Mafalda Escobar-Henriques

Mitochondria are central hubs for cellular fitness, empowered by plastic remodeling of their shape, proteome composition, and/or metabolic state. MFN2 (mitofusin 2) mediates mitochondrial fusion and ensures adaptations in response to metabolic changes and stresses. Besides this canonical role, MFN2 serves as a communication hub with other organelles. It tethers mitochondria to the endoplasmic reticulum (ER), lipid droplets, and peroxisomes, regulating calcium buffering, apoptosis, lipid biosynthesis, and lipolysis. Dysfunctional MFN2 causes the hereditary neuropathy Charcot–Marie–Tooth type 2A (CMT2A) and is linked to several metabolic diseases. In a recent publication, we described another fusion-independent role of MFN2 in proteostasis and mitophagy. MFN2 binds the chaperone HSPA8/HSC70 (heat shock protein family A [Hsp70] member 8) and the proteasome, a key function in maintaining mitochondrial and cellular protein quality control, which appears to be lost in the context of CMT2A-associated MFN2 variants.

The neuropathy Charcot–Marie–Tooth (CMT) is an incurable disease with a lack of genotype–phenotype correlation. Variants of the mitochondrial protein mitofusin 2 (MFN2), a large GTPase that mediates mitochondrial fusion, are responsible for the subtype CMT type 2A (CMT2A). Interestingly, beyond membrane remodelling, additional roles of MFN2 have been identified, expanding the possibilities to explore its involvement in disease. Here, we investigated how cellular functions of MFN2 are associated with variants present in individuals with CMT2A. Using human cellular models, we observed that cells expressing CMT2A variants display increased endoplasmic reticulum (ER) stress and apoptotic cell death. Increased cleavage of PARP1, caspase 9, caspase 7 and caspase 3, alongside BAX translocation to mitochondria, pointed towards effects on intrinsic apoptosis. Moreover, although disruption of fusion and fission dynamics per se did not correlate with cell death markers, expression of MFN1 or MFN2 alleviated the apoptosis markers of CMT2A variant cell lines. In sum, our results highlight excessive cell death by intrinsic apoptosis as a potential target in CMT2A disease.

Mitochondria are essential organelles and their functional state dictates cellular proteostasis. However, little is known about the molecular gatekeepers involved, especially in absence of external stress. Here we identify a role of MFN2 in quality control independent of its function in organellar shape remodeling. MFN2 ablation alters the cellular proteome, marked for example by decreased levels of the import machinery and accumulation of the kinase PINK1. Moreover, MFN2 interacts with the proteasome and cytosolic chaperones, thereby preventing aggregation of newly translated proteins. Similarly to MFN2-KO cells, patient fibroblasts with MFN2-disease variants recapitulate excessive protein aggregation defects. Restoring MFN2 levels re-establishes proteostasis in MFN2-KO cells and rescues fusion defects of MFN1-KO cells. In contrast, MFN1 loss or mitochondrial shape alterations do not alter protein aggregation, consistent with a fusion-independent role of MFN2 in cellular homeostasis. In sum, our findings open new possibilities for therapeutic strategies by modulation of MFN2 levels.

Mitochondria are dynamic cellular compartments that can remodel their own shape and activity. They sense and convert cellular signals into informative triggers, allowing the cell to adapt to its ever-changing needs. We discovered that under stress this adaptation is performed by the E4 enzyme Ufd2/UBE4B, which tags mitochondrial fusion factors called mitofusins, thus signalling their degradation. Our findings highlight therapeutic intervention cues for mitofusin-associated diseases.

In this protocol, we describe the analysis of protein stability over time, using synthesis shutoff. As an example, we express HA-tagged yeast mitofusin Fzo1 in Saccharomyces cerevisiae and inhibit translation via cycloheximide (CHX). Proteasomal inhibition with MG132 is performed, as an optional step, before the addition of CHX. Proteins are extracted via trichloroacetic acid (TCA) precipitation and subsequently separated via SDS-PAGE. Immunoblotting and antibody-decoration are performed to detect Fzo1 using HA-specific antibodies. We have adapted the method of blocking protein translation with cycloheximide to analyze the stability of high molecular weight proteins, including post-translational modifications and their impact on protein turnover., [摘要]在这个协议中，我们描述了蛋白质稳定性随时间的分析，使用合成关闭。例如，我们在酿酒酵母中表达 HA 标记的酵母丝裂融合蛋白 Fzo1，并通过放线菌酮 (CHX) 抑制翻译。在添加 CHX 之前，作为可选步骤，使用 MG132 进行蛋白酶体抑制。通过三氯乙酸 (TCA) 沉淀提取蛋白质，然后通过 SDS-PAGE 分离。使用 HA 特异性抗体进行免疫印迹和抗体修饰以检测 Fzo1。我们采用了用放线菌酮阻断蛋白质翻译的方法来分析高分子量蛋白质的稳定性，包括翻译后修饰及其对蛋白质周转的影响。 [背景] 在阻断翻译后，可以通过跟踪其随时间的变化来检查任何感兴趣的蛋白质的稳定性。不稳定的蛋白质会经历蛋白酶体或液泡降解。因此，蛋白质周转的生理相关性可以通过阻断蛋白酶体、液泡蛋白酶或通过使用其稳定的突变变体来破译。为了绕过抗体与其特异性结合的需要，可以构建和表达目标蛋白质的标记版本。在这里，我们提供了一个详细的协议来评估蛋白质稳定性及其对蛋白酶体的依赖。介绍了一种低成本且可靠的基于蛋白质印迹的测定，受益于蛋白质 Fzo1 的周转特性。酵母丝裂蛋白 Fzo1 是翻译后泛素化的已知靶标（Schuster等，2018），在正常生理条件下会降解（Anton等，2013；Anton等，2011；Anton等，2019） ；Schuster等人，2020 年；Simoes等人，2018 年）。Fzo1 中的残留物和其周转所需的酶已被确定，从而可以使用防止这种降解的相应突变作为周转控制。此外，取决于蛋白酶体的不稳定性增加可能发生在压力条件下或基因改变时。因此，如下文所述并在代表性图中举例说明，Fzo1 代表了蛋白质稳定性分析的一个很好的案例研究示例。我们的协议提供了详细的分步描述，与布坎南等人的常用协议相比，通过无毒制备程序和较少量的放线菌酮来分析蛋白质稳定性。(2016)。众所周知，放线菌酮 (CHX) 通过干扰去乙酰化的 tRNA 并结合 60S 亚基上的核糖体来阻止翻译延伸（Klinge等人，2011 年；Schneider-Poetsch等人，2010 年）。此外，我们使用三氯乙酸 (TCA) 的蛋白质提取协议可用于对小分子量和高分子量蛋白质进行基于蛋白质印迹的分析。

Mitochondria entail an incredible dynamism in their morphology, impacting death signaling and selective elimination of the damaged organelles. In turn, by recycling the superfluous or malfunctioning mitochondria, mostly prevalent during aging, mitophagy contributes to maintain a healthy mitochondrial network. Mitofusins locate at the outer mitochondrial membrane and control the plastic behavior of mitochondria, by mediating fusion events. Besides deciding on mitochondrial interconnectivity, mitofusin 2 regulates physical contacts between mitochondria and the endoplasmic reticulum, but also serves as a decisive docking platform for mitophagy and apoptosis effectors. Thus, mitofusins integrate multiple bidirectional inputs from and into mitochondria and ensure proper energetic and metabolic cellular performance. Here, we review the role of mitofusins and mitophagy at the cross-road between life and apoptotic death decisions. Furthermore, we highlight the impact of this interplay on disease, focusing on how mitofusin 2 and mitophagy affect non-alcoholic fatty liver disease.

Cdc48/p97 is a ring-shaped, ATP-driven hexameric motor, essential for cellular viability. It specifically unfolds and extracts ubiquitylated proteins from membranes or protein complexes, mostly targeting them for proteolytic degradation by the proteasome. Cdc48/p97 is involved in a multitude of cellular processes, reaching from cell cycle regulation to signal transduction, also participating in growth or death decisions. The role of Cdc48/p97 in endoplasmic reticulum-associated degradation (ERAD), where it extracts proteins targeted for degradation from the ER membrane, has been extensively described. Here, we present the roles of Cdc48/p97 in mitochondrial regulation. We discuss mitochondrial quality control surveillance by Cdc48/p97 in mitochondrial-associated degradation (MAD), highlighting the potential pathologic significance thereof. Furthermore, we present the current knowledge of how Cdc48/p97 regulates mitofusin activity in outer membrane fusion and how this may impact on neurodegeneration.