In yeast, cardiolipin unsaturation level plays a key role in mitochondrial function and inner membrane integrity.

Cardiolipin is the signature phospholipid of the mitochondrial inner membrane. It participates in shaping the inner membrane as well as in modulating the activity of many membrane-bound proteins. The acyl chain composition of cardiolipin is finely tuned post-biosynthesis depending on the surrounding phospholipids to produce mature or unsaturated cardiolipin. However, experimental evidence showing that immature and mature cardiolipin are functionally equivalents for mitochondria poses doubts on the relevance of cardiolipin remodeling. In this work, we studied the role of cardiolipin acyl chain composition in mitochondrial bioenergetics, including a detailed bioenergetic profile of yeast mitochondria. Cardiolipin acyl chains were modified by genetic and nutritional manipulation. We found that both the bioenergetic efficiency and osmotic stability of mitochondria are dependent on the unsaturation level of cardiolipin acyl chains. It is proposed that cardiolipin remodeling and, consequently, mature cardiolipins play an important role in mitochondrial inner membrane integrity and functionality.

Cytochrome c modification and oligomerization induced by cardiolipin hydroperoxides in a membrane mimetic model.

Cytochrome c (cytc) is a heme protein of 12 kDa that transfers electrons in the mitochondrial respiratory chain. Increased cytc peroxidase activity leads to cardiolipin (CL) oxidation, a hallmark of early apoptosis stage. Here, we aimed to investigate the interaction between cytc with cardiolipin hydroperoxide (CLOOH) in a mimetic mitochondrial membrane. Cytc-CL peroxidase reaction occurred at faster rates with CLOOH than with H2O2. Moreover, liposomes containing CLOOH promoted increased protein aggregation with minor or no release of cytc from the membrane. Dimeric and trimeric cytc species were observed in the first 15 min, followed by increased formation of high-molecular-weight aggregates afterwards. nLC-MS/MS analysis identified several Lys and His residues covalently modified by lipid aldehydes that showed mass increments corresponding to 4-hydroxynonenal (HNE), 4-oxononenal (ONE), hexanoyl, heptenal and octenal addition. Noteworthy, most modifications were observed at Lys and His residues located at A-site (K73, K87, K88), L-site (H26, H33, and K27) membrane binding sites. Further, dityrosine cross-linked peptides were also characterized at residues Y48-Y74, Y48-Y97 and Y74-Y97. Collectively, our findings show that CLOOH causes irreversible protein damage and crosslinking of cytc in the membrane.

Alterations in lipid metabolism of spinal cord linked to amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is characterized by progressive loss of upper and lower motor neurons leading to muscle paralysis and death. While a link between dysregulated lipid metabolism and ALS has been proposed, lipidome alterations involved in disease progression are still understudied. Using a rodent model of ALS overexpressing mutant human Cu/Zn-superoxide dismutase gene (SOD1-G93A), we performed a comparative lipidomic analysis in motor cortex and spinal cord tissues of SOD1-G93A and WT rats at asymptomatic (~70 days) and symptomatic stages (~120 days). Interestingly, lipidome alterations in motor cortex were mostly related to age than ALS. In contrast, drastic changes were observed in spinal cord of SOD1-G93A 120d group, including decreased levels of cardiolipin and a 6-fold increase in several cholesteryl esters linked to polyunsaturated fatty acids. Consistent with previous studies, our findings suggest abnormal mitochondria in motor neurons and lipid droplets accumulation in aberrant astrocytes. Although the mechanism leading to cholesteryl esters accumulation remains to be established, we postulate a hypothetical model based on neuroprotection of polyunsaturated fatty acids into lipid droplets in response to increased oxidative stress. Implicated in the pathology of other neurodegenerative diseases, cholesteryl esters appear as attractive targets for further investigations.

Lipidomic Analysis Reveals Serum Alteration of Plasmalogens in Patients Infected With ZIKA Virus.

Zika virus (ZIKV) is an arthropod-borne virus (arbovirus) in the Flavivirus genus of the Flaviviridae family. Since the large outbreaks in French Polynesia in 2013-2014 and in Brazil in 2015, ZIKV has been considered a new public health threat. Similar to other related flavivirus, ZIKV is associated with mild and self-limiting symptoms such as rash, pruritus, prostration, headache, arthralgia, myalgia, conjunctivitis, lower back pain and, when present, a short-term low grade fever. In addition, ZIKV has been implicated in neurological complications such as neonatal microcephaly and Guillain-Barré syndrome in adults. Herein, serum lipidomic analysis was used to identify possible alterations in lipid metabolism triggered by ZIKV infection. Patients who presented virus-like symptoms such as fever, arthralgia, headache, exanthema, myalgia and pruritus were selected as the control group. Our study reveals increased levels of several phosphatidylethanolamine (PE) lipid species in the serum of ZIKV patients, the majority of them plasmenyl-phosphatidylethanolamine (pPE) (or plasmalogens) linked to polyunsaturated fatty acids. Constituting up to 20% of total phospholipids in humans, plasmalogens linked to polyunsaturated fatty acids are particularly enriched in neural membranes of the brain. The biosynthesis of plasmalogens requires functional peroxisomes, which are important sites for viral replication, including ZIKV. Thus, increased levels of plasmalogens in serum of ZIKV infected subjects suggest a link between ZIKV life cycle and peroxisomes. Our data provide important insights into specific host cellular lipids that are likely associated with ZIKV replication and may serve as platform for antiviral strategy against ZIKV.

Exploring the role of lipids in protein modification and amyotrophic lateral sclerosis / Explorando o papel dos lipídeos na modificação de proteínas e esclerose lateral amiotrófica

Lipids are a diverse and ubiquitous group of compounds, which have several biological functions such as structural components of cell membranes, energy storage, and participation in signaling pathways. Free radicals or reactive oxygen species could attack polyunsaturated fatty acid esterified to phospholipids generating oxidized products. Once oxidized, lipids are able to modify amino acids residues in proteins leading to modulation signaling pathways and cellular redox balance. Furthermore, alteration of lipid homeostasis is also linked to development and progression of neurodegenerative diseases. The purposes of this study were (i) to investigate the role of lipids in protein aggregation, (ii) to investigate the plasma lipidome of an ALS rat model (SOD1G93A rats), and (iii) to investigate the effect of high-fat diet in plasma lipidome of an ALS rat model. In chapters 1 and 2, the interaction between cytochrome c (cytc) and cardiolipin hydroperoxide (CLOOH), as well as cholesterol hydroperoxide (ChOOH) promoted protein aggregation. Mass spectrometry analysis of tryptic peptides from CLOOH-containing reaction revealed K72 and H26 consistently modified by 4- hydroxynonenal (4-HNE). Further, adduction of K27, K73 and K88 were detected with 4- oxynonenal (4-ONE). For the first time, we characterized the dityrosine cross-linked peptides at Y48-Y74, Y48-97 and Y74-Y97 in oligomeric cytc. Similarly, ChOOH-containing reaction showed dityrosine cross-linked peptides at Y48-Y48, Y48-Y74 and Y48-Y97 in dimeric cytc. In accordance to previous studies, the proposed mechanism under covalent protein oligomerization mediated by lipid hydroperoxide could be related to modification of lysine and tyrosine residues. In chapter 3, we characterized the lipid composition of blood plasma in amyotrophic lateral sclerosis (ALS), since dysregulation of lipid metabolism is increasingly associated with neuropathology. Using untargeted lipidomics approach based on liquid chromatography coupled to mass spectrometry, we found main alterations in triglycerides, phospholipids and sphingolipids in symptomatic ALS rats relative to controls. Additionally, for the first time we reported acylceramides species in the plasma. In order to investigate the source of these lipid alterations, we analyzed the lipid content of fractioned lipoproteins. Triglycerides and phospholipids were found in very low-density lipoprotein (VLDL), while acylceramides and hexosylceramides were found enriched in high-density lipoprotein (HDL). In chapter 4, high-fat diet containing lard or high-fish oil as much as 60% of total lipids has both the largest change on plasma lipid composition. Overall survival was not statistically different when compared to control diet. Increased levels of acylceramides, hexosylceramides and acylcarnitines were observed in ALS rats fed a control diet or high-fat diet in comparison to WT controls. Importantly, untargeted lipidomic analysis of blood plasma highlighted acylceramide d18:1/24:1+20:4 as potential biomarkers of ALS progression. Thus, our lipidomic analysis provides a novel insight into the molecular level event driving molecular dysregulation in ALS. Additional research is needed to determine the effect of plasma lipid alteration on motor neuron process and energetic metabolism. Collectively, our findings reinforce the idea that lipids play a relevant role in modulating cellular processes linked to protein aggregation and neurodegeneration

Os lipídeos são moléculas que possuem várias funções biológicas importantes, atuando como componente de membranas celulares, servindo com fonte de reserva de energia e participando de vias de sinalização. Os ácidos graxos poli-insaturados esterificados aos fosfolipídeos, por exemplo, são potenciais alvos para o ataque de radicais livres gerando produtos oxidados que são capazes de modificar resíduos de aminoácidos em proteínas levando a modulação das vias de sinalização e balanço redox. Por outro lado, alteração na homeostase do metabolismo dos lipídeos está relacionada ao desenvolvimento e progressão de doenças neurodegenerativas. Tendo em vista a importância dos lipídeos nos processos biológicos, os objetivos desse estudo foram (i) investigar o papel dos lipídeos na agregação proteica (capítulo 1 e 2), (ii) investigar as alterações na composição lipídica do plasma de rato modelo SOD1G93A de esclerose lateral amiotrófica (ELA) (capítulo 3) e (iii) investigar o efeito da suplementação de dietas hiperlipídicas na composição lipídica do plasma de rato modelo SOD1G93A (capítulo 4). No capítulo 1 e 2, a interação do citocromo c (citc) com hidroperóxido de cardiolipina (CLOOH) e hidroperóxido de colesterol (ChOOH) promove a agregação covalente do citc. Análise por nLC-MS/MS dos peptídeos digeridos identificou resíduos de lisina (K72) e histidina (H26) modificado por 4-hidroxininenal (4-HNE), enquanto os resíduos K27, K73 e K88 foram modificados por 4-oxinonenal (4-ONE). Pela primeira vez, nós caracterizamos ditirosinas (Y48-Y74, Y48-97 e Y74-Y97) na reação do citc com CLOOH. Também foram caracterizadas ditirosinas envolvendo os resíduos Y48-Y48, Y48-Y74 e Y48-Y97 na reação com ChOOH. Esses resultados corroboram com estudos anteriores que sugerem um mecanismo de agregação proteica envolvendo a perda da carga positiva de lisina e formação de ditirosina pela combinação de radicais de tirosil. No capítulo 3, a análise da composição lipídica do plasma de ratos SOD1G93A utilizando LC-MS/MS revelou alterações significativas na composição de triglicérides, glicerofosfolipídeos e esfingolipídeos em ratos sintomáticos comparado com os assintomáticos. É importante destacar que pela primeira vez acilceramidas foram identificadas em plasma de rato modelo para ALS. Análise da composição lipídica de lipoproteínas isoladas, maior fonte de lipídeos circulantes no plasma, mostraram alterações de triglicérides e glicerofosfolipídeos em VLDL. As acilceramidas e as hexosilceramidas, por sua vez, foram encontradas em maior abundância em HDL. No capítulo 4, a suplementação com dietas hiperlipídicas (rica em banha de porco e óleo de peixe) alterou significativamente o perfil lipídico do plasma em relação a doença. Contudo, não foi observado aumento significativo na sobrevida dos ratos ALS comparado com dieta controle. Independente da dieta, a concentração plasmática de acilcarnitina, hexosilceramidas e acilceramidas foram significativamente aumentadas em ratos ALS comparado com WT. A análise do perfil lipídico do plasma mostrou que a acilceramida d18:1/24:1+20:4 pode ser um potencial marcador de progressão da ALS. Dessa forma, os resultados mostrados fornecem uma visão enriquecedora sobre o evento a nível molecular que conduz a desregulação lipídica na ELA. Coletivamente, nossos resultados reforçam a importância dos lipídeos na modulação dos processos celulares ligados a agregação de proteínas e na neurodegeneração