inhibition or knockdown of CTSB protects cells from necroptosis. Furthermore, induced polymerization of the MLKL N-terminal domain (NTD) also triggers LMP, leading to CTSB release and subsequent cell death. These findings clearly establish the critical role of MLKL polymerization induced lysosomal membrane permeabilization (MPI-LMP) in the process of necroptosis.

MLKL polymerization-induced lysosomal membrane permeabilization promotes necroptosis.

Receptor-interacting protein kinase 1 (RIPK1) plays a crucial role in controlling inflammation and cell death. Its function is tightly controlled through post-translational modifications, enabling its dynamic switch between promoting cell survival and triggering cell death. Phosphorylation of RIPK1 at various sites serves as a critical mechanism for regulating its activity, exerting either activating or inhibitory effects. Perturbations in RIPK1 phosphorylation status have profound implications for the development of severe inflammatory diseases in humans. This review explores the intricate regulation of RIPK1 phosphorylation and dephosphorylation and highlights the potential of targeting RIPK1 phosphorylation as a promising therapeutic strategy for mitigating human diseases.

Regulation of RIPK1 Phosphorylation: Implications for Inflammation, Cell Death, and Therapeutic Interventions.

In mammals, global passive demethylation contributes to epigenetic reprogramming during early embryonic development. At this stage, the majority of DNA-methyltransferase 1 (DNMT1) protein is excluded from nucleus, which is considered the primary cause. However, whether the remaining nuclear activity of DNMT1 is regulated by additional mechanisms is unclear. Here, we report that nuclear DNMT1 abundance is finetuned through proteasomal degradation in mouse zygotes. We identify a maternal factor, Pramel15, which targets DNMT1 for degradation via Cullin-RING E3 ligases. Loss of Pramel15 elevates DNMT1 levels in the zygote pronuclei, impairs zygotic DNA demethylation, and causes a stochastic gain of DNA methylation in early embryos. Thus, Pramel15 can modulate the residual level of DNMT1 in the nucleus during zygotic DNA replication, thereby ensuring efficient DNA methylation reprogramming in early embryos.

Pramel15 facilitates zygotic nuclear DNMT1 degradation and DNA demethylation.

The maintenance of skeletal muscle relies on several cellular signalling pathways directly linked to mitochondrial function. Mitochondria support skeletal muscle bioenergetics and are known as a primary source of reactive oxygen species (ROS). Indeed, mitochondrial dysfunction-induced excessive ROS accumulation leads to irreversible molecular damage caused by lipid peroxidation by product 4-hydroxy-trans-2-nonenal (4-HNE), this is able to directly inactivate proteins and DNA, causing mitochondrial and cellular dysfunction. Aldehyde dehydrogenase 2 (ALDH2) is the primary enzyme responsible for counteracting this deleterious cycle by detoxifying aldehydes and converting them into less harmful molecules. In this context, ALDH2 is essential for functional skeletal muscle maintenance. In fact, studies published in the last decade show that ALDH2 overexpression downregulates atrophic genes; meanwhile, the lack of this protein is associated with muscle weakness and atrophy, demonstrating its relevance for maintaining muscle mass. In this context, interventions capable of increasing ALDH2 levels and/or activity are considered promising therapeutic strategies for mitigating oxidative damage, enhancing mitochondrial function, and preserving muscle integrity under adverse conditions.

ArticleTotal : 2	Year
Publication title Cited by 5	2018
Publication title	2025

Article	Year
MLKL forms disulfide bond-dependent amyloid-like polymers to induce necroptosis. Proceedings of the National Academy of Sciences of the United States of America\| PubMed ID: 28827318	2017
Thioredoxin-1 actively maintains the pseudokinase MLKL in a reduced state to suppress disulfide bond-dependent MLKL polymer formation and necroptosis. The Journal of biological chemistry\| PubMed ID: 28878015	2017
RIPK1 dephosphorylation and kinase activation by PPP1R3G/PP1γ promote apoptosis and necroptosis. Nature communications\| PubMed ID: 34862394	2021
MLKL polymerization-induced lysosomal membrane permeabilization promotes necroptosis. Cell death and differentiation\| PubMed ID: 37996483	2024
Regulation of RIPK1 Phosphorylation: Implications for Inflammation, Cell Death, and Therapeutic Interventions. Biomedicines\| PubMed ID: 39062098	2024
Pramel15 facilitates zygotic nuclear DNMT1 degradation and DNA demethylation. Nature communications\| PubMed ID: 39181896	2024
Targeting ALDH2 to promote skeletal muscle health: A novel strategy against mitochondrial dysfunction. The Journal of physiology\| PubMed ID: 40684425	2025

Zhigao Wang

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