Luca Pellegrini

Mitochondrial Rhomboid PARL Regulates Cytochrome c Release during Apoptosis via OPA1-Dependent Cristae Remodeling

Rhomboids, evolutionarily conserved integral membrane proteases, participate in crucial signaling pathways. Presenilin-associated rhomboid-like (PARL) is an inner mitochondrial membrane rhomboid of unknown function, whose yeast ortholog is involved in mitochondrial fusion. Parl-/- mice display normal intrauterine development but from the fourth postnatal week undergo progressive multisystemic atrophy leading to cachectic death. Atrophy is sustained by increased apoptosis, both in and ex vivo. Parl-/- cells display normal mitochondrial morphology and function but are no longer protected against intrinsic apoptotic death stimuli by the dynamin-related mitochondrial protein OPA1. Parl-/- mitochondria display reduced levels of a soluble, intermembrane space (IMS) form of OPA1, and OPA1 specifically targeted to IMS complements Parl-/- cells, substantiating the importance of PARL in OPA1 processing. Parl-/- mitochondria undergo faster apoptotic cristae remodeling and cytochrome c release. These findings implicate regulated intramembrane proteolysis in controlling apoptosis.

The rhomboids: a nearly ubiquitous family of intramembrane serine proteases that probably evolved by multiple ancient horizontal gene transfers

BackgroundThe rhomboid family of polytopic membrane proteins shows a level of evolutionary conservation unique among membrane proteins. They are present in nearly all the sequenced genomes of archaea, bacteria and eukaryotes, with the exception of several species with small genomes. On the basis of experimental studies with the developmental regulator rhomboid from Drosophila and the AarA protein from the bacterium Providencia stuartii, the rhomboids are thought to be intramembrane serine proteases whose signaling function is conserved in eukaryotes and prokaryotes.ResultsPhylogenetic tree analysis carried out using several independent methods for tree constructions and the corresponding statistical tests suggests that, despite its broad distribution in all three superkingdoms, the rhomboid family was not present in the last universal common ancestor of extant life forms. Instead, we propose that rhomboids evolved in bacteria and have been acquired by archaea and eukaryotes through several independent horizontal gene transfers. In eukaryotes, two distinct, ancient acquisitions apparently gave rise to the two major subfamilies, typified by rhomboid and PARL (presenilins-associated rhomboid-like protein), respectively. Subsequent evolution of the rhomboid family in eukaryotes proceeded by multiple duplications and functional diversification through the addition of extra transmembrane helices and other domains in different orientations relative to the conserved core that harbors the protease activity.ConclusionsAlthough the near-universal presence of the rhomboid family in bacteria, archaea and eukaryotes appears to suggest that this protein is part of the heritage of the last universal common ancestor, phylogenetic tree analysis indicates a likely bacterial origin with subsequent dissemination by horizontal gene transfer. This emphasizes the importance of explicit phylogenetic analysis for the reconstruction of ancestral life forms. A hypothetical scenario for the origin of intracellular membrane proteases from membrane transporters is proposed.

Phosphorylation and cleavage of presenilin-associated rhomboid-like protein (PARL) promotes changes in mitochondrial morphology

Remodeling of mitochondria is a dynamic process coordinated by fusion and fission of the inner and outer membranes of the organelle, mediated by a set of conserved proteins. In metazoans, the molecular mechanism behind mitochondrial morphology has been recruited to govern novel functions, such as development, calcium signaling, and apoptosis, which suggests that novel mechanisms should exist to regulate the conserved membrane fusion/fission machinery. Here we show that phosphorylation and cleavage of the vertebrate-specific Pβ domain of the mammalian presenilin-associated rhomboid-like (PARL) protease can influence mitochondrial morphology. Phosphorylation of three residues embedded in this domain, Ser-65, Thr-69, and Ser-70, impair a cleavage at position Ser77–Ala78 that is required to initiate PARL-induced mitochondrial fragmentation. Our findings reveal that PARL phosphorylation and cleavage impact mitochondrial dynamics, providing a blueprint to study the molecular evolution of mitochondrial morphology.

The coming of age of the mitochondria-ER contact: a matter of thickness.

The sites of near-contact between the mitochondrion and the endoplasmic reticulum (ER) have earned a lot of attention due to their key role in the maintenance of lipid and calcium (Ca2+) homeostasis, in the initiation of autophagy and mitochondrial division, and in sensing metabolic shifts. At these sites, typically called MAMs (mitochondria-associated ER membranes) or MERCs (mitochondria–ER contacts), the organelles juxtapose at a distance that can range from ~10 to ~50 nm. The multifunctional role of this subcellular compartment is puzzling; further, recent studies have shown that mitochondria–ER contacts are highly plastic structures that remodel upon metabolic transitions and that their activity in controlling lipid homeostasis could be involved in Alzheimer’s disease pathogenesis. This review aims at integrating the functions of this subcellular compartment to its most characterizing and unexplored structural parameter, their ‘thickness’: that is, the width of the cleft that separates the cytosolic face of the outer mitochondrial membrane from that of the ER. We describe and discuss the reasons why the thickness of a MERC should be considered a regulated structural parameter of the cell that defines and controls its function. Further, we propose a MERC classification that will help organize the expanding field of MERCs biology and of their role in cell physiology and human disease.

A Mitofusin-2-dependent inactivating cleavage of Opa1 links changes in mitochondria cristae and ER contacts in the postprandial liver.

Significance We provide, to our knowledge, the first in vivo quantitative description of the adaptive response of the mitochondrial reticulum to the metabolic transition occurring in the liver in the hours after feeding. When nutrients become limiting, mitochondria size, cristae density, and respiratory capacity drop, but mitochondria–ER contacts, which control calcium and lipids fluxes between these organelles, double. A proteolytic inactivation of Optic atrophy 1 (Opa1), a major regulator of fusion and cristae architecture, accompanies these changes and found to depend on Mitofusin-2, a key regulator of mitochondria–ER contact biogenesis. Thus, mitochondria adapt to nutrient depletion by coupling the molecular machineries that organize cristae architecture and mitochondria–ER contact assembly, which were previously thought to operate independently of each other. Hepatic metabolism requires mitochondria to adapt their bioenergetic and biosynthetic output to accompany the ever-changing anabolic/catabolic state of the liver cell, but the wiring of this process is still largely unknown. Using a postprandial mouse liver model and quantitative cryo-EM analysis, we show that when the hepatic mammalian target of rapamycin complex 1 (mTORC1) signaling pathway disengages, the mitochondria network fragments, cristae density drops by 30%, and mitochondrial respiratory capacity decreases by 20%. Instead, mitochondria–ER contacts (MERCs), which mediate calcium and phospholipid fluxes between these organelles, double in length. These events are associated with the transient expression of two previously unidentified C-terminal fragments (CTFs) of Optic atrophy 1 (Opa1), a mitochondrial GTPase that regulates cristae biogenesis and mitochondria dynamics. Expression of Opa1 CTFs in the intermembrane space has no effect on mitochondria morphology, supporting a model in which they are intermediates of an Opa1 degradation program. Using an in vitro assay, we show that these CTFs indeed originate from the cleavage of Opa1 at two evolutionarily conserved consensus sites that map within critical folds of the GTPase. This processing of Opa1, termed C-cleavage, is mediated by the activity of a cysteine protease whose activity is independent from that of Oma1 and presenilin-associated rhomboid-like (PARL), two known Opa1 regulators. However, C-cleavage requires Mitofusin-2 (Mfn2), a key factor in mitochondria–ER tethering, thereby linking cristae remodeling to MERC assembly. Thus, in vivo, mitochondria adapt to metabolic shifts through the parallel remodeling of the cristae and of the MERCs via a mechanism that degrades Opa1 in an Mfn2-dependent pathway.

The dynamin GTPase OPA1: More than mitochondria? ☆

The studies addressing the molecular mechanisms governing mitochondrial fusion and fission have brought to light a small group of dynamin-like GTPases (Guanosine-Triphosphate hydrolase) as central regulators of mitochondrial morphology and cristae remodeling, apoptosis, calcium signaling, and metabolism. One of them is the mammalian OPA1 (Optic atrophy 1) protein, which resides inside the mitochondrion anchored to the inner membrane and, in a cleaved form, is associated to oligomeric complexes, in the intermembrane space of the organelle. Here, we review the studies that have made OPA1 emerge as the best understood regulator of mitochondrial inner membrane fusion and cristae remodeling. Further, we re-examine the findings behind the recent claim that OPA1 mediates adrenergic control of lipolysis by functioning as a cytosolic A-kinase anchoring protein (AKAP), on the hemimembrane that envelops the lipid droplet. This article is part of a Special Issue entitled: Mitochondrial dynamics and physiology.

Extracellular chelation of zinc does not affect hippocampal excitability and seizure-induced cell death in rats

In the nervous system, zinc can influence synaptic responses and at extreme concentrations contributes to epileptic and ischaemic neuronal injury. Zinc can originate from synaptic vesicles, the extracellular space and from intracellular stores. In this study, we aimed to determine which of these zinc pools is responsible for the increased hippocampal excitability observed in zinc‐depleted animals or following zinc chelation. Also, we investigated the source of intracellularly accumulating zinc in vulnerable neurons. Our data show that membrane‐permeable and membrane‐impermeable zinc chelators had little or no effect on seizure activity in the CA3 region. Furthermore, extracellular zinc chelation could not prevent the accumulation of lethal concentrations of zinc in dying neurons following epileptic seizures. At the electron microscopic level, zinc staining significantly increased at the presynaptic membrane of mossy fibre terminals in kainic acid‐treated animals. These data indicate that intracellular but not extracellular zinc chelators could influence neuronal excitability and seizure‐induced zinc accumulation observed in the cytosol of vulnerable neurons.

Cell type‐specific action of seizure‐induced intracellular zinc accumulation in the rat hippocampus

Increased levels of intracellular zinc have been implicated in neuronal cell death in ischaemia, epilepsy and traumatic brain damage. However, decreases in zinc levels also lead to increased neuronal death and lowered seizure threshold. In the present study we investigated the physiological role of zinc in neurodegeneration and protection following epileptic seizures. Cells located in the strata oriens and lucidum of the CA3 region accumulated high concentrations of zinc and died. A decrease in zinc level could prevent the death of these neurones after seizures. Most of these cells were GABAergic interneurones. In contrast, neurones in the CA3 pyramidal cell layer accumulated moderate amounts of zinc and survived. Zinc chelation led to an increase in the mortality rate of these cells. Furthermore, in these cells low concentrations of intracellular zinc activated Akt (protein kinase B), thus providing protection against neurodegeneration. These results demonstrate that intracellularly accumulated zinc can be neurotoxic or neuroprotective depending on its concentration. This dual action is cell type specific.

Calcium regulation of mitochondria motility and morphology

In the Fifties, electron microscopy studies on neuronal cells showed that mitochondria typically cluster at synaptic terminals, thereby introducing the concept that proper mitochondria trafficking and partitioning inside the cell could provide functional support to the execution of key physiological processes. Today, the notion that a central event in the life of every eukaryotic cell is to configure, maintain, and reorganize the mitochondrial network at sites of high energy demand in response to environmental and cellular cues is well established, and the challenge ahead is to define the underlying molecular mechanisms and regulatory pathways. Recent pioneering studies have further contributed to place mitochondria at the center of the cell biology by showing that the machinery governing remodeling of mitochondria shape and structure regulates the functional output of the organelle as the powerhouse of the cell, the gateway to programmed cell death, and the platform for Ca(2+) signaling. Thus, a raising issue is to identify the cues integrating mitochondria trafficking and dynamics into cell physiology and metabolism. Given the versatile function of calcium as a second messenger and of the role of mitochondria as a major calcium store, evidences are emerging linking Ca(2+) transients to the modulation of mitochondrial activities. This review focuses on calcium as a switch controlling mitochondria motility and morphology in steady state, stressed, and pathological conditions.

Optic Atrophy 1 Is Epistatic to the Core MICOS Component MIC60 in Mitochondrial Cristae Shape Control

Summary The mitochondrial contact site and cristae organizing system (MICOS) and Optic atrophy 1 (OPA1) control cristae shape, thus affecting mitochondrial function and apoptosis. Whether and how they physically and functionally interact is unclear. Here, we provide evidence that OPA1 is epistatic to MICOS in the regulation of cristae shape. Proteomic analysis identifies multiple MICOS components in native OPA1-containing high molecular weight complexes disrupted during cristae remodeling. MIC60, a core MICOS protein, physically interacts with OPA1, and together, they control cristae junction number and stability, OPA1 being epistatic to MIC60. OPA1 defines cristae width and junction diameter independently of MIC60. Our combination of proteomics, biochemistry, genetics, and electron tomography provides a unifying model for mammalian cristae biogenesis by OPA1 and MICOS.