Oligomeric assembly of the C-terminal and transmembrane region of SARS-CoV-2 nsp3.

As for all single-stranded, positive-sense RNA (+RNA) viruses, intracellular RNA synthesis relies on extensive remodeling of host cell membranes that leads to the formation of specialized structures. In the case of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) coronavirus causing COVID-19, endoplasmic reticulum membranes are modified, resulting in the formation of double-membrane vesicles (DMVs), which contain the viral dsRNA intermediate and constitute membrane-bound replication organelles. The non-structural and transmembrane protein nsp3 is a key player in the biogenesis of DMVs and, therefore, represents an interesting antiviral target. However, as an integral transmembrane protein, it is challenging to express for structural biology. The C-terminus of nsp3 encompasses all the membrane-spanning, -interacting, and -remodeling elements. By using a cell-free expression system, we successfully produced the C-terminal region of nsp3 (nsp3C) and reconstituted purified nsp3C into phospholipid nanodiscs, opening the way for structural studies. Negative-stain transmission electron microscopy revealed the presence of nsp3C oligomers very similar to the region abutting and spanning the membrane on the cytosolic side of DMVs in a recent subtomogram average of the SARS-CoV-2 nsp3-4 pore (1). AlphaFold-predicted structural models fit particularly well with our experimental data and support a pore-forming hexameric assembly. Altogether, our data give unprecedented clues to understand the structural organization of nsp3, the principal component that shapes the molecular pore that spans the DMVs and is required for the export of RNA in vivo. IMPORTANCE: Membrane remodeling is at the heart of intracellular replication for single-stranded, positive-sense RNA viruses. In the case of coronaviruses, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), this leads to the formation of a network of double-membrane vesicles (DMVs). Targeting DMV biogenesis offers promising prospects for antiviral therapies. This requires a better understanding of the molecular mechanisms and proteins involved. Three non-structural proteins (nsp3, nsp4, and nsp6) direct the intracellular membrane rearrangements upon SARS-CoV-2 infection. All of them contain transmembrane helices. The nsp3 component, the largest and multi-functional protein of the virus, plays an essential role in this process. Aiming to understand its structural organization, we used a cell-free protein synthesis assay to produce and reconstitute the C-terminal part of nsp3 (nsp3C) including transmembrane domains into phospholipid nanodiscs. Our work reveals the oligomeric organization of one key player in the biogenesis of SARS-CoV-2 DMVs, providing basis for the design of future antiviral strategies.

Discriminating Susceptibility of Xanthine Oxidoreductase Family to Metals.

The xanthine oxidoreductase (XOR) family are metal-containing enzymes that use the molybdenum cofactor (Moco), 2Fe-2S clusters, and flavin adenine dinucleotide (FAD) for their catalytic activity. This large molybdoenzyme family includes xanthine, aldehyde, and CO dehydrogenases. XORs are widely distributed from bacteria to humans due to their key roles in the catabolism of purines, aldehydes, drugs, and xenobiotics, as well as interconversions between CO and CO2. Assessing the effect of excess metals on the Rubrivivax gelatinosus bacterium, we found that exposure to copper (Cu) or cadmium (Cd) caused a dramatic decrease in the activity of a high-molecular-weight soluble complex exhibiting nitroblue tetrazolium reductase activity. Mass spectrometry and genetic analyses showed that the complex corresponds to a putative CO dehydrogenase (pCOD). Using mutants that accumulate either Cu+ or Cd2+ in the cytoplasm, we show that Cu+ or Cd2+ is a potent inhibitor of XORs (pCOD and the xanthine dehydrogenase [XDH]) in vivo. This is the first in vivo demonstration that Cu+ affects Moco-containing enzymes. The specific inhibitory effect of these compounds on the XOR activity is further supported in vitro by direct addition of competing metals to protein extracts. Moreover, emphasis is given on the inhibitory effect of Cu on bovine XOR, showing that the XOR family could be a common target of Cu. Given the conservation of XOR structure and function across the tree of life, we anticipate that our findings could be transferable to other XORs and organisms. IMPORTANCE The high toxicity of Cu, Cd, Pb, As, and other metals arises from their ability to cross membranes and target metalloenzymes in the cytoplasm. Identifying these targets provides insights into the toxicity mechanisms. The vulnerability of metalloenzymes arises from the accessibility of their cofactors to ions. Accordingly, many enzymes whose cofactors are solvent exposed are likely to be targets of competing metals. Here, we describe for the first time, with in vivo and in vitro experiments, a direct effect of excess Cu on the xanthine oxidoreductase family (XOR/XDH/pCOD). We show that toxic metal affects these Moco enzymes, and we suggest that access to the Moco center by Cu ions could explain the Cu inhibition of XORs in living organisms. Human XOR activity is associated with hyperuricemia, xanthinuria, gout arthritis, and other diseases. Our findings in vivo highlight XOR as a Cu target and thus support the potential use of Cu in metal-based therapeutics against these diseases.

Additive effects of metal excess and superoxide, a highly toxic mixture in bacteria.

Heavy metal contamination is a serious environmental problem. Understanding the toxicity mechanisms may allow to lower concentration of metals in the metal-based antimicrobial treatments of crops, and reduce metal content in soil and groundwater. Here, we investigate the interplay between metal efflux systems and the superoxide dismutase (SOD) in the purple bacterium Rubrivivax gelatinosus and other bacteria through analysis of the impact of metal accumulation. Exposure of the Cd2+ -efflux mutant ΔcadA to Cd2+ caused an increase in the amount and activity of the cytosolic Fe-Sod SodB, thereby suggesting a role of SodB in the protection against Cd2+ . In support of this conclusion, inactivation of sodB gene in the ΔcadA cells alleviated detoxification of superoxide and enhanced Cd2+ toxicity. Similar findings were described in the Cu+ -efflux mutant with Cu+ . Induction of the Mn-Sod or Fe-Sod in response to metals in other bacteria, including Escherichia coli, Pseudomonas aeruginosa, Pseudomonas putida, Vibrio cholera and Bacillus subtilis, was also shown. Both excess Cd2+ or Cu+ and superoxide can damage [4Fe-4S] clusters. The additive effect of metal and superoxide on the [4Fe-4S] could therefore explain the hypersensitive phenotype in mutants lacking SOD and the efflux ATPase. These findings underscore that ROS defence system becomes decisive for bacterial survival under metal excess.

Increasing the copper sensitivity of microorganisms by restricting iron supply, a strategy for bio-management practices.

Pollution by copper (Cu2+ ) extensively used as antimicrobial in agriculture and farming represents a threat to the environment and human health. Finding ways to make microorganisms sensitive to lower metal concentrations could help decreasing the use of Cu2 + in agriculture. In this respect, we showed that limiting iron (Fe) uptake makes bacteria much more susceptible to Cu2 + or Cd2+ poisoning. Using efflux mutants of the purple bacterium Rubrivivax gelatinosus, we showed that Cu+ and Cd2+ resistance relies on the expression of the Fur-regulated FbpABC and Ftr iron transporters. To support this conclusion, inactivation of these Fe-importers in the Cu+ or Cd2+ -ATPase efflux mutants gave rise to hypersensitivity towards these ions. Moreover, in metal overloaded cells the expression of FbpA, the periplasmic iron-binding component of the ferric ion transport FbpABC system was induced, suggesting that cells perceived an 'iron-starvation' situation and responded to it by inducing Fe-importers. In this context, the Fe-Sod activity increased in response to Fe homoeostasis dysregulation. Similar results were obtained for Vibrio cholerae and Escherichia coli, suggesting that perturbation of Fe-homoeostasis by metal excess appeared as an adaptive response commonly used by a variety of bacteria. The presented data support a model in which metal excess induces Fe-uptake to support [4Fe-4S] synthesis and thereby induce ROS detoxification system.

Cadmium and Copper Cross-Tolerance. Cu+ Alleviates Cd2 + Toxicity, and Both Cations Target Heme and Chlorophyll Biosynthesis Pathway in Rubrivivax gelatinosus.

Cadmium, although not redox active is highly toxic. Yet, the underlying mechanisms driving toxicity are still to be characterized. In this study, we took advantage of the purple bacterium Rubrivivax gelatinosus strain with defective Cd2 +-efflux system to identify targets of this metal. Exposure of the ΔcadA strain to Cd2 + causes a decrease in the photosystem amount and in the activity of respiratory complexes. As in case of Cu+ toxicity, the data indicated that Cd2 + targets the porphyrin biosynthesis pathway at the level of HemN, a S-adenosylmethionine and CxxxCxxC coordinated [4Fe-4S] containing enzyme. Cd2 + exposure therefore results in a deficiency in heme and chlorophyll dependent proteins and metabolic pathways. Given the importance of porphyrin biosynthesis, HemN represents a key metal target to account for toxicity. In the environment, microorganisms are exposed to mixture of metals. Nevertheless, the biological effects of such mixtures, and the toxicity mechanisms remain poorly addressed. To highlight a potential cross-talk between Cd2 + and Cu+ -efflux systems, we show (i) that Cd2 + induces the expression of the Cd2 +-efflux pump CadA and the Cu+ detoxification system CopA and CopI; and (ii) that Cu+ ions improve tolerance towards Cd2 +, demonstrating thus that metal mixtures could also represent a selective advantage in the environment.

Silver and Copper Acute Effects on Membrane Proteins and Impact on Photosynthetic and Respiratory Complexes in Bacteria.

Silver (Ag+) and copper (Cu+) ions have been used for centuries in industry, as well as antimicrobial agents in agriculture and health care. Nowadays, Ag+ is also widely used in the field of nanotechnology. Yet, the underlying mechanisms driving toxicity of Ag+ ions in vivo are poorly characterized. It is well known that exposure to excess metal impairs the photosynthetic apparatus of plants and algae. Here, we show that the light-harvesting complex II (LH2) is the primary target of Ag+ and Cu+ exposure in the purple bacterium Rubrivivax gelatinosus Ag+ and Cu+ specifically inactivate the 800-nm absorbing bacteriochlorophyll a (B800), while Ni2+ or Cd2+ treatment had no effect. This was further supported by analyses of CuSO4- or AgNO3-treated membrane proteins. Indeed, this treatment induced changes in the LH2 absorption spectrum related to the disruption of the interaction of B800 molecules with the LH2 protein. This caused the release of B800 molecules and subsequently impacted the spectral properties of the carotenoids within the 850-nm absorbing LH2. Moreover, previous studies have suggested that Ag+ can affect the respiratory chain in mitochondria and bacteria. Our data demonstrated that exposure to Ag+, both in vivo and in vitro, caused a decrease of cytochrome c oxidase and succinate dehydrogenase activities. Ag+ inhibition of these respiratory complexes was also observed in Escherichia coli, but not in Bacillus subtilisIMPORTANCE The use of metal ions represents a serious threat to the environment and to all living organisms because of the acute toxicity of these ions. Nowadays, silver nanoparticles are one of the most widely used nanoparticles in various industrial and health applications. The antimicrobial effect of nanoparticles is in part related to the released Ag+ ions and their ability to interact with bacterial membranes. Here, we identify, both in vitro and in vivo, specific targets of Ag+ ions within the membrane of bacteria. This include complexes involved in photosynthesis, but also complexes involved in respiration.

A unifying mechanism for the biogenesis of membrane proteins co-operatively integrated by the Sec and Tat pathways.

The majority of multi-spanning membrane proteins are co-translationally inserted into the bilayer by the Sec pathway. An important subset of membrane proteins have globular, cofactor-containing extracytoplasmic domains requiring the dual action of the co-translational Sec and post-translational Tat pathways for integration. Here, we identify further unexplored families of membrane proteins that are dual Sec-Tat-targeted. We establish that a predicted heme-molybdenum cofactor-containing protein, and a complex polyferredoxin, each require the concerted action of two translocases for their assembly. We determine that the mechanism of handover from Sec to Tat pathway requires the relatively low hydrophobicity of the Tat-dependent transmembrane domain. This, coupled with the presence of C-terminal positive charges, results in abortive insertion of this transmembrane domain by the Sec pathway and its subsequent release at the cytoplasmic side of the membrane. Together, our data points to a simple unifying mechanism governing the assembly of dual targeted membrane proteins.

ND3, ND1 and 39kDa subunits are more exposed in the de-active form of bovine mitochondrial complex I.

An intriguing feature of mitochondrial complex I from several species is the so-called A/D transition, whereby the idle enzyme spontaneously converts from the active (A) form to the de-active (D) form. The A/D transition plays an important role in tissue response to the lack of oxygen and hypoxic deactivation of the enzyme is one of the key regulatory events that occur in mitochondria during ischaemia. We demonstrate for the first time that the A/D conformational change of complex I does not affect the macromolecular organisation of supercomplexes in vitro as revealed by two types of native electrophoresis. Cysteine 39 of the mitochondrially-encoded ND3 subunit is known to become exposed upon de-activation. Here we show that even if complex I is a constituent of the I+III2+IV (S1) supercomplex, cysteine 39 is accessible for chemical modification in only the D-form. Using lysine-specific fluorescent labelling and a DIGE-like approach we further identified two new subunits involved in structural rearrangements during the A/D transition: ND1 (MT-ND1) and 39kDa (NDUFA9). These results clearly show that structural rearrangements during de-activation of complex I include several subunits located at the junction between hydrophilic and hydrophobic domains, in the region of the quinone binding site. De-activation of mitochondrial complex I results in concerted structural rearrangement of membrane subunits which leads to the disruption of the sealed quinone chamber required for catalytic turnover.

Characterisation of the active/de-active transition of mitochondrial complex I.

Oxidation of NADH in the mitochondrial matrix of aerobic cells is catalysed by mitochondrial complex I. The regulation of this mitochondrial enzyme is not completely understood. An interesting characteristic of complex I from some organisms is the ability to adopt two distinct states: the so-called catalytically active (A) and the de-active, dormant state (D). The A-form in situ can undergo de-activation when the activity of the respiratory chain is limited (i.e. in the absence of oxygen). The mechanisms and driving force behind the A/D transition of the enzyme are currently unknown, but several subunits are most likely involved in the conformational rearrangements: the accessory subunit 39kDa (NDUFA9) and the mitochondrially encoded subunits, ND3 and ND1. These three subunits are located in the region of the quinone binding site. The A/D transition could represent an intrinsic mechanism which provides a fast response of the mitochondrial respiratory chain to oxygen deprivation. The physiological role of the accumulation of the D-form in anoxia is most probably to protect mitochondria from ROS generation due to the rapid burst of respiration following reoxygenation. The de-activation rate varies in different tissues and can be modulated by the temperature, the presence of free fatty acids and divalent cations, the NAD(+)/NADH ratio in the matrix, the presence of nitric oxide and oxygen availability. Cysteine-39 of the ND3 subunit, exposed in the D-form, is susceptible to covalent modification by nitrosothiols, ROS and RNS. The D-form in situ could react with natural effectors in mitochondria or with pharmacological agents. Therefore the modulation of the re-activation rate of complex I could be a way to ameliorate the ischaemia/reperfusion damage. This article is part of a Special Issue entitled: 18th European Bioenergetic Conference. Guest Editors: Manuela Pereira and Miguel Teixeira.

Mitochondrial energetics is impaired in vivo in aged skeletal muscle.

With aging, most skeletal muscles undergo a progressive loss of mass and strength, a process termed sarcopenia. Aging-related defects in mitochondrial energetics have been proposed to be causally involved in sarcopenia. However, changes in muscle mitochondrial oxidative phosphorylation with aging remain a highly controversial issue, creating a pressing need for integrative approaches to determine whether mitochondrial bioenergetics are impaired in aged skeletal muscle. To address this issue, mitochondrial bioenergetics was first investigated in vivo in the gastrocnemius muscle of adult (6 months) and aged (21 months) male Wistar rats by combining a modular control analysis approach with (31) P magnetic resonance spectroscopy measurements of energetic metabolites. Using this innovative approach, we revealed that the in vivo responsiveness ('elasticity') of mitochondrial oxidative phosphorylation to contraction-induced increase in ATP demand is significantly reduced in aged skeletal muscle, a reduction especially pronounced under low contractile activities. In line with this in vivo aging-related defect in mitochondrial energetics, we found that the mitochondrial affinity for ADP is significantly decreased in mitochondria isolated from aged skeletal muscle. Collectively, the results of this study demonstrate that mitochondrial bioenergetics are effectively altered in vivo in aged skeletal muscle and provide a novel cellular basis for this phenomenon.

Molecular mechanism and physiological role of active-deactive transition of mitochondrial complex I.

The unique feature of mitochondrial complex I is the so-called A/D transition (active-deactive transition). The A-form catalyses rapid oxidation of NADH by ubiquinone (k ~104 min-1) and spontaneously converts into the D-form if the enzyme is idle at physiological temperatures. Such deactivation occurs in vitro in the absence of substrates or in vivo during ischaemia, when the ubiquinone pool is reduced. The D-form can undergo reactivation given both NADH and ubiquinone availability during slow (k ~1-10 min-1) catalytic turnover(s). We examined known conformational differences between the two forms and suggested a mechanism exerting A/D transition of the enzyme. In addition, we discuss the physiological role of maintaining the enzyme in the D-form during the ischaemic period. Accumulation of the D-form of the enzyme would prevent reverse electron transfer from ubiquinol to FMN which could lead to superoxide anion generation. Deactivation would also decrease the initial burst of respiration after oxygen reintroduction. Therefore the A/D transition could be an intrinsic protective mechanism for lessening oxidative damage during the early phase of reoxygenation. Exposure of Cys39 of mitochondrially encoded subunit ND3 makes the D-form susceptible for modification by reactive oxygen species and nitric oxide metabolites which arrests the reactivation of the D-form and inhibits the enzyme. The nature of thiol modification defines deactivation reversibility, the reactivation timescale, the status of mitochondrial bioenergetics and therefore the degree of recovery of the ischaemic tissues after reoxygenation.

The mitochondrial ADP/ATP carrier (SLC25 family): pathological implications of its dysfunction.

In aerobic eukaryotic cells, the high energy metabolite ATP is generated mainly within the mitochondria following the process of oxidative phosphorylation. The mitochondrial ATP is exported to the cytoplasm using a specialized transport protein, the ADP/ATP carrier, to provide energy to the cell. Any deficiency or dysfunction of this membrane protein leads to serious consequences on cell metabolism and can cause various diseases such as muscular dystrophy. Described as a decisive player in the programmed cell death, it was recently shown to play a role in cancer. The objective of this review is to summarize the current knowledge of the involvement of the ADP/ATP carrier, encoded by the SLC25A4, SLC25A5, SLC25A6 and SLC25A31 genes, in human diseases and of the efforts made at designing different model systems to study this carrier and the associated pathologies through biochemical, genetic, and structural approaches.

Mitochondrial ADP/ATP carrier: preventing conformational changes by point mutations inactivates nucleotide transport activity.

The mitochondrial ADP/ATP carrier (Ancp) is a paradigm of the mitochondrial carrier family (MCF); its members allow metabolic fluxes between mitochondria and the cytosol. The members of the MCF share numerous structural and functional characteristics. Ancp is very specifically inhibited by two classes of compounds, which stabilize the carrier in two different conformations involved in nucleotide transport. Resolution of the atomic structure of the bovine Ancp, in complex with one of its specific inhibitors, is that of the carrier open toward the intermembrane space. To gain insights into the interconversion from one conformation to the other, we introduced point mutations in the yeast carrier at positions Cys73 in the first matrix loop and Tyr97 and Gly298 in transmembrane helices 2 and 6. We demonstrate in this paper that they impair stabilization of the carrier in one conformation or the other, resulting in an almost complete inactivation of nucleotide transport in both cases. The results are discussed on the basis of the atomic structure of the conformation open to the cytosol. These mutant proteins could afford convenient tools for undertaking structural studies of both conformations of the yeast carrier.

The transmembrane prolines of the mitochondrial ADP/ATP carrier are involved in nucleotide binding and transport and its biogenesis.

The mitochondrial ADP/ATP carrier (Ancp) is a paradigm of the mitochondrial carrier family, which allows cross-talk between mitochondria, where cell energy is mainly produced, and cytosol, where cell energy is mainly consumed. The members of this family share numerous structural and functional characteristics. Resolution of the atomic structure of the bovine Ancp, in a complex with one of its specific inhibitors, revealed interesting features and suggested the involvement of some particular residues in the movements of the protein to perform translocation of nucleotides from one side of the membrane to the other. They correspond to three prolines located in the odd-numbered transmembrane helices (TMH), Pro-27, Pro-132, and Pro-229. The corresponding residues of the yeast Ancp (Pro-43, Ser-147, and Pro-247) were mutated into alanine or leucine, one at a time and analysis of the various mutants evidenced a crucial role of Pro-43 and Pro-247 during nucleotide transport. Beside, replacement of Ser-147 with proline does not inactivate Ancp and this is discussed in view of the conservation of the three prolines at equivalent positions in the Ancp sequences. These prolines belong to the signature sequences of the mitochondrial carriers and we propose they play a dual role in the mitochondrial ADP/ATP carrier function and biogenesis. Unexpectedly their mutations cause more general effects on mitochondrial biogenesis and morphology, as evidenced by measurements of respiratory rates, cytochrome contents, and also clearly highlighted by fluorescence microscopy.