Mitofusin 1 silencing decreases the senescent associated secretory phenotype, promotes immune cell recruitment and delays melanoma tumor growth after chemotherapy.

Cellular senescence is a therapy endpoint in melanoma, and the senescence-associated secretory phenotype (SASP) can affect tumor growth and microenvironment, influencing treatment outcomes. Metabolic interventions can modulate the SASP, and mitochondrial energy metabolism supports resistance to therapy in melanoma. In a previous report we showed that senescence, induced by the DNA methylating agent temozolomide, increased the level of fusion proteins mitofusin 1 and 2 in melanoma, and silencing Mfn1 or Mfn2 expression reduced interleukin-6 secretion by senescent cells. Here we expanded these observations evaluating the secretome of senescent melanoma cells using shotgun proteomics, and explored the impact of silencing Mfn1 on the SASP. A significant increase in proteins reported to reduce the immune response towards the tumor was found in the media of senescent cells. The secretion of several of these immunomodulatory proteins was affected by Mfn1 silencing, among them was galectin-9. In agreement, tumors lacking mitofusin 1 responded better to treatment with the methylating agent dacarbazine, tumor size was reduced and a higher immune cell infiltration was detected in the tumor. Our results highlight mitochondrial dynamic proteins as potential pharmacological targets to modulate the SASP in the context of melanoma treatment.

CD300f immune receptor contributes to healthy aging by regulating inflammaging, metabolism, and cognitive decline.

Emerging evidence suggests that immune receptors may participate in many aging-related processes such as energy metabolism, inflammation, and cognitive decline. CD300f, a TREM2-like lipid-sensing immune receptor, is an exceptional receptor as it integrates activating and inhibitory cell-signaling pathways that modulate inflammation, efferocytosis, and microglial metabolic fitness. We hypothesize that CD300f can regulate systemic aging-related processes and ultimately healthy lifespan. We closely followed several cohorts of two strains of CD300f-/- and WT mice of both sexes for 30 months and observed an important reduction in lifespan and healthspan in knockout mice. This was associated with systemic inflammaging, increased cognitive decline, reduced brain glucose uptake observed by 18FDG PET scans, enrichment in microglial aging/neurodegeneration phenotypes, proteostasis alterations, senescence, increased frailty, and sex-dependent systemic metabolic changes. Moreover, the absence of CD300f altered macrophage immunometabolic phenotype. Taken together, we provide strong evidence suggesting that myeloid cell CD300f immune receptor contributes to healthy aging.

Computational and mitochondrial functional studies of novel compound heterozygous variants in SPATA5 gene support a causal link with epileptogenic encephalopathy.

The SPATA5 gene encodes a 892 amino-acids long protein that has a putative mitochondrial targeting sequence and has been proposed to function in maintenance of mitochondrial function and integrity during mouse spermatogenesis. Several studies have associated homozygous or compound heterozygous mutations in SPATA5 gene to microcephaly, intellectual disability, seizures and hearing loss. This suggests a role of the SPATA5 gene also in neuronal development. Recently, our group presented results validating the use of blood cells for the assessment of mitochondrial function for diagnosis and follow-up of mitochondrial disease, minimizing the need for invasive procedures such as muscle biopsy. In this study, we were able to diagnose a patient with epileptogenic encephalopathy using next generation sequencing. We found two novel compound heterozygous variants in SPATA5 that are most likely causative. To analyze the impact of SPATA5 mutations on mitochondrial functional studies directly on the patients' mononuclear cells and platelets were undertaken. Oxygen consumption rates in platelets and PBMCs were impaired in the patient when compared to a healthy control. Also, a decrease in mitochondrial mass was observed in the patient monocytes with respect to the control. This suggests a true pathogenic effect of the mutations in mitochondrial function, especially in energy production and possibly biogenesis, leading to the observed phenotype.

De novo sequencing and construction of a unique antibody for the recognition of alternative conformations of cytochrome c in cells.

Cytochrome c (cyt c) can undergo reversible conformational changes under biologically relevant conditions. Revealing these alternative cyt c conformers at the cell and tissue level is challenging. A monoclonal antibody (mAb) identifying a key conformational change in cyt c was previously reported, but the hybridoma was rendered nonviable. To resurrect the mAb in a recombinant form, the amino-acid sequences of the heavy and light chains were determined by peptide mapping-mass spectrometry-bioinformatic analysis and used to construct plasmids encoding the full-length chains. The recombinant mAb (R1D3) was shown to perform similarly to the original mAb in antigen-binding assays. The mAb bound to a variety of oxidatively modified cyt c species (e.g., nitrated at Tyr74 or oxidized at Met80), which lose the sixth heme ligation (Fe-Met80); it did not bind to several cyt c phospho- and acetyl-mimetics. Peptide competition assays together with molecular dynamic studies support that R1D3 binds a neoepitope within the loop 40-57. R1D3 was employed to identify alternative conformations of cyt c in cells under oxidant- or senescence-induced challenge as confirmed by immunocytochemistry and immunoaffinity studies. Alternative conformers translocated to the nuclei without causing apoptosis, an observation that was further confirmed after pinocytic loading of oxidatively modified cyt c to B16-F1 cells. Thus, alternative cyt c conformers, known to gain peroxidatic function, may represent redox messengers at the cell nuclei. The availability and properties of R1D3 open avenues of interrogation regarding the presence and biological functions of alternative conformations of cyt c in mammalian cells and tissues.

Differential hepatic mitochondrial function and gluconeogenic gene expression in 2 Holstein strains in a pasture-based system.

The objective of this study was to assess hepatic ATP synthesis in Holstein cows of North American and New Zealand origins and the gluconeogenic pathway, one of the pathways with the highest ATP demands in the ruminant liver. Autumn-calving Holstein cows of New Zealand and North American origins were managed in a pasture-based system with supplementation of concentrate that represented approximately 33% of the predicted dry matter intake during 2017, 2018, and 2019, and hepatic biopsies were taken during mid-lactation at 174 ± 23 days in milk. Cows of both strains produced similar levels of solids-corrected milk, and no differences in body condition score were found. Plasma glucose concentrations were higher for cows of New Zealand versus North American origin. Hepatic mitochondrial function evaluated measuring oxygen consumption rates showed that mitochondrial parameters related to ATP synthesis and maximum respiratory rate were increased for cows of New Zealand compared with North American origin. However, hepatic gene expression of pyruvate carboxylase, phosphoenolpyruvate carboxykinase, and pyruvate dehydrogenase kinase was increased in North American compared with New Zealand cows. These results altogether suggest an increased activity of the tricarboxylic cycle in New Zealand cows, leading to increased ATP synthesis, whereas North American cows pull tricarboxylic cycle intermediates toward gluconeogenesis. The fact that this occurs during mid-lactation could account for the increased persistency of North American cows, especially in a pasture-based system. In addition, we observed an augmented mitochondrial density in New Zealand cows, which could be related to feed efficiency mechanisms. In sum, our results contribute to the elucidation of hepatic molecular mechanisms in dairy cows in production systems with higher inclusion of pastures.

Human papillomavirus type 18 E5 oncoprotein cooperates with E6 and E7 in promoting cell viability and invasion and in modulating the cellular redox state.

BACKGROUND: High-risk human papillomaviruses (HR-HPVs) are the etiological agents of cervical cancer. Among them, types 16 and 18 are the most prevalent worldwide. The HPV genome encodes three oncoproteins (E5, E6, and E7) that possess a high transformation potential in culture cells when transduced simultaneously. In the present study, we analysed how these oncoproteins cooperate to boost key cancer cell features such as uncontrolled cell proliferation, invasion potential, and cellular redox state imbalance. Oxidative stress is known to contribute to the carcinogenic process, as reactive oxygen species (ROS) constitute a potentially harmful by-product of many cellular reactions, and an efficient clearance mechanism is therefore required. Cells infected with HR-HPVs can adapt to oxidative stress conditions by upregulating the formation of endogenous antioxidants such as catalase, glutathione (GSH), and peroxiredoxin (PRX). OBJECTIVES: The primary aim of this work was to study how these oncoproteins cooperate to promote the development of certain cancer cell features such as uncontrolled cell proliferation, invasion potential, and oxidative stress that are known to aid in the carcinogenic process. METHODS: To perform this study, we generated three different HaCaT cell lines using retroviral transduction that stably expressed combinations of HPV-18 oncogenes that included HaCaT E5-18, HaCaT E6/E7-18, and HaCaT E5/E6/E7-18. FINDINGS: Our results revealed a statistically significant increment in cell viability as measured by MTT assay, cell proliferation, and invasion assays in the cell line containing the three viral oncogenes. Additionally, we observed that cells expressing HPV-18 E5/E6/E7 exhibited a decrease in catalase activity and a significant augmentation of GSH and PRX1 levels relative to those of E5, E6/E7, and HaCaT cells. MAIN CONCLUSIONS: This study demonstrates for the first time that HPV-18 E5, E6, and E7 oncoproteins can cooperate to enhance malignant transformation.

A novel form of Deleted in breast cancer 1 (DBC1) lacking the N-terminal domain does not bind SIRT1 and is dynamically regulated in vivo.

The protein Deleted in Breast Cancer-1 is a regulator of several transcription factors and epigenetic regulators, including HDAC3, Rev-erb-alpha, PARP1 and SIRT1. It is well known that DBC1 regulates its targets, including SIRT1, by protein-protein interaction. However, little is known about how DBC1 biological activity is regulated. In this work, we show that in quiescent cells DBC1 is proteolytically cleaved, producing a protein (DN-DBC1) that misses the S1-like domain and no longer binds to SIRT1. DN-DBC1 is also found in vivo in mouse and human tissues. Interestingly, DN-DBC1 is cleared once quiescent cells re-enter to the cell cycle. Using a model of liver regeneration after partial hepatectomy, we found that DN-DBC1 is down-regulated in vivo during regeneration. In fact, WT mice show a decrease in SIRT1 activity during liver regeneration, coincidentally with DN-DBC1 downregulation and the appearance of full length DBC1. This effect on SIRT1 activity was not observed in DBC1 KO mice. Finally, we found that DBC1 KO mice have altered cell cycle progression and liver regeneration after partial hepatectomy, suggesting that DBC1/DN-DBC1 transitions play a role in normal cell cycle progression in vivo after cells leave quiescence. We propose that quiescent cells express DN-DBC1, which either replaces or coexist with the full-length protein, and that restoring of DBC1 is required for normal cell cycle progression in vitro and in vivo. Our results describe for the first time in vivo a naturally occurring form of DBC1, which does not bind SIRT1 and is dynamically regulated, thus contributing to redefine the knowledge about its function.

Particles from the Echinococcus granulosus Laminated Layer Inhibit CD40 Upregulation in Dendritic Cells by Interfering with Akt Activation.

The larval stage of the cestode Echinococcus granulosus causes cystic echinococcosis in humans and livestock. This larva is protected by the millimeter-thick, mucin-based laminated layer (LL), from which materials have to be shed to allow parasite growth. We previously reported that dendritic cells (DCs) respond to microscopic pieces of the mucin gel of the LL (pLL) with unconventional maturation phenotypes, in the absence or presence of Toll-like receptor (TLR) agonists, including lipopolysaccharide (LPS). We also reported that the presence of pLL inhibited the activating phosphorylation of the phosphatidylinositol 3-kinase (PI3K) effector Akt induced by granulocyte-macrophage colony-stimulating factor or interleukin-4. We now show that the inhibitory effect of pLL extends to LPS as a PI3K activator, and results in diminished phosphorylation of GSK3 downstream from Akt. Functionally, the inhibition of Akt and GSK3 phosphorylation are linked to the blunted upregulation of CD40, a major feature of the unconventional maturation phenotype. Paradoxically, all aspects of unconventional maturation induced by pLL depend on PI3K class I. Additional components of the phagocytic machinery are needed, but phagocytosis of pLL particles is not required. These observations hint at a DC response mechanism related to receptor-independent mechanisms proposed for certain crystalline and synthetic polymer-based particles; this would fit the previously reported lack of detection of molecular-level motifs necessary of the effects of pLL on DCs. Finally, we report that DCs exposed to pLL are able to condition DCs not exposed to the material so that these cannot upregulate CD40 in full in response to LPS.

Mitofusins modulate the increase in mitochondrial length, bioenergetics and secretory phenotype in therapy-induced senescent melanoma cells.

Cellular senescence is an endpoint of chemotherapy, and targeted therapies in melanoma and the senescence-associated secretory phenotype (SASP) can affect tumor growth and microenvironment, influencing treatment outcomes. Metabolic interventions can modulate the SASP, and an enhanced mitochondrial energy metabolism supports resistance to therapy in melanoma cells. Herein, we assessed the mitochondrial function of therapy-induced senescent melanoma cells obtained after exposing the cells to temozolomide (TMZ), a methylating chemotherapeutic agent. Senescence induction in melanoma was accompanied by a substantial increase in mitochondrial basal, ATP-linked, and maximum respiration rates and in coupling efficiency, spare respiratory capacity, and respiratory control ratio. Further examinations revealed an increase in mitochondrial mass and length. Alterations in mitochondrial function and morphology were confirmed in isolated senescent cells, obtained by cell-size sorting. An increase in mitofusin 1 and 2 (MFN1 and 2) expression and levels was observed in senescent cells, pointing to alterations in mitochondrial fusion. Silencing mitofusin expression with short hairpin RNA (shRNA) prevented the increase in mitochondrial length, oxygen consumption rate and secretion of interleukin 6 (IL-6), a component of the SASP, in melanoma senescent cells. Our results represent the first in-depth study of mitochondrial function in therapy-induced senescence in melanoma. They indicate that senescence increases mitochondrial mass, length and energy metabolism; and highlight mitochondria as potential pharmacological targets to modulate senescence and the SASP.

Respiratory analysis of coupled mitochondria in cryopreserved liver biopsies.

The aim of this work was to develop a cryopreservation method of small liver biopsies for in situ mitochondrial function assessment. Herein we describe a detailed protocol for tissue collection, cryopreservation, high-resolution respirometry using complex I and II substrates, calculation and interpretation of respiratory parameters. Liver biopsies from cow and rat were sequentially frozen in a medium containing dimethylsulfoxide as cryoprotectant and stored for up to 3 months at -80 °C. Oxygen consumption rate studies of fresh and cryopreserved samples revealed that most respiratory parameters remained unchanged. Additionally, outer mitochondrial membrane integrity was assessed adding cytochrome c, proving that our cryopreservation method does not harm mitochondrial structure. In sum, we present a reliable way to cryopreserve small liver biopsies without affecting mitochondrial function. Our protocol will enable the transport and storage of samples, extending and facilitating mitochondrial function analysis of liver biopsies.

Interplay between oxidant species and energy metabolism.

It has long been recognized that energy metabolism is linked to the production of reactive oxygen species (ROS) and critical enzymes allied to metabolic pathways can be affected by redox reactions. This interplay between energy metabolism and ROS becomes most apparent during the aging process and in the onset and progression of many age-related diseases (i.e. diabetes, metabolic syndrome, atherosclerosis, neurodegenerative diseases). As such, the capacity to identify metabolic pathways involved in ROS formation, as well as specific targets and oxidative modifications is crucial to our understanding of the molecular basis of age-related diseases and for the design of novel therapeutic strategies. Herein we review oxidant formation associated with the cell's energetic metabolism, key antioxidants involved in ROS detoxification, and the principal targets of oxidant species in metabolic routes and discuss their relevance in cell signaling and age-related diseases.

Oncogene-induced senescence results in marked metabolic and bioenergetic alterations.

Oncogene-induced senescence (OIS) is characterized by permanent growth arrest and the acquisition of a secretory, pro-inflammatory state. Increasingly, OIS is viewed as an important barrier to tumorgenesis. Surprisingly, relatively little is known about the metabolic changes that accompany and therefore may contribute to OIS. Here, we have performed a metabolomic and bioenergetic analysis of Ras-induced senescence. Profiling approximately 300 different intracellular metabolites reveals that cells that have undergone OIS develop a unique metabolic signature that differs markedly from cells undergoing replicative senescence. A number of lipid metabolites appear uniquely increased in OIS cells, including a marked increase in the level of certain intracellular long chain fatty acids. Functional studies reveal that this alteration in the metabolome reflects substantial changes in overall lipid metabolism. In particular, Ras-induced senescent cells manifest a decline in lipid synthesis and a significant increase in fatty acid oxidation. Increased fatty acid oxidation results in an unexpectedly high rate of basal oxygen consumption in cells that have undergone OIS. Pharmacological or genetic inhibition of carnitine palmitoyltransferase 1, the rate-limiting step in mitochondrial fatty acid oxidation, restores a pre-senescent metabolic rate and, surprisingly, selectively inhibits the secretory, pro-inflammatory state that accompanies OIS. Thus, Ras-induced senescent cells demonstrate profound alterations in their metabolic and bioenergetic profiles, particularly with regards to the levels, synthesis and oxidation of free fatty acids. Furthermore, the inflammatory phenotype that accompanies OIS appears to be related to these underlying changes in cellular metabolism.

Thiol-sensitive mutant forms of human SOD2, L60F, and I58T: the role of Cys140.

We previously described a coding mutation (L60F) in the mitochondrial superoxide dismutase (SOD2) gene of the human T cell leukemia-derived cell line Jurkat. In cell extracts the L60F mutant enzyme showed unusual inhibition by thiol reagents not seen in wild-type enzyme. Here we compare the properties of purified recombinant L60F SOD2 with a previously described SOD2 mutant, I58T. Both mutant proteins display a weakened dimer-dimer interaction and thermal instability at 55 degrees C. Both I58T and L60F lose activity at 37 degrees C in the presence of 5 mM N-ethylmaleimide, whereas the wild-type SOD2 does not. Each subunit contains one exposed, reactive cysteine residue at position 196 and a second cysteine residue at 140, which is buried and unreactive in the wild-type tetramer. We propose that the mutant enzymes, which exist largely as dimers, allow both cysteine residues to react with thiol reagents. When the cysteine residue at 140 was changed to serine by site-directed mutagenesis, both double mutants I58T/C140S and L60F/C140S lost their increased thiol sensitivity. The evolutionary significance of Cys140 is discussed.

Bmi1 regulates mitochondrial function and the DNA damage response pathway.

Mice deficient in the Polycomb repressor Bmi1 develop numerous abnormalities including a severe defect in stem cell self-renewal, alterations in thymocyte maturation and a shortened lifespan. Previous work has implicated de-repression of the Ink4a/Arf (also known as Cdkn2a) locus as mediating many of the aspects of the Bmi1(-/-) phenotype. Here we demonstrate that cells derived from Bmi1(-/-) mice also have impaired mitochondrial function, a marked increase in the intracellular levels of reactive oxygen species and subsequent engagement of the DNA damage response pathway. Furthermore, many of the deficiencies normally observed in Bmi1(-/-) mice improve after either pharmacological treatment with the antioxidant N-acetylcysteine or genetic disruption of the DNA damage response pathway by Chk2 (also known as Chek2) deletion. These results demonstrate that Bmi1 has an unexpected role in maintaining mitochondrial function and redox homeostasis and indicate that the Polycomb family of proteins can coordinately regulate cellular metabolism with stem and progenitor cell function.

Prevention of peroxynitrite-induced apoptosis of motor neurons and PC12 cells by tyrosine-containing peptides.

Although peroxynitrite stimulates apoptosis in many cell types, whether peroxynitrite acts directly as an oxidant or the induction of apoptosis is because of the radicals derived from peroxynitrite decomposition remains unknown. Before undergoing apoptosis because of trophic factor deprivation, primary motor neuron cultures become immunoreactive for nitrotyrosine. We show here using tyrosine-containing peptides that free radical processes mediated by peroxynitrite decomposition products were required for triggering apoptosis in primary motor neurons and in PC12 cells cultures. The same concentrations of tyrosine-containing peptides required to prevent the nitration and apoptosis of motor neurons induced by trophic factor deprivation and of PC12 cells induced by peroxynitrite also prevented peroxynitrite-mediated nitration of motor neurons, brain homogenates, and PC12 cells. The heat shock protein 90 chaperone was nitrated in both trophic factor-deprived motor neurons and PC12 cells incubated with peroxynitrite. Tyrosine-containing peptides did not affect the induction of PC12 cell death by hydrogen peroxide. Tyrosine-containing peptides should protect by scavenging peroxynitrite-derived radicals and not by direct reactions with peroxynitrite as they neither increase the rate of peroxynitrite decomposition nor decrease the bimolecular peroxynitrite-mediated oxidation of thiols. These results reveal an important role for free radical-mediated nitration of tyrosine residues, in apoptosis induced by endogenously produced and exogenously added peroxynitrite; moreover, tyrosine-containing peptides may offer a novel strategy to neutralize the toxic effects of peroxynitrite.

Tyrosine nitration by superoxide and nitric oxide fluxes in biological systems: modeling the impact of superoxide dismutase and nitric oxide diffusion.

Tyrosine nitration is a posttranslational modification observed in many pathologic states that can be associated with peroxynitrite (ONOO(-)) formation. However, in vitro, peroxynitrite-dependent tyrosine nitration is inhibited when its precursors, superoxide (O(2)*(-)) and nitric oxide ((*)NO), are formed at ratios (O(2)*(-)/(*)NO) different from one, severely questioning the use of 3-nitrotyrosine as a biomarker of peroxynitrite-mediated oxidations. We herein hypothesize that in biological systems the presence of superoxide dismutase (SOD) and the facile transmembrane diffusion of (*)NO preclude accumulation of O(2)*(-) and (*)NO radicals under flux ratios different from one, preventing the secondary reactions that result in the inhibition of 3-nitrotyrosine formation. Using an array of reactions and kinetic constants, computer-assisted simulations were performed in order to assess the flux of 3-nitrotyrosine formation (J(NO(2(-))Y)) during exposure to simultaneous fluxes of superoxide (J(O(2)*(-))) and nitric oxide (J((*)NO)), varying the radical flux ratios (J(O(2)*(-))/ J((*)NO)), in the presence of carbon dioxide. With a basic set of reactions, J(NO(2(-))Y) as a function of radical flux ratios rendered a bell-shape profile, in complete agreement with previous reports. However, when superoxide dismutation by SOD and (*)NO decay due to diffusion out of the compartment were incorporated in the model, a quite different profile of J(NO(2(-))Y) as a function of the radical flux ratio was obtained: despite the fact that nitration yields were much lower, the bell-shape profile was lost and the extent of tyrosine nitration was responsive to increases in either O(2)*(-) or (*)NO, in agreement with in vivo observations. Thus, the model presented herein serves to reconcile the in vitro and in vivo evidence on the role of peroxynitrite in promoting tyrosine nitration.

Peroxynitrite reactions and formation in mitochondria.

Mitochondria constitute a primary locus for the intracellular formation and reactions of peroxynitrite, and these interactions are recognized to contribute to the biological and pathological effects of both nitric oxide ((*)NO) and peroxynitrite. Extra- or intramitochondrially formed peroxynitrite can diffuse through mitochondrial compartments and undergo fast direct and free radical-dependent target molecule reactions. These processes result in oxidation, nitration, and nitrosation of critical components in the matrix, inner and outer membrane, and intermembrane space. Mitochondrial scavenging and repair systems for peroxynitrite-dependent oxidative modifications operate but they can be overwhelmed under enhanced cellular (*)NO formation as well as under conditions that lead to augmented superoxide formation by the electron transport chain. Peroxynitrite can lead to alterations in mitochondrial energy and calcium homeostasis and promote the opening of the permeability transition pore. The effects of peroxynitrite in mitochondrial physiology can be largely rationalized based on the reactivities of peroxynitrite and peroxynitrite-derived carbonate, nitrogen dioxide, and hydroxyl radicals with critical protein amino acids and transition metal centers of key mitochondrial proteins. In this review we analyze (i) the existing evidence for the intramitochondrial formation and reactions of peroxynitrite, (ii) the key reactions and fate of peroxynitrite in mitochondria, and (iii) their impact in mitochondrial physiology and signaling of cell death.