LUBAC assembles a ubiquitin signaling platform at mitochondria for signal amplification and transport of NF-κB to the nucleus.

Mitochondria are increasingly recognized as cellular hubs to orchestrate signaling pathways that regulate metabolism, redox homeostasis, and cell fate decisions. Recent research revealed a role of mitochondria also in innate immune signaling; however, the mechanisms of how mitochondria affect signal transduction are poorly understood. Here, we show that the NF-κB pathway activated by TNF employs mitochondria as a platform for signal amplification and shuttling of activated NF-κB to the nucleus. TNF treatment induces the recruitment of HOIP, the catalytic component of the linear ubiquitin chain assembly complex (LUBAC), and its substrate NEMO to the outer mitochondrial membrane, where M1- and K63-linked ubiquitin chains are generated. NF-κB is locally activated and transported to the nucleus by mitochondria, leading to an increase in mitochondria-nucleus contact sites in a HOIP-dependent manner. Notably, TNF-induced stabilization of the mitochondrial kinase PINK1 furthermore contributes to signal amplification by antagonizing the M1-ubiquitin-specific deubiquitinase OTULIN. Overall, our study reveals a role for mitochondria in amplifying TNF-mediated NF-κB activation, both serving as a signaling platform, as well as a transport mode for activated NF-κB to the nuclear.

An in vitro vesicle formation assay reveals cargo clients and factors that mediate vesicular trafficking.

The fidelity of protein transport in the secretory pathway relies on the accurate sorting of proteins to their correct destinations. To deepen our understanding of the underlying molecular mechanisms, it is important to develop a robust approach to systematically reveal cargo proteins that depend on specific sorting machinery to be enriched into transport vesicles. Here, we used an in vitro assay that reconstitutes packaging of human cargo proteins into vesicles to quantify cargo capture. Quantitative mass spectrometry (MS) analyses of the isolated vesicles revealed cytosolic proteins that are associated with vesicle membranes in a GTP-dependent manner. We found that two of them, FAM84B (also known as LRAT domain containing 2 or LRATD2) and PRRC1, contain proline-rich domains and regulate anterograde trafficking. Further analyses revealed that PRRC1 is recruited to endoplasmic reticulum (ER) exit sites, interacts with the inner COPII coat, and its absence increases membrane association of COPII. In addition, we uncovered cargo proteins that depend on GTP hydrolysis to be captured into vesicles. Comparing control cells with cells depleted of the cargo receptors, SURF4 or ERGIC53, we revealed specific clients of each of these two export adaptors. Our results indicate that the vesicle formation assay in combination with quantitative MS analysis is a robust and powerful tool to uncover novel factors that mediate vesicular trafficking and to uncover cargo clients of specific cellular factors.

Caspase-3/GSDME-mediated pyroptosis leads to osteogenic dysfunction of osteoblast-like cells.

OBJECTIVE: Cell pyroptosis is implicated in progressive bone loss in dental inflammatory diseases. We induced caspase-3/Gasdermin E (GSDME)-mediated pyroptosis in osteoblast-like cells and evaluated the effects on osteogenesis. MATERIALS AND METHODS: Osteoblast-like cells were treated with various concentrations of sodium butyrate (NaB) to identify the most appropriate for inducing caspase-3/GSDME-mediated pyroptosis. Cells were divided into control, NaB and NaB+Ac-DEVD-CHO (specific caspase-3 inhibitor) groups. Pyroptosis level was evaluated by immunofluorescence, morphological observation, flow cytometry, lactate dehydrogenase (LDH) release assays, mRNA and protein levels of pyroptosis-related markers. Then, inflammation level, osteoprotegerin (OPG) and receptor activator of nuclear factor-κB ligand (RANKL) expression and osteogenic function were detected. RESULTS: Treatment with 10 mM NaB increased caspase-3 expression, GSDME cleavage, LDH release and the number of pyroptotic cells, with morphologic changes, indicating GSDME-mediated pyroptosis induction. The pyroptosis-related changes were abolished by caspase-3 inhibition. Caspase-3/GSDME-mediated pyroptosis triggered the expression of inflammatory cytokines and RANKL, downregulated alkaline phosphatase (ALP) activity, mineralisation level, mRNA and protein levels of multiple osteogenic markers. These effects were partly reversed by Ac-DEVD-CHO. CONCLUSION: Caspase-3/GSDME-mediated pyroptosis induced by NaB activated the inflammatory response, reduced osteogenic differentiation and disturbed OPG/RANKL axis, leading to osteogenic dysfunction in osteoblast-like cells.

Digitally designed occlusion of an implant-supported crown considering physiological tooth displacement under occlusal loading.

Physiological natural tooth displacement under occlusal loading can influence intraoral occlusal contacts. However, gypsum casts and digital scans cannot simulate the physiological tooth displacement under occlusal loading. The occlusal design of the implant-supported crowns has been based mainly on the experience of dental laboratory technicians, lacking accuracy and individualization. Therefore, a digital technique that considers physiological tooth displacement is presented to design the occlusion of implant-supported single crowns.

Protein quality control by the proteasome and autophagy: A regulatory role of ubiquitin and liquid-liquid phase separation.

Degradation of dysfunctional, damaged, or misfolded proteins is a crucial component of the protein quality control network to maintain cellular proteostasis. Dysfunction in proteostasis regulation due to imbalances in protein synthesis, folding, and degradation challenges the integrity of the cellular proteome and favors the accumulation of aggregated proteins that can damage cells by a loss of their functions and/or a gain of adverse functions. Ubiquitination is an essential player in proteostasis regulation but also in orchestrating signaling pathways in response to various stress conditions. Both cellular degradation systems, the proteasome and autophagy, employ ubiquitin for selection and targeting of substrates to the degradative machineries. Here we summarize the manifold functions of ubiquitin in protein degradation and discuss its emerging role in the formation of biomolecular condensates through liquid-liquid phase separation, which allows spatiotemporal regulation of protein quality control.