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1.
Pathogens ; 11(7)2022 Jul 19.
Artigo em Inglês | MEDLINE | ID: mdl-35890050

RESUMO

Conventionally, hyperimmune globulin drugs manufactured from pooled immunoglobulins from vaccinated or convalescent donors have been used in treating infections where no treatment is available. This is especially important where multi-epitope neutralization is required to prevent the development of immune-evading viral mutants that can emerge upon treatment with monoclonal antibodies. Using microfluidics, flow sorting, and a targeted integration cell line, a first-in-class recombinant hyperimmune globulin therapeutic against SARS-CoV-2 (GIGA-2050) was generated. Using processes similar to conventional monoclonal antibody manufacturing, GIGA-2050, comprising 12,500 antibodies, was scaled-up for clinical manufacturing and multiple development/tox lots were assessed for consistency. Antibody sequence diversity, cell growth, productivity, and product quality were assessed across different manufacturing sites and production scales. GIGA-2050 was purified and tested for good laboratory procedures (GLP) toxicology, pharmacokinetics, and in vivo efficacy against natural SARS-CoV-2 infection in mice. The GIGA-2050 master cell bank was highly stable, producing material at consistent yield and product quality up to >70 generations. Good manufacturing practices (GMP) and development batches of GIGA-2050 showed consistent product quality, impurity clearance, potency, and protection in an in vivo efficacy model. Nonhuman primate toxicology and pharmacokinetics studies suggest that GIGA-2050 is safe and has a half-life similar to other recombinant human IgG1 antibodies. These results supported a successful investigational new drug application for GIGA-2050. This study demonstrates that a new class of drugs, recombinant hyperimmune globulins, can be manufactured consistently at the clinical scale and presents a new approach to treating infectious diseases that targets multiple epitopes of a virus.

2.
Biochim Biophys Acta Biomembr ; 1861(5): 1030-1036, 2019 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-30876942

RESUMO

Miniature membranes comprised of tetramyristoylcardiolipin (CL) and apolipoprotein (apo) A-I, termed nanodisks (ND), are stable, aqueous soluble, reconstituted high density lipoproteins. When CL ND, but not dimyristoylphosphatidylcholine (PC) ND, were incubated with CaCl2, a concentration dependent increase in sample turbidity occurred, consistent with CL undergoing a bilayer to non-bilayer transition. To assess the cation specificity of this reaction, CL ND were incubated with various mono- and divalent cations. Whereas monovalent cations had no discernable effect, MgCl2 and SrCl2 induced a response similar to CaCl2. When ND were formulated using different weight ratios of CL and PC, those possessing 100% CL or 75% CL remained susceptible to CaCl2 induced sample turbidity development while ND possessing 50% CL displayed reduced susceptibility. ND comprised of 25% CL and 75% PC were unaffected by CaCl2 under these conditions. SDS PAGE analysis of insoluble material generated by incubation of CL ND with CaCl2 revealed that nearly all apoA-I was recovered in the insoluble fraction along with CL. One h after addition of EDTA to CaCl2-treated CL ND, sample clarity was restored. Collectively, the data are consistent with a model wherein Ca2+ forms a bidentate interaction with anionic phosphates in the polar head group of CL. As phosphate group repositioning occurs to maximize Ca2+ binding, CL acyl chains reposition, accentuating the conical shape of CL to an extent that is incompatible with the ND bilayer structure.


Assuntos
Cloreto de Cálcio/química , Cardiolipinas/química , Nanopartículas/química , Conformação Molecular , Solubilidade , Água/química
3.
J Biomed Res ; 32(2): 107-112, 2018 03 26.
Artigo em Inglês | MEDLINE | ID: mdl-29336355

RESUMO

Barth syndrome (BTHS) is a mitochondrial disorder characterized by cardiomyopathy and skeletal muscle weakness. Disease results from mutations in the tafazzin (TAZ) gene, encoding a phospholipid transacylase. Defective tafazzin activity results in an aberrant cardiolipin (CL) profile. The feasibility of restoring the intracellular CL profile was tested by in vivo administration of exogenous CL in nanodisk (ND) delivery particles. Ninety mg/kg CL (as ND) was administered to doxycycline-inducible taz shRNA knockdown (KD) mice once a week. After 10 weeks of CL-ND treatment, the mice were sacrificed and tissues harvested. Liquid chromatography-mass spectrometry of extracted lipids revealed that CL-ND administration failed to alter the CL profile of taz KD or WT mice. Thus, although CL-ND were previously shown to be an effective means of delivering CL to cultured cells, this effect does not extend to an in vivo setting. We conclude that CL-ND administration is not a suitable therapy option for BTHS.

4.
Protein Expr Purif ; 134: 18-24, 2017 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-28336201

RESUMO

Apolipoprotein (apo) A-I is the major protein component of high-density lipoprotein (HDL) and plays key roles in the Reverse Cholesterol Transport pathway. In the past decade, reconstituted HDL (rHDL) has been employed as a therapeutic agent for treatment of atherosclerosis. The ability of rHDL to promote cholesterol efflux from peripheral cells has been documented to reduce the size of atherosclerotic plaque lesions. However, development of apoA-I rHDL-based therapeutics for human use requires a cost effective process to generate an apoA-I product that meets "Good Manufacturing Practice" standards. Methods available for production and isolation of unmodified recombinant human apoA-I at scale are cumbersome, laborious and complex. To overcome this obstacle, a streamlined two-step procedure has been devised for isolation of recombinant untagged human apoA-I from E. coli that takes advantage of its ability to re-fold to a native conformation following denaturation. Heat treatment of a sonicated E. coli supernatant fraction induced precipitation of a large proportion of host cell proteins (HCP), yielding apoA-I as the major soluble protein. Reversed-phase HPLC of this material permitted recovery of apoA-I largely free of HCP and endotoxin. Purified apoA-I possessed α-helix secondary structure, formed rHDL upon incubation with phospholipid and efficiently promoted cholesterol efflux from cholesterol loaded J774 macrophages.


Assuntos
Apolipoproteína A-I , Escherichia coli/metabolismo , Redobramento de Proteína , Apolipoproteína A-I/biossíntese , Apolipoproteína A-I/química , Apolipoproteína A-I/isolamento & purificação , Apolipoproteína A-I/farmacocinética , Transporte Biológico Ativo/efeitos dos fármacos , Linhagem Celular , Colesterol/metabolismo , Escherichia coli/química , Escherichia coli/genética , Humanos , Macrófagos/metabolismo , Estrutura Secundária de Proteína , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/farmacologia
5.
Biochim Biophys Acta Biomembr ; 1859(6): 1156-1163, 2017 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-28336315

RESUMO

A fundamental question in cell biology, under investigation for over six decades, is the structural organization of mitochondrial cristae. Long known to harbor electron transport chain proteins, crista membrane integrity is key to establishment of the proton gradient that drives oxidative phosphorylation. Visualization of cristae morphology by electron microscopy/tomography has provided evidence that cristae are tube-like extensions of the mitochondrial inner membrane (IM) that project into the matrix space. Reconciling ultrastructural data with the lipid composition of the IM provides support for a continuously curved cylindrical bilayer capped by a dome-shaped tip. Strain imposed by the degree of curvature is relieved by an asymmetric distribution of phospholipids in monolayer leaflets that comprise cristae membranes. The signature mitochondrial lipid, cardiolipin (~18% of IM phospholipid mass), and phosphatidylethanolamine (34%) segregate to the negatively curved monolayer leaflet facing the crista lumen while the opposing, positively curved, matrix-facing monolayer leaflet contains predominantly phosphatidylcholine. Associated with cristae are numerous proteins that function in distinctive ways to establish and/or maintain their lipid repertoire and structural integrity. By combining unique lipid components with a set of protein modulators, crista membranes adopt and maintain their characteristic morphological and functional properties. Once established, cristae ultrastructure has a direct impact on oxidative phosphorylation, apoptosis, fusion/fission as well as diseases of compromised energy metabolism.


Assuntos
Cardiolipinas/metabolismo , Mitocôndrias/metabolismo , Membranas Mitocondriais/metabolismo , Proteínas Mitocondriais/genética , Fosforilação Oxidativa , Aciltransferases , Proteínas Sanguíneas/genética , Proteínas Sanguíneas/metabolismo , GTP Fosfo-Hidrolases/genética , GTP Fosfo-Hidrolases/metabolismo , Regulação da Expressão Gênica , Humanos , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Mitocôndrias/ultraestrutura , Dinâmica Mitocondrial , Membranas Mitocondriais/ultraestrutura , Proteínas Mitocondriais/metabolismo , Fosfatidilcolinas/metabolismo , Fosfatidiletanolaminas/metabolismo , Proibitinas , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
6.
Lipids ; 52(2): 99-108, 2017 02.
Artigo em Inglês | MEDLINE | ID: mdl-28070695

RESUMO

The Barth syndrome (BTHS) is caused by an inborn error of metabolism that manifests characteristic phenotypic features including altered mitochondrial membrane phospholipids, lactic acidosis, organic acid-uria, skeletal muscle weakness and cardiomyopathy. The underlying cause of BTHS has been definitively traced to mutations in the tafazzin (TAZ) gene locus on chromosome X. TAZ encodes a phospholipid transacylase that promotes cardiolipin acyl chain remodeling. Absence of tafazzin activity results in cardiolipin molecular species heterogeneity, increased levels of monolysocardiolipin and lower cardiolipin abundance. In skeletal muscle and cardiac tissue mitochondria these alterations in cardiolipin perturb the inner membrane, compromising electron transport chain function and aerobic respiration. Decreased electron flow from fuel metabolism via NADH ubiquinone oxidoreductase activity leads to a buildup of NADH in the matrix space and product inhibition of key TCA cycle enzymes. As TCA cycle activity slows pyruvate generated by glycolysis is diverted to lactic acid. In turn, Cori cycle activity increases to supply muscle with glucose for continued ATP production. Acetyl CoA that is unable to enter the TCA cycle is diverted to organic acid waste products that are excreted in urine. Overall, reduced ATP production efficiency in BTHS is exacerbated under conditions of increased energy demand. Prolonged deficiency in ATP production capacity underlies cell and tissue pathology that ultimately is manifest as dilated cardiomyopathy.


Assuntos
Síndrome de Barth/metabolismo , Cardiolipinas/metabolismo , Miocárdio/metabolismo , Fatores de Transcrição/genética , Aciltransferases , Trifosfato de Adenosina/metabolismo , Síndrome de Barth/genética , Síndrome de Barth/fisiopatologia , Transporte de Elétrons , Humanos , Mitocôndrias/metabolismo , Mutação
7.
J Inherit Metab Dis ; 39(5): 749-756, 2016 09.
Artigo em Inglês | MEDLINE | ID: mdl-27091556

RESUMO

3-methylglutaconic acid (3MGA)-uria occurs in numerous inborn errors of metabolism (IEM) associated with compromised mitochondrial energy metabolism. This organic acid arises from thioester cleavage of 3-methylglutaconyl CoA (3MG CoA), an intermediate in leucine catabolism. In individuals harboring mutations in 3MG CoA hydratase (i.e., primary 3MGA-uria), dietary leucine is the source of 3MGA. In secondary 3MGA-uria, however, no leucine metabolism defects have been reported. While others have suggested 3MGA arises from aberrant isoprenoid shunting from cytosol to mitochondria, an alternative route posits that 3MG CoA arises in three steps from mitochondrial acetyl CoA. Support for this biosynthetic route in IEMs is seen by its regulated occurrence in microorganisms. The fungus, Ustilago maydis, the myxobacterium, Myxococcus xanthus and the marine cyanobacterium, Lyngbya majuscule, generate 3MG CoA (or acyl carrier protein derivative) in the biosynthesis of iron chelating siderophores, iso-odd chain fatty acids and polyketide/nonribosomal peptide products, respectively. The existence of this biosynthetic machinery in these organisms supports a model wherein, under conditions of mitochondrial dysfunction, accumulation of acetyl CoA in the inner mitochondrial space as a result of inefficient fuel utilization drives de novo synthesis of 3MG CoA. Since humans lack the downstream biosynthetic capability of the organisms mentioned above, as 3MG CoA levels rise, thioester hydrolysis yields 3MGA, which is excreted in urine as unspent fuel. Understanding the metabolic origins of 3MGA may increase its utility as a biomarker.


Assuntos
Metabolismo Energético/fisiologia , Glutaratos/metabolismo , Mitocôndrias/metabolismo , Doenças Mitocondriais/metabolismo , Acetilcoenzima A/metabolismo , Humanos
8.
Biochem Biophys Res Commun ; 464(2): 580-5, 2015 Aug 21.
Artigo em Inglês | MEDLINE | ID: mdl-26164234

RESUMO

The concentration and composition of cardiolipin (CL) in mitochondria are altered in age-related heart disease, Barth Syndrome, and other rare genetic disorders, resulting in mitochondrial dysfunction. To explore whether exogenous CL can be delivered to cells, CL was combined with apolipoprotein A-I to generate water-soluble, nanoscale complexes termed nanodisks (ND). Mass spectrometry of HL60 myeloid progenitor cell extracts revealed a 30-fold increase in cellular CL content following incubation with CL-ND. When CL-ND containing a fluorescent CL analogue was employed, confocal microscopy revealed CL localization to mitochondria. The ability of CL-ND to elicit a physiological response was examined in an HL60 cell culture model of Barth Syndrome neutropenia. siRNA knockdown of the phospholipid transacylase, tafazzin (TAZ), induced apoptosis in these cells. When TAZ knockdown cells were incubated with CL-ND, the apoptotic response was attenuated. Thus, CL-ND represent a potential intervention strategy for replenishment of CL in Barth Syndrome, age-related heart disease, and other disorders characterized by depletion of this key mitochondrial phospholipid.


Assuntos
Apoptose/efeitos dos fármacos , Cardiolipinas/metabolismo , Mitocôndrias/metabolismo , Células Progenitoras Mieloides/patologia , Fatores de Transcrição/genética , Aciltransferases , Anexina A5/metabolismo , Apoptose/genética , Técnicas de Silenciamento de Genes , Células HL-60 , Células HeLa , Humanos , Ligação Proteica
9.
Proc Natl Acad Sci U S A ; 111(50): 17833-8, 2014 Dec 16.
Artigo em Inglês | MEDLINE | ID: mdl-25453101

RESUMO

The mammalian target of rapamycin (mTOR) kinase is a master regulator of cellular, developmental, and metabolic processes. Deregulation of mTOR signaling is implicated in numerous human diseases including cancer and diabetes. mTOR functions as part of either of the two multisubunit complexes, mTORC1 and mTORC2, but molecular details about the assembly and oligomerization of mTORCs are currently lacking. We use the single-molecule pulldown (SiMPull) assay that combines principles of conventional pulldown assays with single-molecule fluorescence microscopy to investigate the stoichiometry and assembly of mTORCs. After validating our approach with mTORC1, confirming a dimeric assembly as previously reported, we show that all major components of mTORC2 exist in two copies per complex, indicating that mTORC2 assembles as a homodimer. Interestingly, each mTORC component, when free from the complexes, is present as a monomer and no single subunit serves as the dimerizing component. Instead, our data suggest that dimerization of mTORCs is the result of multiple subunits forming a composite surface. SiMPull also allowed us to distinguish complex disassembly from stoichiometry changes. Physiological conditions that abrogate mTOR signaling such as nutrient deprivation or energy stress did not alter the stoichiometry of mTORCs. On the other hand, rapamycin treatment leads to transient appearance of monomeric mTORC1 before complete disruption of the mTOR-raptor interaction, whereas mTORC2 stoichiometry is unaffected. These insights into assembly of mTORCs may guide future mechanistic studies and exploration of therapeutic potential.


Assuntos
Complexos Multiproteicos/química , Serina-Treonina Quinases TOR/química , Proteínas de Bactérias , Western Blotting , Dimerização , Células HEK293 , Humanos , Imunoprecipitação , Proteínas Luminescentes , Alvo Mecanístico do Complexo 1 de Rapamicina , Alvo Mecanístico do Complexo 2 de Rapamicina , Microscopia de Fluorescência , Modelos Moleculares , Complexos Multiproteicos/metabolismo , Sirolimo/farmacologia , Serina-Treonina Quinases TOR/metabolismo , Proteína Vermelha Fluorescente
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