70 results on '"Nathan N. Alder"'
Search Results
2. Interactions of amyloidogenic proteins with mitochondrial protein import machinery in aging-related neurodegenerative diseases
- Author
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Ashley L. Reed, Wayne Mitchell, Andrei T. Alexandrescu, and Nathan N. Alder
- Subjects
mitochondria ,amyloids ,neurodegeneration ,protein import ,cryptic targeting ,targeting signals ,Physiology ,QP1-981 - Abstract
Most mitochondrial proteins are targeted to the organelle by N-terminal mitochondrial targeting sequences (MTSs, or “presequences”) that are recognized by the import machinery and subsequently cleaved to yield the mature protein. MTSs do not have conserved amino acid compositions, but share common physicochemical properties, including the ability to form amphipathic α-helical structures enriched with basic and hydrophobic residues on alternating faces. The lack of strict sequence conservation implies that some polypeptides can be mistargeted to mitochondria, especially under cellular stress. The pathogenic accumulation of proteins within mitochondria is implicated in many aging-related neurodegenerative diseases, including Alzheimer’s, Parkinson’s, and Huntington’s diseases. Mechanistically, these diseases may originate in part from mitochondrial interactions with amyloid-β precursor protein (APP) or its cleavage product amyloid-β (Aβ), α-synuclein (α-syn), and mutant forms of huntingtin (mHtt), respectively, that are mediated in part through their associations with the mitochondrial protein import machinery. Emerging evidence suggests that these amyloidogenic proteins may present cryptic targeting signals that act as MTS mimetics and can be recognized by mitochondrial import receptors and transported into different mitochondrial compartments. Accumulation of these mistargeted proteins could overwhelm the import machinery and its associated quality control mechanisms, thereby contributing to neurological disease progression. Alternatively, the uptake of amyloidogenic proteins into mitochondria may be part of a protein quality control mechanism for clearance of cytotoxic proteins. Here we review the pathomechanisms of these diseases as they relate to mitochondrial protein import and effects on mitochondrial function, what features of APP/Aβ, α-syn and mHtt make them suitable substrates for the import machinery, and how this information can be leveraged for the development of therapeutic interventions.
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- 2023
- Full Text
- View/download PDF
3. Phosphatidylethanolamine made in the inner mitochondrial membrane is essential for yeast cytochrome bc 1 complex function
- Author
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Elizabeth Calzada, Erica Avery, Pingdewinde N. Sam, Arnab Modak, Chunyan Wang, J. Michael McCaffery, Xianlin Han, Nathan N. Alder, and Steven M. Claypool
- Subjects
Science - Abstract
Phosphatidylethanolamine (PE) is synthesized by four separate pathways, although surprisingly, perturbing mitochondrial PE synthesis compromises mitochondrial function. Here, the authors show that mitochondrial PE synthesis is required for Complex III function and challenge PE trafficking dogma.
- Published
- 2019
- Full Text
- View/download PDF
4. Structure-activity relationships of mitochondria-targeted tetrapeptide pharmacological compounds
- Author
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Wayne Mitchell, Jeffrey D Tamucci, Emery L Ng, Shaoyi Liu, Alexander V Birk, Hazel H Szeto, Eric R May, Andrei T Alexandrescu, and Nathan N Alder
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mitochondria ,peptide therapeutics ,membrane interactions ,NMR structure ,structure-activity relationship ,cardiolipin ,Medicine ,Science ,Biology (General) ,QH301-705.5 - Abstract
Mitochondria play a central role in metabolic homeostasis, and dysfunction of this organelle underpins the etiology of many heritable and aging-related diseases. Tetrapeptides with alternating cationic and aromatic residues such as SS-31 (elamipretide) show promise as therapeutic compounds for mitochondrial disorders. In this study, we conducted a quantitative structure-activity analysis of three alternative tetrapeptide analogs, benchmarked against SS-31, that differ with respect to aromatic side chain composition and sequence register. We present the first structural models for this class of compounds, obtained with Nuclear Magnetic Resonance (NMR) and molecular dynamics approaches, showing that all analogs except for SS-31 form compact reverse turn conformations in the membrane-bound state. All peptide analogs bound cardiolipin-containing membranes, yet they had significant differences in equilibrium binding behavior and membrane interactions. Notably, analogs had markedly different effects on membrane surface charge, supporting a mechanism in which modulation of membrane electrostatics is a key feature of their mechanism of action. The peptides had no strict requirement for side chain composition or sequence register to permeate cells and target mitochondria in mammalian cell culture assays. All four peptides were pharmacologically active in serum withdrawal cell stress models yet showed significant differences in their abilities to restore mitochondrial membrane potential, preserve ATP content, and promote cell survival. Within our peptide set, the analog containing tryptophan side chains, SPN10, had the strongest impact on most membrane properties and showed greatest efficacy in cell culture studies. Taken together, these results show that side chain composition and register influence the activity of these mitochondria-targeted peptides, helping provide a framework for the rational design of next-generation therapeutics with enhanced potency.
- Published
- 2022
- Full Text
- View/download PDF
5. Reduction of elevated proton leak rejuvenates mitochondria in the aged cardiomyocyte
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Huiliang Zhang, Nathan N Alder, Wang Wang, Hazel Szeto, David J Marcinek, and Peter S Rabinovitch
- Subjects
aging ,mitochondria ,SS-31 ,proton leak ,cardiomyocyte ,Medicine ,Science ,Biology (General) ,QH301-705.5 - Abstract
Aging-associated diseases, including cardiac dysfunction, are increasingly common in the population. However, the mechanisms of physiologic aging in general, and cardiac aging in particular, remain poorly understood. Age-related heart impairment is lacking a clinically effective treatment. Using the model of naturally aging mice and rats, we show direct evidence of increased proton leak in the aged heart mitochondria. Moreover, our data suggested ANT1 as the most likely site of mediating increased mitochondrial proton permeability in old cardiomyocytes. Most importantly, the tetra-peptide SS-31 prevents age-related excess proton entry, decreases the mitochondrial flash activity and mitochondrial permeability transition pore opening, rejuvenates mitochondrial function by direct association with ANT1 and the mitochondrial ATP synthasome, and leads to substantial reversal of diastolic dysfunction. Our results uncover the excessive proton leak as a novel mechanism of age-related cardiac dysfunction and elucidate how SS-31 can reverse this clinically important complication of cardiac aging.
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- 2020
- Full Text
- View/download PDF
6. Conserved cardiolipin-mitochondrial ADP/ATP carrier interactions assume distinct structural and functional roles that are clinically relevant
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Nanami Senoo, Dinesh K. Chinthapalli, Matthew G. Baile, Vinaya K. Golla, Bodhisattwa Saha, Oluwaseun B. Ogunbona, James A. Saba, Teona Munteanu, Yllka Valdez, Kevin Whited, Dror Chorev, Nathan N. Alder, Eric R. May, Carol V. Robinson, and Steven M. Claypool
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Article - Abstract
SummaryThe mitochondrial phospholipid cardiolipin (CL) promotes bioenergetics via oxidative phosphorylation (OXPHOS). Three tightly bound CLs are evolutionarily conserved in the ADP/ATP carrier (AAC in yeast; adenine nucleotide translocator, ANT in mammals) which resides in the inner mitochondrial membrane and exchanges ADP and ATP to enable OXPHOS. Here, we investigated the role of these buried CLs in the carrier using yeast Aac2 as a model. We introduced negatively charged mutations into each CL-binding site of Aac2 to disrupt the CL interactions via electrostatic repulsion. While all mutations disturbing the CL-protein interaction destabilized Aac2 monomeric structure, transport activity was impaired in a pocket-specific manner. Finally, we determined that a disease-associated missense mutation in one CL-binding site in ANT1 compromised its structure and transport activity, resulting in OXPHOS defects. Our findings highlight the conserved significance of CL in AAC/ANT structure and function, directly tied to specific lipid-protein interactions.
- Published
- 2023
7. Associations of Mitochondrial Function, Stress, and Neurodevelopmental Outcomes in Early Life: A Systematic Review
- Author
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Tingting Zhao, Nathan N. Alder, Angela R. Starkweather, Ming-Hui Chen, Adam P. Matson, Wanli Xu, Jeremy L. Balsbaugh, and Xiaomei Cong
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Developmental Neuroscience ,Neurology - Abstract
Early life stress is commonly experienced by infants, especially preterm infants, and may impact their neurodevelopmental outcomes in their early and later lives. Mitochondrial function/dysfunction may play an important role underlying the linkage of prenatal and postnatal stress and neurodevelopmental outcomes in infants. This review aimed to provide insights on the relationship between early life stress and neurodevelopment and the mechanisms of mitochondrial function/dysfunction that contribute to the neuropathology of stress. The Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) statement was used to develop this systematic review. PubMed, Scopus, PsycINFO, and Biosis databases were searched for primary research articles published between 2010 and 2021 that examined the relationships among mitochondrial function/dysfunction, infant stress, and neurodevelopment. Thirty studies were identified. There is evidence to support that mitochondrial function/dysfunction mediates the relationship between prenatal and postnatal stress and neurodevelopmental outcomes in infants. Maternal transgenerational transmission of mitochondrial bioenergetic patterns influenced prenatal stress induced neurodevelopmental outcomes and behavioral changes in infants. Multiple functionally relevant mitochondrial proteins, genes, and polymorphisms were associated with stress exposure. This is the first review of the role that mitochondrial function/dysfunction plays in the association between stress and neurodevelopmental outcomes in full-term and preterm infants. Although multiple limitations were found based on the lack of data on the influence of biological sex, and due to invasive sampling, and lack of longitudinal data, many genes and proteins associated with mitochondrial function/dysfunction were found to influence neurodevelopmental outcomes in the early life of infants.
- Published
- 2022
8. In organello real-time NMR metabolomics of mitochondria: An S. cerevisiae model of Barth syndrome displays perturbed metabolism
- Author
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Antonio J. Rua, Wayne Mitchell, Nathan N. Alder, and Andrei T. Alexandrescu
- Subjects
Biophysics - Published
- 2023
9. The mitochondria-targeted peptide SS-31 binds lipid bilayers and modulates surface electrostatics as a key component of its mechanism of action
- Author
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Wayne Mitchell, Nicholas A. Eddy, Xianlin Han, Jeffrey D. Tamucci, Murugappan Sathappa, Eric R. May, Kevin J. Boyd, Nathan N. Alder, Hazel H. Szeto, Adrian Coscia, Emily A. Ng, and Meixia Pan
- Subjects
0301 basic medicine ,Lipid Bilayers ,Static Electricity ,Phospholipid ,Peptide ,Saccharomyces cerevisiae ,Mitochondrion ,bioenergetics ,Biochemistry ,lipid structure ,03 medical and health sciences ,chemistry.chemical_compound ,elamipretide ,Cardiolipin ,drug action ,Szeto-Schiller peptide ,Lipid bilayer ,Molecular Biology ,peptide therapeutic ,inner membrane ,chemistry.chemical_classification ,030102 biochemistry & molecular biology ,SS-31 ,Cell Biology ,Elamipretide ,electrostatics ,Mitochondria ,030104 developmental biology ,Membrane ,chemistry ,peptides ,Biophysics ,Calcium ,cardiolipin ,Oligopeptides ,membrane biophysics ,Membrane biophysics ,Molecular Biophysics - Abstract
Mitochondrial dysfunction underlies many heritable diseases, acquired pathologies, and aging-related declines in health. Szeto–Schiller (SS) peptides comprise a class of amphipathic tetrapeptides that are efficacious toward a wide array of mitochondrial disorders and are believed to target mitochondrial membranes because they are enriched in the anionic phospholipid cardiolipin (CL). However, little is known regarding how SS peptides interact with or alter the physical properties of lipid bilayers. In this study, using biophysical and computational approaches, we have analyzed the interactions of the lead compound SS-31 (elamipretide) with model and mitochondrial membranes. Our results show that this polybasic peptide partitions into the membrane interfacial region with an affinity and a lipid binding density that are directly related to surface charge. We found that SS-31 binding does not destabilize lamellar bilayers even at the highest binding concentrations; however, it did cause saturable alterations in lipid packing. Most notably, SS-31 modulated the surface electrostatics of both model and mitochondrial membranes. We propose nonexclusive mechanisms by which the tuning of surface charge could underpin the mitoprotective properties of SS-31, including alteration of the distribution of ions and basic proteins at the interface, and/or modulation of bilayer physical properties. As a proof of concept, we show that SS-31 alters divalent cation (calcium) distribution within the interfacial region and reduces the energetic burden of calcium stress in mitochondria. The mechanistic details of SS-31 revealed in this study will help inform the development of future compound variants with enhanced efficacy and bioavailability.
- Published
- 2020
10. Decision letter: Towards a molecular mechanism underlying mitochondrial protein import through the TOM and TIM23 complexes
- Author
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Raffaele Ieva and Nathan N Alder
- Published
- 2021
11. Structure-activity relationships of mitochondria-targeted tetrapeptide pharmacological compounds
- Author
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Wayne Mitchell, Jeffrey D Tamucci, Emery L Ng, Shaoyi Liu, Alexander V Birk, Hazel H Szeto, Eric R May, Andrei T Alexandrescu, and Nathan N Alder
- Subjects
Mammals ,Structure-Activity Relationship ,Mitochondrial Diseases ,General Immunology and Microbiology ,Cardiolipins ,General Neuroscience ,Animals ,Humans ,General Medicine ,Peptides ,General Biochemistry, Genetics and Molecular Biology ,Mitochondria - Abstract
Mitochondria play a central role in metabolic homeostasis, and dysfunction of this organelle underpins the etiology of many heritable and aging-related diseases. Tetrapeptides with alternating cationic and aromatic residues such as SS-31 (elamipretide) show promise as therapeutic compounds for mitochondrial disorders. In this study, we conducted a quantitative structure-activity analysis of three alternative tetrapeptide analogs, benchmarked against SS-31, that differ with respect to aromatic side chain composition and sequence register. We present the first structural models for this class of compounds, obtained with Nuclear Magnetic Resonance (NMR) and molecular dynamics approaches, showing that all analogs except for SS-31 form compact reverse turn conformations in the membrane-bound state. All peptide analogs bound cardiolipin-containing membranes, yet they had significant differences in equilibrium binding behavior and membrane interactions. Notably, analogs had markedly different effects on membrane surface charge, supporting a mechanism in which modulation of membrane electrostatics is a key feature of their mechanism of action. The peptides had no strict requirement for side chain composition or sequence register to permeate cells and target mitochondria in mammalian cell culture assays. All four peptides were pharmacologically active in serum withdrawal cell stress models yet showed significant differences in their abilities to restore mitochondrial membrane potential, preserve ATP content, and promote cell survival. Within our peptide set, the analog containing tryptophan side chains, SPN10, had the strongest impact on most membrane properties and showed greatest efficacy in cell culture studies. Taken together, these results show that side chain composition and register influence the activity of these mitochondria-targeted peptides, helping provide a framework for the rational design of next-generation therapeutics with enhanced potency.
- Published
- 2021
12. Structure-Activity Relationships in the Design of Mitochondria-Targeted Peptide Therapeutics
- Author
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Andrei T. Alexandrescu, Wayne Mitchell, Eric R. May, Nathan N. Alder, Hazel H. Szeto, Jeffrey D. Tamucci, Emery L. Ng, and Shaoyi Liu
- Subjects
Turn (biochemistry) ,chemistry.chemical_classification ,Membrane ,Mechanism of action ,Tetrapeptide ,Chemistry ,Organelle ,medicine ,Biophysics ,Rational design ,Peptide ,medicine.symptom ,Elamipretide - Abstract
Mitochondria play a central role in metabolic homeostasis; hence, dysfunction of this organelle underpins the etiology of many heritable and aging-related diseases. Mitochondria-targeted tetrapeptides with alternating cationic and aromatic residues, such as SS-31 (Elamipretide), show promise as therapeutic compounds. In this study, we conducted a quantitative structure-activity analysis of three alternative tetrapeptide analogs that differed with respect to aromatic side chain composition and sequence register, benchmarked against SS-31. Using NMR and molecular dynamics approaches, we obtained the first structural models for this class of compounds, showing that all analogs except for SS-31 form compact reverse turn conformations in the membrane-bound state. All peptide analogs bound cardiolipin-containing membranes, yet they had significant differences in equilibrium binding behavior and membrane interactions. Notably, the analogs had markedly different effects on membrane surface charge, supporting a mechanism in which modulation of membrane electrostatics is a key feature of their mechanism of action. All peptide analogs preserved survival and energy metabolism more effectively than SS-31 in cell stress models. Within our peptide set, the analog containing tryptophan side chains, SPN10, had the strongest impact on most membrane properties and showed greatest efficacy in cell culture studies. Taken together, these results show that side chain composition and register strongly influence the activity of these mitochondria-targeted peptides. Furthermore, this work helps provide a framework for the rational design of next-generation therapeutics with enhanced potency.
- Published
- 2021
13. Astaxanthin attenuates the increase in mitochondrial respiration during the activation of hepatic stellate cells
- Author
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Seung-Hyun Hong, Pujan Joshi, Minkyung Bae, Sung I. Koo, Young-Ki Park, Ji-Young Lee, Dong-Guk Shin, Yoojin Lee, and Nathan N. Alder
- Subjects
0301 basic medicine ,Endocrinology, Diabetes and Metabolism ,Clinical Biochemistry ,Mitochondria, Liver ,Xanthophylls ,DNA, Mitochondrial ,Biochemistry ,Article ,Transforming Growth Factor beta1 ,Extracellular matrix ,03 medical and health sciences ,chemistry.chemical_compound ,0302 clinical medicine ,Astaxanthin ,Respiration ,Hepatic Stellate Cells ,medicine ,Extracellular ,Animals ,Humans ,Glycolysis ,Molecular Biology ,Cells, Cultured ,Liver injury ,Nutrition and Dietetics ,Chemistry ,medicine.disease ,Cell biology ,Mice, Inbred C57BL ,030104 developmental biology ,Gene Expression Regulation ,030220 oncology & carcinogenesis ,Cell Transdifferentiation ,Hepatic stellate cell ,Energy Metabolism ,Flux (metabolism) - Abstract
Upon liver injury, quiescent hepatic stellate cells (qHSCs) transdifferentiate to myofibroblast-like activated HSCs (aHSCs), which are primarily responsible for the accumulation of extracellular matrix proteins during the development of liver fibrosis. Therefore, aHSCs may exhibit different energy metabolism from that of qHSCs to meet their high energy demand. We previously demonstrated that astaxanthin (ASTX), a xanthophyll carotenoid, prevents the activation of HSCs. The objective of this study was to determine if ASTX can exert its antifibrogenic effect by attenuating any changes in energy metabolism during HSC activation. To characterize the energy metabolism of qHSCs and aHSCs, mouse primary HSCs were cultured on uncoated plastic dishes for 7 days for spontaneous activation in the presence or absence of 25 μM ASTX. qHSCs (1 day after isolation) and aHSCs treated with or without ASTX for 7 days were used to determine parameters related to mitochondrial respiration using a Seahorse XFe24 Extracellular Flux analyzer. aHSCs had significantly higher basal respiration, maximal respiration, ATP production, spare respiratory capacity and proton leak than those of qHSCs. However, ASTX prevented most of the changes occurring during HSC activation and improved mitochondrial cristae structure with decreased cristae junction width, lumen width and the area in primary mouse aHSCs. Furthermore, qHSCs isolated from ASTX-fed mice had lower mitochondrial respiration and glycolysis than control qHSCs. Our findings suggest that ASTX may exert its antifibrogenic effect by attenuating the changes in energy metabolism during HSC activation.
- Published
- 2019
14. Phosphatidylethanolamine made in the inner mitochondrial membrane is essential for yeast cytochrome bc 1 complex function
- Author
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J. Michael McCaffery, Steven M. Claypool, Arnab Modak, Chunyan Wang, Elizabeth Calzada, Erica Avery, Pingdewinde N. Sam, Xianlin Han, and Nathan N. Alder
- Subjects
0301 basic medicine ,Cytochrome ,Protein subunit ,Science ,General Physics and Astronomy ,02 engineering and technology ,Mitochondrion ,General Biochemistry, Genetics and Molecular Biology ,03 medical and health sciences ,chemistry.chemical_compound ,Endomembrane system ,Inner mitochondrial membrane ,lcsh:Science ,Phosphatidylethanolamine ,Multidisciplinary ,biology ,Chemistry ,General Chemistry ,021001 nanoscience & nanotechnology ,Cell biology ,030104 developmental biology ,Coenzyme Q – cytochrome c reductase ,biology.protein ,lcsh:Q ,0210 nano-technology ,Phosphatidylserine decarboxylase - Abstract
Of the four separate PE biosynthetic pathways in eukaryotes, one occurs in the mitochondrial inner membrane (IM) and is executed by phosphatidylserine decarboxylase (Psd1). Deletion of Psd1 is lethal in mice and compromises mitochondrial function. We hypothesize that this reflects inefficient import of non-mitochondrial PE into the IM. Here, we test this by re-wiring PE metabolism in yeast by re-directing Psd1 to the outer mitochondrial membrane or the endomembrane system and show that PE can cross the IMS in both directions. Nonetheless, PE synthesis in the IM is critical for cytochrome bc1 complex (III) function and mutations predicted to disrupt a conserved PE-binding site in the complex III subunit, Qcr7, impair complex III activity similar to PSD1 deletion. Collectively, these data challenge the current dogma of PE trafficking and demonstrate that PE made in the IM by Psd1 support the intrinsic functionality of complex III. Phosphatidylethanolamine (PE) is synthesized by four separate pathways, although surprisingly, perturbing mitochondrial PE synthesis compromises mitochondrial function. Here, the authors show that mitochondrial PE synthesis is required for Complex III function and challenge PE trafficking dogma.
- Published
- 2019
15. Mitochondrial compartmentalization: emerging themes in structure and function
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Joseph C. Iovine, Steven M. Claypool, and Nathan N. Alder
- Subjects
Mitochondrial Proteins ,Mitochondrial structure ,Spatial segregation ,Evolutionary biology ,Mitochondrial Membranes ,Morphogenesis ,Mitochondrion ,Compartmentalization (psychology) ,Biology ,Molecular Biology ,Biochemistry ,Structure and function ,Mitochondria - Abstract
Within cellular structures, compartmentalization is the concept of spatial segregation of macromolecules, metabolites, and biochemical pathways. Therefore, this concept bridges organellar structure and function. Mitochondria are morphologically complex, partitioned into several subcompartments by a topologically elaborate two-membrane system. They are also dynamically polymorphic, undergoing morphogenesis events with an extent and frequency that is only now being appreciated. Thus, mitochondrial compartmentalization is something that must be considered both spatially and temporally. Here, we review new developments in how mitochondrial structure is established and regulated, the factors that underpin the distribution of lipids and proteins, and how they spatially demarcate locations of myriad mitochondrial processes. Consistent with its pre-eminence, disturbed mitochondrial compartmentalization contributes to the dysfunction associated with heritable and aging-related diseases.
- Published
- 2021
16. Author response: Reduction of elevated proton leak rejuvenates mitochondria in the aged cardiomyocyte
- Author
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Peter S. Rabinovitch, Huiliang Zhang, Wang Wang, Nathan N. Alder, David J. Marcinek, and Hazel H. Szeto
- Subjects
Leak ,Proton ,Chemistry ,medicine.medical_treatment ,medicine ,Biophysics ,Mitochondrion ,Reduction (orthopedic surgery) - Published
- 2020
17. Characterization of styrene-maleic acid copolymer membrane solubilization using reductionist and mitochondrial systems
- Author
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Joseph C. Iovine, Steven M. Claypool, and Nathan N. Alder
- Subjects
Biophysics - Published
- 2022
18. QSAR investigation into SS peptides' interaction with lipid bilayers and their effects on transmembrane ion leakage in the presence of transmembrane potentials
- Author
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Jeffrey D. Tamucci, Wayne Mitchell, Emery L. Ng, Shaoyi Liu, Hazel H. Szeto, Andrei T. Alexandrescu, Nathan N. Alder, and Eric R. May
- Subjects
Biophysics - Published
- 2022
19. Investigating the efficacy of mitochondria-targeted therapeutics in barth syndrome models
- Author
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Emery L. Ng, Nicholas A. Eddy, Hazel H. Szeto, and Nathan N. Alder
- Subjects
Biophysics - Published
- 2022
20. Abstract 250: Acting Instructor
- Author
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David J. Marcinek, Hazel H. Szeto, Wang Wang, Huiliang Zhang, Nathan N. Alder, and Peter S. Rabinovitch
- Subjects
Physiology ,Cardiology and Cardiovascular Medicine - Abstract
Rational: Aging-associated diseases, including cardiac dysfunction, are increasingly common in the population. However, the mechanisms of physiologic aging in general, and cardiac aging in particular, remain poorly understood. While effective medical interventions are available for some kinds of heart failure, one age-related impairment, diastolic dysfunction in Heart Failure with Preserved Ejection Fraction (HFpEF) is lacking a clinically effective treatment. Methods and Results: Using the pH indicator cpYFP in the model of naturally aging mice and rats, we show direct evidence of increased mitochondrial proton leak in aged heart mitochondria following a pH gradient stress. Furthermore, we identified Adenine Nucleotide Translocator 1 (ANT1) as mediating the increased proton permeability of old cardiomyocytes. Most importantly, acute (2 hours) in vitro treatment with the tetra-peptide drug SS-31 (elamipretide) reverses age-related excess proton entry, decreases the mitochondrial flash activity and mitochondrial permeability transition pore (mPTP) opening and rejuvenates mitochondrial function. Moreover, we show that SS-31 benefits the old mitochondria by direct association with ANT1 and stabilization of the mitochondrial ATP synthasome, leading to substantial reversal of diastolic dysfunction. Conclusion: Our results uncover excessive mitochondrial proton leak as a novel mechanism of age-related cardiac dysfunction and elucidate how SS-31 is able to reverse this clinically important complication of cardiac aging.
- Published
- 2020
21. Reduction of Elevated Proton Leak Rejuvenates Mitochondria in the Aged Cardiomyocyte
- Author
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David J. Marcinek, Wang Wang, Peter S. Rabinovitch, Hazel H. Szeto, Huiliang Zhang, and Nathan N. Alder
- Subjects
0301 basic medicine ,Leak ,Health (social science) ,Mouse ,cardiomyocyte ,Mitochondrion ,Health Professions (miscellaneous) ,Mitochondria, Heart ,Membrane Potentials ,Abstracts ,chemistry.chemical_compound ,Adenosine Triphosphate ,0302 clinical medicine ,Medicine ,Myocytes, Cardiac ,Biology (General) ,AcademicSubjects/SOC02600 ,Cells, Cultured ,Cellular Senescence ,0303 health sciences ,education.field_of_study ,SS-31 ,General Neuroscience ,MPTP ,030302 biochemistry & molecular biology ,Age Factors ,General Medicine ,Hydrogen-Ion Concentration ,3. Good health ,mitochondria ,Cardiology ,Protons ,Oligopeptides ,Research Article ,medicine.medical_specialty ,QH301-705.5 ,Science ,Population ,Diastole ,Mice, Transgenic ,General Biochemistry, Genetics and Molecular Biology ,Cardiac dysfunction ,03 medical and health sciences ,Internal medicine ,Animals ,Effective treatment ,Life-span and Life-course Studies ,education ,030304 developmental biology ,Session 3023 (Paper) ,General Immunology and Microbiology ,Mitochondrial Permeability Transition Pore ,business.industry ,aging ,Adenine Nucleotide Translocator 1 ,Cell Biology ,Elamipretide ,medicine.disease ,Rats, Inbred F344 ,Cardiovascular Health and Disease (paper) ,Mice, Inbred C57BL ,030104 developmental biology ,Endocrinology ,Mitochondrial permeability transition pore ,chemistry ,Heart failure ,Rat ,Energy Metabolism ,Reactive Oxygen Species ,business ,Heart failure with preserved ejection fraction ,030217 neurology & neurosurgery ,proton leak - Abstract
Aging-associated diseases, including cardiac dysfunction, are increasingly common in the population. However, the mechanisms of physiologic aging in general, and cardiac aging in particular, remain poorly understood. While effective medical interventions are available for some kinds of heart failure, one age-related impairment, diastolic dysfunction in Heart Failure with Preserved Ejection Fraction (HFpEF) is lacking a clinically effective treatment. Using the model of naturally aging mice and rats, we show direct evidence of increased proton leak in the aged heart mitochondria. Moreover, we identified ANT1 as mediating the increased proton permeability of old cardiomyocytes. Most importantly, the tetra-peptide drug SS-31 (elamipretide) prevents age-related excess proton entry, decreases the mitochondrial flash activity and mitochondrial permeability transition pore (mPTP) opening and rejuvenates mitochondrial function by direct association with ANT1 and the mitochondrial ATP synthasome. Our results uncover a novel mechanism of age-related cardiac dysfunction and elucidate how SS-31 is able to reverse this clinically important complication of cardiac aging. Significance Aging is the greatest risk factor for cardiac dysfunction, including Heart Failure with Preserved Ejection Fraction (HFpEF). Unfortunately, the mechanisms of cardiac aging remain elusive, and there are no effective pharmacologic therapies for HFpEF. Here, we show direct evidence of increased proton leak in aged cardiac mitochondria and have identified ANT1 as mediating the increased proton permeability of old cardiomyocytes. Moreover, the mitochondrial-targeted tetra-peptide SS-31 (elamipretide) prevents the age-related excess proton entry and rejuvenates mitochondrial function by direct association with ANT1 and the mitochondrial ATP synthasome, resulting in alleviation of diastolic dysfunction in old mice. Our results unmask a novel mechanism of cardiac aging and elucidate how SS-31 reverses this clinically important complication of aging.
- Published
- 2020
22. Molecular Mechanism of Action of Mitochondrial Therapeutic SS-31 (Elamipretide): Membrane Interactions and Effects on Surface Electrostatics
- Author
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Jeffrey D. Tamucci, Emily A. Ng, Xianlin Han, Nicholas A. Eddy, Kevin J. Boyd, Eric R. May, Nathan N. Alder, Hazel H. Szeto, Meixia Pan, Adrian Coscia, Wayne Mitchell, and Murugappan Sathappa
- Subjects
0303 health sciences ,Mitochondrial disease ,Phospholipid ,Mitochondrion ,Elamipretide ,medicine.disease ,03 medical and health sciences ,chemistry.chemical_compound ,0302 clinical medicine ,Membrane ,Mechanism of action ,chemistry ,medicine ,Cardiolipin ,Biophysics ,medicine.symptom ,Lipid bilayer ,030217 neurology & neurosurgery ,030304 developmental biology - Abstract
Mitochondrial dysfunction includes heritable diseases, acquired pathologies, and age-related declines in health. Szeto-Schiller (SS) peptides comprise a class of amphipathic tetrapeptides that have demonstrated efficacy in treating a wide array of mitochondrial disorders, and are believed to target mitochondrial membranes due to their enrichment in the anionic phospholipid cardiolipin (CL). However, little is known regarding how SS peptides interact with or alter the physical properties of lipid bilayers. In this study, we have analyzed the interactions of the lead compound SS-31 (Elamipretide) with model and mitochondrial membranes using biophysical and computational approaches. Our results show that this polybasic peptide partitions into the membrane interfacial region with affinity and binding density that are directly related to surface charge. SS-31 binding does not destabilize lamellar bilayers even at the highest binding concentrations; however, it does cause saturable alterations in lipid packing. Most notably, SS-31 modulates the surface electrostatic properties of model and mitochondrial membranes, which could play a significant role in the mitoprotective properties of this compound. As a proof of concept, we show that SS-31 alters ion distribution at the membrane interface with implications for maintaining mitochondrial membranes subject to divalent cation (calcium) stress. Taken together, these results support a mechanism of action in which SS peptides interact with lipid bilayers and alter the biophysical (primarily electrostatic) properties of mitochondrial membranes as their primary mechanism of action. Understanding this molecular mechanism is key to the development of future compound variants with enhanced efficacy.SignificanceSzeto-Schiller (SS) peptides are among the most promising therapeutic compounds for mitochondrial dysfunction. However, the molecular target(s) and the mechanism of action of SS peptides are poorly understood. In this study, we evaluate the interaction of the lead compound SS-31 (Elamipretide) with mitochondrial and synthetic model membranes using a host of biophysical techniques. Our results show that SS-31 membrane interaction is driven largely by the negative surface charge of mitochondrial membranes and that SS-31 alters lipid bilayer properties, most notably electrostatics at the membrane interface. This work supports a mechanism in which SS peptides act on a key physical property of mitochondrial membranes rather than with a specific protein complex, consistent with the exceptionally broad therapeutic efficacy of these compounds.
- Published
- 2019
23. Abstract 287: Altered Mitochondrial Flash Activity and mPTP Opening in the Aged Heart Are Reversed by Elamipretide Treatment
- Author
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Hazel H. Szeto, Huiliang Zhang, Nathan N. Alder, and Peter S. Rabinovitch
- Subjects
medicine.medical_specialty ,chemistry.chemical_compound ,Flash (photography) ,Endocrinology ,chemistry ,Physiology ,Internal medicine ,MPTP ,medicine ,Elamipretide ,Cardiology and Cardiovascular Medicine - Abstract
Rationale: The mitochondrial theory of aging pinpoints mitochondria as the major reactive oxygen species (ROS) production site and target of oxidative stress during aging. Mitochondrial flash (mitoflash) is recently discovered excitable event that is a manifestation of ROS production combined with pH change and coupled with mitochondrial permeability transition pore (mPTP) opening. Our preliminary data shown that 8-week SS-31 peptide (elamipretide) protects the functional performance of the aged mouse heart. This study aims to investigate mitoflash activity and mPTP opening in the aged heart, especially whether and how SS-31 protects the heart function involving modulation of these activities. Methods and Results: By using confocal microscopy imaging on the isolated rat cardiomyocytes with overexpression of mitochondrial targeted cpYFP, we found mitoflash activity in the cells from old (26 mo) was higher than that in young (5 mo) rat cells (2.2 ± 0.2 in old vs 1.3 ± 0.2 in young, /1000μm 2 /100s, n=27-64, pin vitro treatment of the old cardiomyocytes with SS-31 100 nm decreased proton leak. Moreover, SS-31 (100 nM) decreased the superoxide production as measured by ratio of MitoSOX Red to MitoTrackerGreen. Conclusion: These results indicate that SS-31 directly protects cardiac aging through rapid rejuvenation of mitochondrial respiration in cardiomyocytes, and in particular, by reducing proton leak, mitochondrial flash activity, and decreasing the mPTP opening. This study helps to uncover the mechanism of the cardiac aging and the protective effect from SS-31 treatment.
- Published
- 2019
24. Fluorescence Spectroscopy and Its Applications in Analysing Biomolecular Processes
- Author
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Nathan N. Alder
- Subjects
chemistry.chemical_compound ,Fluorophore ,Quenching (fluorescence) ,Chemistry ,Energy transfer ,Steady state (chemistry) ,Anisotropy ,Photochemistry ,Spectroscopy ,Fluorescence ,Fluorescence spectroscopy - Published
- 2019
25. Computational Investigation into the Effect of SS-31 on Membrane Ion Distributions, Pore Formation and Ion Leakage in the Presence of Transmembrane Potentials
- Author
-
Eric R. May, Jeffrey D. Tamucci, and Nathan N. Alder
- Subjects
Membrane ,Materials science ,Biophysics ,Transmembrane protein ,Ion ,Leakage (electronics) - Published
- 2021
26. The ionization properties of cardiolipin and its variants in model bilayers
- Author
-
Murugappan Sathappa and Nathan N. Alder
- Subjects
0301 basic medicine ,Cardiolipins ,Lipid Bilayers ,Static Electricity ,Phospholipid ,Biophysics ,Biochemistry ,Article ,03 medical and health sciences ,chemistry.chemical_compound ,Cardiolipin ,Inner mitochondrial membrane ,Lipid bilayer ,Ions ,030102 biochemistry & molecular biology ,Bilayer ,Cell Biology ,Hydrogen-Ion Concentration ,030104 developmental biology ,Membrane ,Models, Chemical ,chemistry ,Membrane curvature ,lipids (amino acids, peptides, and proteins) - Abstract
The anionic phospholipid cardiolipin has an unusual dimeric structure with a two-phosphate headgroup and four acyl chains. Cardiolipin is present in energy-transducing membranes that maintain electrochemical gradients, including most bacterial plasma membranes and the mitochondrial inner membrane, where it mediates respiratory complex assembly and activation, among many other roles. Dysfunctional biogenesis of cardiolipin is implicated in the pathogenesis of several diseases including Barth syndrome. Because cardiolipin is a dominant anionic lipid in energy-conserving membranes, its headgroup is a major contributor to surface charge density and the bilayer electrostatic profile. However, the proton dissociation behavior of its headgroup remains controversial. In one model, the pKa values of the phosphates differ by several units and the headgroup exists as a monoanion at physiological pH. In another model, both phosphates ionize as strong acids with low pKa values and the headgroup exists in dianionic form at physiological pH. Using independent electrokinetic and spectroscopic approaches, coupled with analysis using Gouy–Chapman–Stern formalism, we have analyzed the ionization properties of cardiolipin within biologically relevant lipid bilayer model systems. We show that both phosphates of the cardiolipin headgroup show strong ionization behavior with low pKa values. Moreover, cardiolipin variants lacking structural features proposed to be required to maintain disparate pKa values – namely the secondary hydroxyl on the central glycerol or a full complement of four acyl chains – were shown to have ionization behavior identical to intact cardiolipin. Hence, these results indicate that within the physiological pH range, the cardiolipin headgroup is fully ionized as a dianion. We discuss the implications of these results with respect to the role of cardiolipin in defining membrane surface potential, activating respiratory complexes, and modulating membrane curvature.
- Published
- 2016
- Full Text
- View/download PDF
27. Functional Assembly of the Mitochondrial Protein Transport Machinery
- Author
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Pablo M. Peixoto, Nathan N. Alder, and Arnab Modak
- Subjects
Chemistry ,Genetics ,Molecular Biology ,Biochemistry ,Mitochondrial protein ,Biotechnology ,Cell biology - Published
- 2020
28. Phosphatidylethanolamine made in the inner mitochondrial membrane is essential for yeast cytochrome bc
- Author
-
Elizabeth, Calzada, Erica, Avery, Pingdewinde N, Sam, Arnab, Modak, Chunyan, Wang, J Michael, McCaffery, Xianlin, Han, Nathan N, Alder, and Steven M, Claypool
- Subjects
Electron Transport Complex IV ,Electron Transport Complex III ,Saccharomyces cerevisiae Proteins ,Phosphatidylethanolamines ,Mitochondrial Membranes ,Mutation ,Reproducibility of Results ,Saccharomyces cerevisiae ,Endoplasmic Reticulum ,Aerobiosis ,Article - Abstract
Of the four separate PE biosynthetic pathways in eukaryotes, one occurs in the mitochondrial inner membrane (IM) and is executed by phosphatidylserine decarboxylase (Psd1). Deletion of Psd1 is lethal in mice and compromises mitochondrial function. We hypothesize that this reflects inefficient import of non-mitochondrial PE into the IM. Here, we test this by re-wiring PE metabolism in yeast by re-directing Psd1 to the outer mitochondrial membrane or the endomembrane system and show that PE can cross the IMS in both directions. Nonetheless, PE synthesis in the IM is critical for cytochrome bc1 complex (III) function and mutations predicted to disrupt a conserved PE-binding site in the complex III subunit, Qcr7, impair complex III activity similar to PSD1 deletion. Collectively, these data challenge the current dogma of PE trafficking and demonstrate that PE made in the IM by Psd1 support the intrinsic functionality of complex III., Phosphatidylethanolamine (PE) is synthesized by four separate pathways, although surprisingly, perturbing mitochondrial PE synthesis compromises mitochondrial function. Here, the authors show that mitochondrial PE synthesis is required for Complex III function and challenge PE trafficking dogma.
- Published
- 2018
29. Biophysical Approaches Toward Understanding the Molecular Mechanism of Action of the Mitochondrial Therapeutic SS-31 (Elamipretide)
- Author
-
Emily A. Ng, Nathan N. Alder, Jeffrey D. Tamucci, Nicholas A. Eddy, Eric R. May, Kevin J. Boyd, Hazel H. Szeto, and Wayne Mitchell
- Subjects
Action (philosophy) ,Chemistry ,Biophysics ,Molecular mechanism ,Elamipretide ,Neuroscience - Published
- 2019
30. Molecular Dynamics Analysis of Cardiolipin and Monolysocardiolipin on Bilayer Properties
- Author
-
Eric R. May, Kevin J. Boyd, and Nathan N. Alder
- Subjects
0301 basic medicine ,Membranes ,Chemistry ,Hydrogen bond ,Cardiolipins ,Monolysocardiolipin ,Bilayer ,Lipid Bilayers ,Biophysics ,Molecular Conformation ,Barth syndrome ,Molecular Dynamics Simulation ,medicine.disease ,Protein–protein interaction ,03 medical and health sciences ,chemistry.chemical_compound ,Molecular dynamics ,030104 developmental biology ,Cardiolipin ,medicine ,Lysophospholipids ,Inner mitochondrial membrane - Abstract
The mitochondrial lipid cardiolipin (CL) contributes to the spatial protein organization and morphological character of the inner mitochondrial membrane. Monolysocardiolipin (MLCL), an intermediate species in the CL remodeling pathway, is enriched in the multisystem disease Barth syndrome. Despite the medical relevance of MLCL, a detailed molecular description that elucidates the structural and dynamic differences between CL and MLCL has not been conducted. To this end, we performed comparative atomistic molecular dynamics studies on bilayers consisting of pure CL or MLCL to elucidate similarities and differences in their molecular and bulk bilayer properties. We describe differential headgroup dynamics and hydrogen bonding patterns between the CL variants and show an increased cohesiveness of MLCL's solvent interfacial region, which may have implications for protein interactions. Finally, using the coarse-grained Martini model, we show that substitution of MLCL for CL in bilayers mimicking mitochondrial composition induces drastic differences in bilayer mechanical properties and curvature-dependent partitioning behavior. Together, the results of this work reveal differences between CL and MLCL at the molecular and mesoscopic levels that may underpin the pathomechanisms of defects in cardiolipin remodeling.
- Published
- 2017
31. Cardiolipin mediates membrane and channel interactions of the mitochondrial TIM23 protein import complex receptor Tim50
- Author
-
Ketan Malhotra, Shivangi Nangia, Tyler H. Daman, Arnab Modak, Victoria L. Robinson, Umut Günsel, Eric R. May, Nathan N. Alder, and Dejana Mokranjac
- Subjects
Models, Molecular ,0301 basic medicine ,Cardiolipins ,Protein Conformation ,Protein subunit ,Lipid Bilayers ,Gene Expression ,Plasma protein binding ,Biology ,Mitochondrial Membrane Transport Proteins ,Models, Biological ,Biochemistry ,Structure-Activity Relationship ,03 medical and health sciences ,chemistry.chemical_compound ,Protein structure ,Cardiolipin ,Receptor ,Lipid bilayer ,Research Articles ,Multidisciplinary ,Cell Membrane ,SciAdv r-articles ,Life Sciences ,Biological Transport ,Biological membrane ,Recombinant Proteins ,Mitochondria ,Transport protein ,Cell biology ,030104 developmental biology ,chemistry ,Proteolysis ,lipids (amino acids, peptides, and proteins) ,Protein Binding ,Research Article - Abstract
Cardiolipin mediates dynamic receptor-channel interactions within the mitochondrial TIM23 protein import complex., The phospholipid cardiolipin mediates the functional interactions of proteins that reside within energy-conserving biological membranes. However, the molecular basis by which this lipid performs this essential cellular role is not well understood. We address this role of cardiolipin using the multisubunit mitochondrial TIM23 protein transport complex as a model system. The early stages of protein import by this complex require specific interactions between the polypeptide substrate receptor, Tim50, and the membrane-bound channel-forming subunit, Tim23. Using analyses performed in vivo, in isolated mitochondria, and in reductionist nanoscale model membrane systems, we show that the soluble receptor domain of Tim50 interacts with membranes and with specific sites on the Tim23 channel in a manner that is directly modulated by cardiolipin. To obtain structural insights into the nature of these interactions, we obtained the first small-angle x-ray scattering-based structure of the soluble Tim50 receptor in its entirety. Using these structural insights, molecular dynamics simulations combined with a range of biophysical measurements confirmed the role of cardiolipin in driving the association of the Tim50 receptor with lipid bilayers with concomitant structural changes, highlighting the role of key structural elements in mediating this interaction. Together, these results show that cardiolipin is required to mediate specific receptor-channel associations in the TIM23 complex. Our results support a new working model for the dynamic structural changes that occur within the complex during transport. More broadly, this work strongly advances our understanding of how cardiolipin mediates interactions among membrane-associated proteins.
- Published
- 2017
32. Buckling Under Pressure: Curvature-Based Lipid Segregation and Stability Modulation in Cardiolipin-Containing Bilayers
- Author
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Kevin J. Boyd, Eric R. May, and Nathan N. Alder
- Subjects
0301 basic medicine ,Cardiolipins ,Lipid Bilayers ,Molecular Dynamics Simulation ,Article ,03 medical and health sciences ,chemistry.chemical_compound ,Molecular dynamics ,Electrochemistry ,Cardiolipin ,Pressure ,General Materials Science ,Lipid bilayer phase behavior ,Lipid bilayer ,Inner mitochondrial membrane ,Spectroscopy ,Phosphatidylethanolamine ,Bilayer ,Proteins ,Surfaces and Interfaces ,Condensed Matter Physics ,Crystallography ,030104 developmental biology ,chemistry ,Membrane curvature ,Mitochondrial Membranes ,Biophysics ,lipids (amino acids, peptides, and proteins) - Abstract
Mitochondrial metabolic function is affected by the morphology and protein organization of the mitochondrial inner membrane. Cardiolipin (CL) is a unique tetra-acyl lipid that is involved in the maintenance of the highly curved shape of the mitochondrial inner membrane as well as spatial organization of the proteins necessary for respiration and oxidative phosphorylation. Cardiolipin has been suggested to self-organize into lipid domains due to its inverted conical molecular geometry, though the driving forces for this organization are not fully understood. In this work, we use coarse-grained molecular dynamics simulations to study the mechanical properties and lipid dynamics in heterogeneous bilayers both with and without CL, as a function of membrane curvature. We find that incorporation of CL increases bilayer deformability and that CL becomes highly enriched in regions of high negative curvature. We further show that another mitochondrial inverted conical lipid, phosphatidylethanolamine (PE), does not partition or increase the deformability of the membrane in a significant manner. Therefore, CL appears to possess some unique characteristics that cannot be inferred simply from molecular geometry considerations.
- Published
- 2017
33. Reconstitution of Mitochondrial Membrane Proteins into Nanodiscs by Cell-Free Expression
- Author
-
Ketan Malhotra and Nathan N. Alder
- Subjects
0301 basic medicine ,Scaffold protein ,Lipid Bilayers ,Synthetic membrane ,In Vitro Techniques ,Article ,Workflow ,03 medical and health sciences ,Lipid bilayer ,Inner mitochondrial membrane ,Nanodisc ,Triticum ,030102 biochemistry & molecular biology ,Cell-Free System ,Chemistry ,Membrane Proteins ,Lipid metabolism ,Nanostructures ,030104 developmental biology ,Membrane protein ,Mitochondrial Membrane Protein ,Protein Biosynthesis ,Mitochondrial Membranes ,Biophysics ,lipids (amino acids, peptides, and proteins) ,Protein Binding - Abstract
The isolation and characterization of mitochondrial membrane proteins is technically challenging because they natively reside within the specialized environment of the lipid bilayer, an environment that must be recapitulated to some degree during reconstitution to ensure proper folding, stability, and function. Here we describe protocols for the assembly of a membrane protein into lipid bilayer nanodiscs in a series of cell-free reactions. Cell-free expression of membrane proteins circumvents problems attendant with in vivo expression such as cytotoxicity, low expression levels, and the formation of inclusion bodies. Nanodiscs are artificial membrane systems comprised of discoidal lipid bilayer particles bound by annuli of amphipathic scaffold protein that shield lipid acyl chains from water. They are therefore excellent platforms for membrane protein reconstitution and downstream solution-based biochemical and biophysical analysis. This chapter details the procedures for the reconstitution of a mitochondrial membrane protein into nanodiscs using two different types of approaches: cotranslational and posttranslational assembly. These strategies are broadly applicable for different mitochondrial membrane proteins. They are also applicable for the use of nanodiscs with distinct lipid compositions that are biomimetic for different mitochondrial membranes and that recapitulate lipid profiles associated with pathological disorders in lipid metabolism.
- Published
- 2017
34. Mitochondrial Ca2+ influx targets cardiolipin to disintegrate respiratory chain complex II for cell death induction
- Author
-
Stefan Grimm, Christine T. Schwall, Evangelos Pazarentzos, Christoph Datler, Ming-Shih Hwang, and Nathan N. Alder
- Subjects
Programmed cell death ,Cardiolipins ,Respiratory chain ,Mitochondrion ,Biology ,medicine.disease_cause ,chemistry.chemical_compound ,Organelle ,Cardiolipin ,medicine ,Humans ,Molecular Biology ,chemistry.chemical_classification ,Original Paper ,Reactive oxygen species ,Cell Death ,Electron Transport Complex II ,Cell Biology ,Mitochondria ,Cell biology ,Oxidative Stress ,chemistry ,Calcium ,Cell fractionation ,Reactive Oxygen Species ,Oxidative stress - Abstract
Massive Ca(2+) influx into mitochondria is critically involved in cell death induction but it is unknown how this activates the organelle for cell destruction. Using multiple approaches including subcellular fractionation, FRET in intact cells, and in vitro reconstitutions, we show that mitochondrial Ca(2+) influx prompts complex II of the respiratory chain to disintegrate, thereby releasing an enzymatically competent sub-complex that generates excessive reactive oxygen species (ROS) for cell death induction. This Ca(2+)-dependent dissociation of complex II is also observed in model membrane systems, but not when cardiolipin is replaced with a lipid devoid of Ca(2+) binding. Cardiolipin is known to associate with complex II and upon Ca(2+) binding coalesces into separate homotypic clusters. When complex II is deprived of this lipid, it disintegrates for ROS formation and cell death. Our results reveal Ca(2+) binding to cardiolipin for complex II disintegration as a pivotal step for oxidative stress and cell death induction.
- Published
- 2014
35. Unremodeled and Remodeled Cardiolipin Are Functionally Indistinguishable in Yeast
- Author
-
Nathan N. Alder, Xianlin Han, J. Michael McCaffery, Erin N. Pryce, Murugappan Sathappa, Ya-Wen Lu, Kevin Whited, Matthew G. Baile, and Steven M. Claypool
- Subjects
Saccharomyces cerevisiae Proteins ,Cardiolipins ,Membrane lipids ,Saccharomyces cerevisiae ,Oxidative phosphorylation ,Mitochondrion ,Biology ,Biochemistry ,chemistry.chemical_compound ,Biosynthesis ,Membrane Biology ,Cardiolipin ,medicine ,Humans ,Molecular Biology ,Barth syndrome ,Cell Biology ,biology.organism_classification ,medicine.disease ,Mitochondria ,chemistry ,Barth Syndrome ,Membrane biogenesis ,lipids (amino acids, peptides, and proteins) ,Acyltransferases ,Gene Deletion ,Transcription Factors - Abstract
After biosynthesis, an evolutionarily conserved acyl chain remodeling process generates a final highly homogeneous and yet tissue-specific molecular form of the mitochondrial lipid cardiolipin. Hence, cardiolipin molecules in different organisms, and even different tissues within the same organism, contain a distinct collection of attached acyl chains. This observation is the basis for the widely accepted paradigm that the acyl chain composition of cardiolipin is matched to the unique mitochondrial demands of a tissue. For this hypothesis to be correct, cardiolipin molecules with different acyl chain compositions should have distinct functional capacities, and cardiolipin that has been remodeled should promote cardiolipin-dependent mitochondrial processes better than its unremodeled form. However, functional disparities between different molecular forms of cardiolipin have never been established. Here, we interrogate this simple but crucial prediction utilizing the best available model to do so, Saccharomyces cerevisiae. Specifically, we compare the ability of unremodeled and remodeled cardiolipin, which differ markedly in their acyl chain composition, to support mitochondrial activities known to require cardiolipin. Surprisingly, defined changes in the acyl chain composition of cardiolipin do not alter either mitochondrial morphology or oxidative phosphorylation. Importantly, preventing cardiolipin remodeling initiation in yeast lacking TAZ1, an ortholog of the causative gene in Barth syndrome, ameliorates mitochondrial dysfunction. Thus, our data do not support the prevailing hypothesis that unremodeled cardiolipin is functionally distinct from remodeled cardiolipin, at least for the functions examined, suggesting alternative physiological roles for this conserved pathway.
- Published
- 2014
36. The Styrene-Maleic Acid Copolymer Extracts Active Complexes from Native Biomembranes
- Author
-
Ketan Malhotra, Ashley R. Long, Anthony Watts, Arlene D. Albert, Nathan N. Alder, and Catherine C. O'Brien
- Subjects
education.field_of_study ,Chemistry ,Population ,Biophysics ,equipment and supplies ,Transmembrane protein ,Membrane ,Biochemistry ,Membrane protein ,Amphiphile ,Copolymer ,education ,Lipid bilayer ,Inner mitochondrial membrane - Abstract
Amphipathic polymers have been widely used to maintain the solubility of membrane proteins and complexes following detergent solubilization. However, their ability to extract proteins directly from lipid bilayers has remained largely unexplored. Here we show that a copolymer composed of styrene and maleic acid pendant groups (SMA) extracts proteins form native membranes and reconstitutes them into polymer-bound lipoprotein particles. First, we found that the SMA copolymer disrupted the membranes of intact mitochondria in a concentration-dependent and saturable manner. This was evidenced by the collapse of the transmembrane electric field of the inner mitochondrial membrane and by the solubilization of mitochondrial membrane proteins, both of which were mediated by the SMA copolymer in a manner similar to that mediated by nonionic detergents. Second, following incubation of the SMA copolymer with mitochondrial membranes and chromatographic separation, we observed by transmission electron microscopy that the resulting polymer-bound particles were a monodisperse population of discoids. The dimensions of these particles were similar to those previously reported for particles derived from liposomes or proteoliposomes of synthetic phospholipids (Lipodisqs®). Finally, using mitochondrial respiratory Complex IV (cytochrome c oxidase) as a model enzyme, we demonstrate that the SMA copolymer can extract even large, multicomponent complexes from native lipid bilayers and maintain them in a fully functional state amenable to solution-based biophysical studies. This novel approach to membrane protein reconstitution obviates the requirement for detergents and is therefore better suited to preserving native annular lipids and protein stability in comparison with traditional solubilization techniques.
- Published
- 2016
37. The stability and activity of respiratory Complex II is cardiolipin-dependent
- Author
-
Victoria Greenwood, Nathan N. Alder, and Christine T. Schwall
- Subjects
Cardiolipins ,Phospholipid ,Biophysics ,Oxidative phosphorylation ,Biology ,Biochemistry ,03 medical and health sciences ,chemistry.chemical_compound ,0302 clinical medicine ,Enzyme Stability ,Cardiolipin ,Inner mitochondrial membrane ,Lipid bilayer ,POPC ,Nanodisc ,030304 developmental biology ,chemistry.chemical_classification ,0303 health sciences ,Reactive oxygen species ,Electron Transport Complex II ,ROS ,Phosphatidylglycerols ,Cell Biology ,3. Good health ,Mitochondria ,chemistry ,Complex II ,Reactive Oxygen Species ,030217 neurology & neurosurgery - Abstract
Respiratory Complex II of the mitochondrial inner membrane serves as a link between the tricarboxylic acid cycle and the electron transport chain. Complex II dysfunction has been implicated in a wide range of heritable mitochondrial diseases, including cancer, by a mechanism that likely involves the production of reactive oxygen species (ROS). Using Complex II enzymes reconstituted into nanoscale lipid bilayers (nanodiscs) with varying lipid composition, we demonstrate for the first time that the phospholipid environment, specifically the presence of cardiolipin, is critical for the assembly and enzymatic activity of the complex, as well as in the curtailment of ROS production.
- Published
- 2012
- Full Text
- View/download PDF
38. Lipid-Dependence of the Membrane Interactions of the Tim23 Channel Subunit of the Mitochondrial Protein Import Machinery
- Author
-
Kevin J. Boyd, Eric R. May, Nathan N. Alder, and Melissa K. Skoryk
- Subjects
Vesicle-associated membrane protein 8 ,Membrane ,Chemistry ,Protein subunit ,Translocase of the inner membrane ,Biophysics ,Mitochondrial protein import ,Mitochondrion ,Intermembrane space ,Inner mitochondrial membrane ,Cell biology - Abstract
The TIM23 complex of the mitochondrial inner membrane mediates the translocation and integration of most nuclear-encoded polypeptides that reside within the mitochondrion. The central subunit of this complex is Tim23, a voltage-gated channel-forming protein that constitutes part of the polypeptide-conducting channel. Tim23 has a bipartite domain organization with a carboxy-terminal integral membrane domain and an amino-terminal intrinsically disordered region that resides in the intermembrane space (IMS).
- Published
- 2017
39. Tyrosine Phosphorylation as a Conformational Switch
- Author
-
Nahum Meller, Olga Vinogradova, Tatiana V. Byzova, Lalit Deshmukh, and Nathan N. Alder
- Subjects
Tyrosine phosphorylation ,Cell Biology ,Biology ,Biochemistry ,Cell biology ,Phosphorylation cascade ,chemistry.chemical_compound ,Protein structure ,chemistry ,Cell surface receptor ,Phosphorylation ,Protein phosphorylation ,Integrin, beta 6 ,Signal transduction ,Molecular Biology - Abstract
Reversible protein phosphorylation is vital for many fundamental cellular processes. The actual impact of adding and removing phosphate group(s) is 3-fold: changes in the local/global geometry, alterations in the electrostatic potential and, as the result of both, modified protein-target interactions. Here we present a comprehensive structural investigation of the effects of phosphorylation on the conformational as well as functional states of a crucial cell surface receptor, αIIbβ3 integrin. We have analyzed phosphorylated (Tyr747 and Tyr759) β3 integrin cytoplasmic tail (CT) primarily by NMR, and our data demonstrate that under both aqueous and membrane-mimetic conditions, phosphorylation causes substantial conformational rearrangements. These changes originate from novel ionic interactions and revised phospholipid binding. Under aqueous conditions, the critical Tyr747 phosphorylation prevents β3CT from binding to its heterodimer partner αIIbCT, thus likely maintaining an activated state of the receptor. This conclusion was tested in vivo and confirmed by integrin-dependent endothelial cells adhesion assay. Under membrane-mimetic conditions, phosphorylation results in a modified membrane embedding characterized by significant changes in the secondary structure pattern and the overall fold of β3CT. Collectively these data provide unique molecular insights into multiple regulatory roles of phosphorylation.
- Published
- 2011
40. Green Proteorhodopsin Reconstituted into Nanoscale Phospholipid Bilayers (Nanodiscs) as Photoactive Monomers
- Author
-
Christine T. Schwall, Nathan N. Alder, Matthew J. Ranaghan, and Robert R. Birge
- Subjects
Rhodopsin ,Proteorhodopsin ,Nanostructure ,biology ,Lipid Bilayers ,Phospholipid ,Bacteriorhodopsin ,General Chemistry ,Photochemical Processes ,Biochemistry ,Article ,Catalysis ,Nanostructures ,chemistry.chemical_compound ,Crystallography ,Colloid and Surface Chemistry ,Monomer ,chemistry ,Rhodopsins, Microbial ,biology.protein ,Lipid bilayer ,Phospholipids ,Nanodisc - Abstract
Over 4000 putative proteorhodopsins (PRs) have been identified throughout the oceans and seas of the Earth. The first of these eubacterial rhodopsins was discovered in 2000 and has expanded the family of microbial proton pumps to all three domains of life. With photophysical properties similar to those of bacteriorhodopsin, an archaeal proton pump, PRs are also generating interest for their potential use in various photonic applications. We perform here the first reconstitution of the minimal photoactive PR structure into nanoscale phospholipid bilayers (nanodiscs) to better understand how protein-protein and protein-lipid interactions influence the photophysical properties of PR. Spectral (steady-state and time-resolved UV-visible spectroscopy) and physical (size-exclusion chromatography and electron microscopy) characterization of these complexes confirms the preparation of a photoactive PR monomer within nanodiscs. Specifically, when embedded within a nanodisc, monomeric PR exhibits a titratable pK(a) (6.5-7.1) and photocycle lifetime (∼100-200 ms) that are comparable to the detergent-solubilized protein. These ndPRs also produce a photoactive blue-shifted absorbance, centered at 377 or 416 nm, that indicates that protein-protein interactions from a PR oligomer are required for a fast photocycle. Moreover, we demonstrate how these model membrane systems allow modulation of the PR photocycle by variation of the discoidal diameter (i.e., 10 or 12 nm), bilayer thickness (i.e., 23 or 26.5 Å), and degree of saturation of the lipid acyl chain. Nanodiscs also offer a highly stable environment of relevance to potential device applications.
- Published
- 2011
41. Quaternary Structure of the Mitochondrial TIM23 Complex Reveals Dynamic Association between Tim23p and Other Subunits
- Author
-
Robert E. Jensen, Ashley I. Buhring, Nathan N. Alder, Arthur E. Johnson, and Jennifer Sutherland
- Subjects
Saccharomyces cerevisiae Proteins ,Translocase of the outer membrane ,Protein subunit ,Saccharomyces cerevisiae ,Biology ,Models, Biological ,Substrate Specificity ,Mitochondrial membrane transport protein ,Mitochondrial Precursor Protein Import Complex Proteins ,Immunoprecipitation ,Protein Structure, Quaternary ,Inner mitochondrial membrane ,Molecular Biology ,Integral membrane protein ,Membrane Potential, Mitochondrial ,Peripheral membrane protein ,Membrane Transport Proteins ,Articles ,Cell Biology ,Mitochondria ,Cell biology ,Protein Subunits ,Protein Transport ,Cross-Linking Reagents ,Translocase of the inner membrane ,biology.protein ,Mutant Proteins ,Protein quaternary structure ,sense organs ,Protein Binding - Abstract
Tim23p is an essential channel-forming component of the multisubunit TIM23 complex of the mitochondrial inner membrane that mediates protein import. Radiolabeled Tim23p monocysteine mutants were imported in vitro, incorporated into functional TIM23 complexes, and subjected to chemical cross-linking. Three regions of proximity between Tim23p and other subunits of the TIM23 complex were identified: Tim17p and the first transmembrane segment of Tim23p; Tim50p and the C-terminal end of the Tim23p hydrophilic region; and the entire hydrophilic domains of Tim23p molecules. These regions of proximity reversibly change in response to changes in membrane potential across the inner membrane and also when a translocating substrate is trapped in the TIM23 complex. These structural changes reveal that the macromolecular arrangement within the TIM23 complex is dynamic and varies with the physiological state of the mitochondrion.
- Published
- 2008
42. The Tim9p/10p and Tim8p/13p Complexes Bind to Specific Sites on Tim23p during Mitochondrial Protein Import
- Author
-
Nathan N. Alder, Arthur E. Johnson, Robert E. Jensen, and Alison J. Davis
- Subjects
Saccharomyces cerevisiae Proteins ,Protein Conformation ,Translocase of the outer membrane ,TIM/TOM complex ,Saccharomyces cerevisiae ,medicine.disease_cause ,Mitochondrial Membrane Transport Proteins ,Substrate Specificity ,Mitochondrial Proteins ,Mitochondrial membrane transport protein ,Mitochondrial Precursor Protein Import Complex Proteins ,Protein targeting ,medicine ,Molecular Biology ,Integral membrane protein ,Binding Sites ,biology ,Peripheral membrane protein ,Membrane Proteins ,Membrane Transport Proteins ,Articles ,Cell Biology ,Translocon ,Mitochondria ,Cell biology ,Protein Transport ,Multiprotein Complexes ,Translocase of the inner membrane ,biology.protein - Abstract
The import of polytopic membrane proteins into the mitochondrial inner membrane (IM) is facilitated by Tim9p/Tim10p and Tim8p/Tim13p protein complexes in the intermembrane space (IMS). These complexes are proposed to act as chaperones by transporting the hydrophobic IM proteins through the aqueous IMS and preventing their aggregation. To examine the nature of this interaction, Tim23p molecules containing a single photoreactive cross-linking probe were imported into mitochondria in the absence of an IM potential where they associated with small Tim complexes in the IMS. On photolysis and immunoprecipitation, a probe located at a particular Tim23p site (27 different locations were examined) was found to react covalently with, in most cases, only one of the small Tim proteins. Tim8p, Tim9p, Tim10p, and Tim13p were therefore positioned adjacent to specific sites in the Tim23p substrate before its integration into the IM. This specificity of binding to Tim23p strongly suggests that small Tim proteins do not function solely as general chaperones by minimizing the exposure of nonpolar Tim23p surfaces to the aqueous medium, but may also align a folded Tim23p substrate in the proper orientation for delivery and integration into the IM at the TIM22 translocon.
- Published
- 2007
43. Cotranslational Membrane Protein Biogenesis at the Endoplasmic Reticulum
- Author
-
Nathan N. Alder and Arthur E. Johnson
- Subjects
Chemistry ,Endoplasmic reticulum ,Membrane Proteins ,Target peptide ,STIM1 ,Intracellular Membranes ,Cell Biology ,Endoplasmic-reticulum-associated protein degradation ,Endoplasmic Reticulum ,Biochemistry ,Membrane contact site ,Cell biology ,Secretory protein ,Protein Biosynthesis ,Microsome ,Animals ,Humans ,Molecular Biology ,Secretory pathway - Published
- 2004
44. Investigation of the Physiochemical Properties of the Phospholipid Cardiolipin: Implications for Oxphos Regulation and Barth Syndrome
- Author
-
Nathan N. Alder, Murugappan Sathappa, and Matthew Greenwood
- Subjects
Cytochrome ,biology ,Cytochrome c ,Biophysics ,Tafazzin ,Phospholipid ,Barth syndrome ,medicine.disease ,chemistry.chemical_compound ,chemistry ,Biochemistry ,Cardiolipin ,biology.protein ,medicine ,Cytochrome c oxidase ,lipids (amino acids, peptides, and proteins) ,Lipid bilayer - Abstract
Cardiolipin (CL) is the signature phospholipid of the mitochondrial inner membrane, which houses respiratory complexes that generate electrochemical ion gradients to drive processes such as ATP synthesis by oxidative phosphorylation. Nascent CL, which is enriched in saturated acyl chains of variable lengths, is subject to a two-stage remodeling process: a deacylase first removes an acyl chain to generate the monolysocardiolpin (MLCL) intermediate, and the transacylase tafazzin regenerates the four-chain lipid with highly unsaturated acyl chains with a high degree of symmetry. Mutations in the TAZ gene that encodes tafazzin underpin Barth syndrome, a multi-system mitochondrial disorder characterized by gross abnormalities in lipid profiles, cardiac and skeletal myopathy, neutropenia, growth retardation and mortality in early childhood. First, we analyze the role of CL in activating OXPHOS complexes using nanoscale lipid bilayers containing headgroup and acyl chain variants of CL. To this end, purified cytochrome c oxidase was reconstituted into nanodiscs of defined lipid composition and assayed for CL-dependent redox activity. Second, we have analyzed the binding characteristics of cytochrome c to bilayers containing CL variants. This work revealed that MLCL, which lacks a lipid tail, does not allow for the hydrophobic interactions and unfolding of cytochrome c, a requisite step in promoting peroxidase activity and of the apoptotic program. Finally, we use a combination of electrokinetic measurements and fluorescence-based approaches to address the ionization properties of the phosphate esters among CL variants. In contrast to the dominant paradigm in which a resonance-stabilized bicyclic headgroup structure promotes two disparate pKa values, we find the CL head group behaves as a strong dibasic acid, and exists as a dianion at physiological pH. Moreover, the ionization properties of the CL head group are similar for all variants examined.
- Published
- 2016
45. Energy use by biological protein transport pathways
- Author
-
Steven M. Theg and Nathan N. Alder
- Subjects
Adenosine Triphosphatases ,Hydrolysis ,Biological membrane ,GTPase ,Guanosine triphosphate ,Models, Biological ,Biochemistry ,Transmembrane protein ,Mitochondria ,Protein Structure, Tertiary ,Cell biology ,Transport protein ,Kinetics ,Protein Transport ,Cytosol ,chemistry.chemical_compound ,Adenosine Triphosphate ,Protein structure ,Bacterial Proteins ,chemistry ,Thermodynamics ,Guanosine Triphosphate ,Molecular Biology ,Adenosine triphosphate - Abstract
The targeting of proteins into and across biological membranes to their correct cellular locations is mediated by a variety of transport pathways. These systems must couple the thermodynamically unfavorable processes of substrate translocation and integration with the expenditure of metabolic energy, using the free energy of ATP and GTP hydrolysis and/or a transmembrane protonmotive force. Several recent advances in our knowledge of the structure and function of these transport systems have provided insights into the mechanisms of energy transduction, force generation and energy use by different protein transport pathways.
- Published
- 2003
46. Energetics of Protein Transport across Biological Membranes
- Author
-
Nathan N. Alder and Steven M. Theg
- Subjects
Chloroplast ,Biochemistry ,Biochemistry, Genetics and Molecular Biology(all) ,Antiporter ,Thylakoid ,Biophysics ,Molecule ,Biological membrane ,Biology ,General Biochemistry, Genetics and Molecular Biology ,Transmembrane protein ,In vitro ,Transport protein - Abstract
Among the pathways for protein translocation across biological membranes, the ΔpH-dependent/Tat system is unusual in its sole reliance upon the transmembrane pH gradient to drive protein transport. The free energy cost of protein translocation via the chloro-plast ΔpH-dependent/Tat pathway was measured by conducting in vitro transport assays with isolated thylakoids while concurrently monitoring energetic parameters. These experiments revealed a substrate-specific energetic barrier to cpTat-mediated transport as well as direct utilization of protons from the gradient, consistent with a H + /protein antiporter mechanism. The magnitude of proton flux was assayed by four independent approaches and averaged 7.9 × 10 4 protons released from the gradient per transported protein. This corresponds to a ΔG transport of 6.9 × 10 5 kJ·mol protein translocated −1 , representing the utilization of an energetic equivalent of 10 4 molecules of ATP. At this cost, we estimate that the ΔpH-dependent/cpTat pathway utilizes approximately 3% of the total energy output of the chloroplast.
- Published
- 2003
- Full Text
- View/download PDF
47. Molecular Dynamics Investigation into the Distribution of Cardiolipin Variants in Flat and Buckled Heterogeneous Bilayers
- Author
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Eric R. May, Kevin J. Boyd, and Nathan N. Alder
- Subjects
Monolysocardiolipin ,Peripheral membrane protein ,Biophysics ,Barth syndrome ,Biology ,medicine.disease ,chemistry.chemical_compound ,Membrane ,chemistry ,Biochemistry ,Membrane curvature ,Cardiolipin ,medicine ,lipids (amino acids, peptides, and proteins) ,Lipid bilayer ,Inner mitochondrial membrane - Abstract
Cardiolipin is a unique lipid molecule, which has an unusual dimeric structure with a dual-phosphate headgroup and four acyl chains. In most eukaryotic membranes cardiolipin is absent or present only at very low concentrations, however in the inner mitochondrial membrane (IMM) cardiolipin constitutes approximately 10-20% the lipid composition. Cardiolipin is believed to play a role the organization and morphology of the IMM, which contains highly curved membrane regions called cristae that protrude into the matrix. Cardiolipin is also involved in cellular respiration and apoptosis, and has been shown to bind preferentially to several mitochondrial integral and peripheral proteins. During its biosynthesis in the IMM, cardiolipin undergoes a remodeling process whereby acyl chains are removed and replaced to produce mature cardiolipin species with highly unsaturated and uniform lipid tail composition. In the mitochondrial disorder Barth syndrome, defects in the remodeling machinery lead to accumulation of the three-tailed cardiolipin variant monolysocardiolipin. The phenotype of Barth syndrome cells includes abnormal mitochondrial morphology and inhibited energy metabolism. In this work we explore the relationship between the spatial distribution of lipid species and membrane curvature in cardiolipin containing heterogeneous membranes using molecular dynamics simulations. We investigate cardiolipin-containing bilayer properties both in flat lamellar environments and in highly curved (buckled) membranes, and demonstrate that clustering of cardiolipin occurs in areas of high negative curvature. We compare this clustering behavior as well as the pressure induced buckling susceptibility and other biophysical membrane properties in several bilayer systems. One goal of this study is to elucidate the effect of an accumulation of monolysocardiolipin on the physical properties of lipid bilayers.
- Published
- 2017
48. Investigation of the Interactions of the SS-31 Peptides with Cardiolipin Variants: A Potential Therapeutic for Barth Syndrome
- Author
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Murugappan Sathappa, Wayne Mitchell, Eric R. May, Kevin J. Boyd, Hazel H. Szeto, Nathan N. Alder, and Adrian Coscia
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biology ,Monolysocardiolipin ,Cytochrome c ,Biophysics ,Tafazzin ,Barth syndrome ,Oxidative phosphorylation ,medicine.disease ,chemistry.chemical_compound ,Biochemistry ,chemistry ,medicine ,biology.protein ,Cardiolipin ,Inner membrane ,Inner mitochondrial membrane - Abstract
Szeto-Schiller (SS) peptides are synthetic tetrapeptides containing an alternating aromatic-cationic motif. They are readily taken up by cells and are targeted to the mitochondrial inner membrane. Among them, SS-31 has been shown to bind to bilayers in a cardiolipin-dependent manner via electrostatic and hydrophobic interactions. As a therapeutic agent, SS-31 leads to clinical improvements in many complex diseases, likely linked to its selective interaction with the cardiolipin-rich inner membrane, protection of mitochondrial ultrastructure, curtailment of ROS, and restoration of oxidative phosphorylation. SS-31 represents a first-in-class cardiolipin-protective therapeutic; however, its molecular mechanism of action remains largely unknown. In this study we have explored the use of SS-31 as a potential therapeutic for Barth syndrome (BTHS), a multisystem disorder characterized by cardiac and skeletal myopathy, neutropenia, growth retardation, and premature death in young males. BTHS is caused by mutations in the gene that encodes the transacylase tafazzin, involved in acyl chain remodeling of cardiolipin following its biosynthesis. Dysfunctional tafazzin results in decreased levels of cardiolipin with abnormal fatty acid tail profiles, and an accumulation of the remodeling intermediate monolysocardiolipin. Using model membrane systems and in organello studies, we show that the binding affinities of SS-31 to bilayers containing cardiolipin and monolysocardiolipin are similar. Moreover, the presence of SS-31 appears to modulate the interaction of the electron carrier cytochrome c with bilayers containing cardiolipin and its variants. Our results suggest that SS-31 may act in part by altering membrane phase behavior. Our empirical results are complemented by computational approaches that address the nature of the SS-31 bilayer interaction. Our results point to the potential use of SS peptides as therapeutics for BTHS and give key mechanistic insights into the nature of their bilayer interactions.
- Published
- 2017
49. Targeting mitochondria with methylene blue protects mice against acetaminophen-induced liver injury
- Author
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Naoki Imaizumi, Kang Kwang Lee, Urs A. Boelsterli, Nathan N. Alder, and Sally R. Chamberland
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Male ,NAPQI ,SDHA ,Drug Evaluation, Preclinical ,Mitochondria, Liver ,Oxidative phosphorylation ,Mitochondrion ,Pharmacology ,Biology ,Necrosis ,Peroxynitrous Acid ,medicine ,Benzoquinones ,Animals ,Enzyme Inhibitors ,Cells, Cultured ,Acetaminophen ,Liver injury ,Hepatology ,Cell Death ,Electron Transport Complex II ,digestive, oral, and skin physiology ,Analgesics, Non-Narcotic ,medicine.disease ,Methylene Blue ,Mice, Inbred C57BL ,Succinate Dehydrogenase ,medicine.anatomical_structure ,Mitochondrial permeability transition pore ,Liver ,Hepatocyte ,Hepatocytes ,Imines ,Chemical and Drug Induced Liver Injury ,medicine.drug - Abstract
Acetaminophen (APAP) overdose is a frequent cause of drug-induced liver injury and the most frequent cause of acute liver failure in the Western world. Previous studies with mouse models have revealed that impairment of mitochondrial respiration is an early event in the pathogenesis, but the exact mechanisms have remained unclear, and therapeutic approaches to specifically target mitochondria have been insufficiently explored. Here, we found that the reactive oxidative metabolite of APAP, N-acetyl-p-benzoquinoneimine (NAPQI), caused the selective inhibition of mitochondrial complex II activity by >90% in both mouse hepatic mitochondria and yeast-derived complexes reconstituted into nanoscale model membranes, as well as the decrease of succinate-driven adenosine triphosphate (ATP) biosynthesis rates. Based on these findings, we hypothesized that methylene blue (MB), a mitochondria-permeant redox-active compound that can act as an alternative electron carrier, protects against APAP-induced hepatocyte injury. We found that MB (
- Published
- 2014
50. Cation-Dependent Behavior of Cardiolipin-Containing Membranes and Implications for Respiratory Complex II Assembly and Activity
- Author
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Nathan N. Alder and Christine T. Schwall
- Subjects
Diphenylhexatriene ,chemistry.chemical_classification ,0303 health sciences ,Chemistry ,Stereochemistry ,Bilayer ,Phospholipid ,Biophysics ,Divalent ,03 medical and health sciences ,chemistry.chemical_compound ,Crystallography ,0302 clinical medicine ,Lamellar phase ,Cardiolipin ,lipids (amino acids, peptides, and proteins) ,Lipid bilayer ,Laurdan ,030217 neurology & neurosurgery ,030304 developmental biology - Abstract
Cardiolipin (CL) is the signature phospholipid of energy-conserving mitochondrial inner membrane. It has a uniquely dimeric structure, with a headgroup composed of two phosphatidyl moieties linked by a glycerol bridge and a hydrophobic domain of four acyl chains. Despite having two phosphodiesters, CL is proposed to be monoanionic at physiological pH due to resonance stabilization of a bicyclic acid-anion structure that traps a proton. Moreover, CL displays phase polymorphism by undergoing a lamellar to inverted hexagonal transition in the presence of divalent cations. We have investigated the influence of divalent cations on lipid bilayers containing CLs with varying headgroups and acyl chains. We analyzed global physical properties of membranes in liposomes and nanodiscs, using fluorescent probes that localize to different bilayer depths. The environment-sensitive probe Laurdan was used to detect changes in hydration level of the interfacial region. With increasing concentrations of divalent cations, we observed a CL-dependent biphasic increase in degrees of lipid packing that is profoundly reduced for CL analogs lacking the resonance-stabilized headgroup or containing the anionic lipid phosphatidylglycerol. Parallel measurements of fluorescence anisotropy using diphenylhexatriene localized in the hydrophobic bilayer interior revealed much less pronounced effects on acyl chain dynamics with identical cation titrations. Finally, based on our recent report that CL is critical for respiratory Complex II structure and function, we investigated effects of divalent cations on activity and integrity of this enzyme. We observed cation-dependent disassembly of Complex II with an attendant loss of its redox activity that shows remarkable correlation with our fluorescence-detected alterations in bilayer structure. Based on these results, we propose a model in which coordination of divalent cations by CL causes specific changes in bilayer structure that are linked with structural alterations in CL-binding protein complexes.
- Published
- 2014
- Full Text
- View/download PDF
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