• Gaspar J, Mathieu J, Alvarez PJJ. A Rapid Platform to Generate Lipofuscin and Screen Therapeutic Drugs for Efficacy in Lipofuscin Removal. Materials, Methods & Technologies, Volume 10, 2016. Read on external site. Categories: LysoSENS

    A Rapid Platform to Generate Lipofuscin and Screen Therapeutic Drugs for Efficacy in Lipofuscin Removal.

    Materials, Methods & Technologies, Volume 10, 2016.

    A Rapid Platform to Generate Lipofuscin and Screen Therapeutic Drugs for Efficacy in Lipofuscin Removal.

    Gaspar J, Mathieu J, Alvarez PJJ.

    Abstract

    Abstract:

    Lipofuscin is a brown-yellow, autofluorescent polymeric material that accumulates in a ceroid manner within postmitotic cells during aging. Lipofuscin accumulation impairs proteosome and lysosome pathways critical to cell health and homeostasis. Therefore, the ability to quickly generate lipofuscin in vitro, and identify drugs that mitigate the accumulation or clear lipofuscin would be of great benefit to aging research. Here, we present a platform to quickly create lipofuscin-loaded (but otherwise healthy) cells and screen drugs for efficacy in lipofuscin removal. The combination of leupeptin, iron (III) chloride and hydrogen peroxide generates significant amounts of lipofuscin within cells while eliminating the need for a 40% hyperoxic chamber. Alternative methods which load fibroblasts with "artificial" lipofuscin obtained via UV-peroxidation of mitochondrial fragments are much more labor-intensive. This method is faster (≤10 days) than most protocols to generate lipofuscin and assess its removal, which typically require 2 to 4 weeks or longer to complete.

  • Mathieu JM, Wang F, Segatori L, Alvarez PJ. Increased resistance to oxysterol cytotoxicity in fibroblasts transfected with a lysosomally targeted Chromobacterium oxidase. Biotechnol Bioeng. 2012 Sep;109(9):2409-15. doi: 10.1002/bit.24506. PubMed: 22447444. Categories: LysoSENS

    Increased resistance to oxysterol cytotoxicity in fibroblasts transfected with a lysosomally targeted Chromobacterium oxidase.

    Biotechnol Bioeng. 2012 Sep;109(9):2409-15. doi: 10.1002/bit.24506.

    Increased resistance to oxysterol cytotoxicity in fibroblasts transfected with a lysosomally targeted Chromobacterium oxidase.

    Mathieu JM, Wang F, Segatori L, Alvarez PJ.

    Abstract

    Abstract:

    7-Ketocholesterol (7KC) is a cytotoxic oxysterol that plays a role in many age-related degenerative diseases. 7KC formation and accumulation often occurs in the lysosome, which hinders enzymatic transformations that reduce its toxicity and increase the sensitivity to lysosomal membrane permeabilization. We assayed the potential to mitigate 7KC cytotoxicity and enhance cell viability by overexpressing 7KC-active enzymes in human fibroblasts. One of the enzymes tested, a cholesterol oxidase engineered for lysosomal targeting, significantly increased cell viability in the short term upon treatment with up to 50 µM 7KC relative to controls. These results suggest targeting the lysosome for optimal treatment of oxysterol-mediated cytotoxicity, and support the use of introducing novel catalytic function into the lysosome for therapeutic and research applications.

  • Wu Y, Zhou J, Fishkin N, Rittmann BE, Sparrow JR. Enzymatic degradation of A2E, a retinal pigment epithelial lipofuscin bisretinoid. J Am Chem Soc. 2011 Feb 2;133(4):849-57. doi: 10.1021/ja107195u. PubMed: 21166406. Categories: LysoSENS

    Enzymatic degradation of A2E, a retinal pigment epithelial lipofuscin bisretinoid.

    J Am Chem Soc. 2011 Feb 2;133(4):849-57. doi: 10.1021/ja107195u.

    Enzymatic degradation of A2E, a retinal pigment epithelial lipofuscin bisretinoid.

    Wu Y, Zhou J, Fishkin N, Rittmann BE, Sparrow JR.

    Abstract

    Abstract:

    Some forms of blinding macular disease are associated with excessive accumulation of bisretinoid lipofuscin in retinal pigment epithelial (RPE) cells of the eye. This material is refractory to lysosomal enzyme degradation. In addition to gene and drug-based therapies, treatments that reverse the accumulation of bisretinoid would be beneficial. Thus, we have examined the feasibility of degrading the bisretinoids by delivery of exogenous enzyme. As proof of principle we report that horseradish peroxidase (HRP) can cleave the RPE bisretinoid A2E. In both cell-free and cell-based assays, A2E levels were decreased in the presence of HRP. HRP-associated cleavage products were detected by ultraperformance liquid chromatography (UPLC) coupled to electrospray ionization mass spectrometry, and the structures of the aldehyde-bearing cleavage products were elucidated by 18O-labeling and 1H NMR spectroscopy and by recording UV−vis absorbance spectra. These findings indicate that RPE bisretinoids such as A2E can be degraded by appropriate enzyme activities.

  • Peto MV. Aluminium and iron in humans: bioaccumulation, pathology, and removal. Rejuvenation Res 2010 Oct;13(5):589-98. PubMed: 21142669. Categories: AmyloSENS, LysoSENS, OncoSENS

    Aluminium and iron in humans: bioaccumulation, pathology, and removal.

    Rejuvenation Res 2010 Oct;13(5):589-98.

    Aluminium and iron in humans: bioaccumulation, pathology, and removal.

    Peto MV.

    Abstract

    Abstract:

    It is well known that exposure to various elements has a noticeable effect on human health. The effect of an element is determined by several characteristics, including its similarity to elements of biological necessity, metabolism, and degree of interaction with physiological processes. This review investigates the scientific literature of iron and aluminium to evaluate the extent to which these elements accumulate and cause pathology in humans. Iron was chosen for review because it is necessary for human life while seemingly having relationships with numerous pathological states such as heart disease, cancer, and impaired insulin sensitivity. Aluminium is reviewed because of its prevalence in daily life, observed interference with several biological processes, controversial relationship with Alzheimer disease, and lack of physiological role. Furthermore, because each of these metals has long been investigated for a possible relationship with various pathological states, a substantial volume of research is available regarding the effects of iron and aluminium in biological systems. For both aluminium and iron, this review focuses on: (1) Evaluating the evidence of toxicity, (2) considering the possibility of bioaccumulation, and (3) exploring methods of managing their accumulation.

  • Mathieu JM, Mohn WW, Eltis LD, LeBlanc JC, Stewart GR, Dresen C, Okamoto K, Alvarez PJ. 7-ketocholesterol catabolism by Rhodococcus jostii RHA1. Appl Environ Microbiol. 2010 Jan;76(1):352-5. doi: 10.1128/AEM.02538-09. PubMed: 19880645. Categories: LysoSENS

    7-ketocholesterol catabolism by Rhodococcus jostii RHA1.

    Appl Environ Microbiol. 2010 Jan;76(1):352-5. doi: 10.1128/AEM.02538-09.

    7-ketocholesterol catabolism by Rhodococcus jostii RHA1.

    Mathieu JM, Mohn WW, Eltis LD, LeBlanc JC, Stewart GR, Dresen C, Okamoto K, Alvarez PJ.

    Abstract

    Abstract:

    Oxysterols from steroid autooxidation have numerous harmful effects, but their biodegradation is poorly understood. Microarrays were used to study mineralization of the most common oxysterol, 7-ketocholesterol (7KC), by Rhodococcus jostii RHA1. Growth on 7KC versus growth on cholesterol resulted in 363 differentially expressed genes, including upregulation of two large gene clusters putatively encoding steroid catabolism. Despite this difference, 7KC degradation required key genes involved in cholesterol degradation, indicating a common catabolic route.

  • Schloendorn J, Webb T, Kemmish K, Hamalainen M, Jackemeyer D, Jiang L, Mathieu J, Rebo J, Sankman J, Sherman L, Tontson L, Qureshi A, Alvarez P, Rittmann B. Medical bioremediation: a concept moving toward reality. Rejuvenation Res. 2009 Dec;12(6):411-9. doi: 10.1089/rej.2009.0917. PubMed: 20041735. Categories: LysoSENS

    Medical bioremediation: a concept moving toward reality.

    Rejuvenation Res. 2009 Dec;12(6):411-9. doi: 10.1089/rej.2009.0917.

    Medical bioremediation: a concept moving toward reality.

    Schloendorn J, Webb T, Kemmish K, Hamalainen M, Jackemeyer D, Jiang L, Mathieu J, Rebo J, Sankman J, Sherman L, Tontson L, Qureshi A, Alvarez P, Rittmann B.

    Abstract

    Abstract:

    A major driver of aging is catabolic insufficiency, the inability of our bodies to break down certain substances that accumulate slowly throughout the life span. Even though substance buildup is harmless while we are young, by old age the accumulations can reach a toxic threshold and cause disease. This includes some of the most prevalent diseases in old age-atherosclerosis and macular degeneration. Atherosclerosis is associated with the buildup of cholesterol and its oxidized derivatives (particularly 7-ketocholesterol) in the artery wall. Age-related macular degeneration is associated with carotenoid lipofuscin, primarily the pyridinium bisretinoid A2E. Medical bioremediation is the concept of reversing the substance accumulations by using enzymes from foreign species to break down the substances into forms that relieve the disease-related effect. We report on an enzyme discovery project to survey the availability of microorganisms and enzymes with these abilities. We found that such microorganisms and enzymes exist. We identified numerous bacteria having the ability to transform cholesterol and 7-ketocholesterol. Most of these species initiate the breakdown by same reaction mechanism as cholesterol oxidase, and we have used this enzyme directly to reduce the toxicity of 7-ketocholesterol, the major toxic oxysterol, to cultured human cells. We also discovered that soil fungi, plants, and some bacteria possess peroxidase and carotenoid cleavage oxygenase enzymes that effectively destroy with varied degrees of efficiency and selectivity the carotenoid lipofuscin found in macular degeneration.

  • Mathieu JM, Schloendorn J, Rittmann BE, Alvarez PJ. Medical bioremediation of age-related diseases. Microb Cell Fact. 2009 Apr 9;8:21. doi: 10.1186/1475-2859-8-21. PubMed: 19358742. Categories: LysoSENS

    Medical bioremediation of age-related diseases.

    Microb Cell Fact. 2009 Apr 9;8:21. doi: 10.1186/1475-2859-8-21.

    Medical bioremediation of age-related diseases.

    Mathieu JM, Schloendorn J, Rittmann BE, Alvarez PJ.

    Abstract

    Abstract:

    Catabolic insufficiency in humans leads to the gradual accumulation of a number of pathogenic compounds associated with age-related diseases, including atherosclerosis, Alzheimer's disease, and macular degeneration. Removal of these compounds is a widely researched therapeutic option, but the use of antibodies and endogenous human enzymes has failed to produce effective treatments, and may pose risks to cellular homeostasis. Another alternative is "medical bioremediation," the use of microbial enzymes to augment missing catabolic functions. The microbial genetic diversity in most natural environments provides a resource that can be mined for enzymes capable of degrading just about any energy-rich organic compound. This review discusses targets for biodegradation, the identification of candidate microbial enzymes, and enzyme-delivery methods.

  • Mathieu J, Schloendorn J, Rittmann BE, Alvarez PJ. Microbial degradation of 7-ketocholesterol. Biodegradation. 2008;19(6):807-13. PubMed: 18344006. Categories: LysoSENS

    Microbial degradation of 7-ketocholesterol.

    Biodegradation. 2008;19(6):807-13.

    Microbial degradation of 7-ketocholesterol.

    Mathieu J, Schloendorn J, Rittmann BE, Alvarez PJ.

    Abstract

    Abstract:

    7-Ketocholesterol (7KC) is an oxidized derivative of cholesterol suspected to be involved in the pathogenesis of atherosclerosis and possibly Alzheimer's disease. While some oxysterols are important biological mediators, 7KC is generally cytotoxic and interferes with cellular homeostasis. Despite recent interest in preventing the accumulation of 7KC in a variety of matrices to avoid adverse biological effects, its microbial degradation has not been previously addressed in the peer-reviewed literature. Thus, the rate and extent of biodegradation of this oxysterol was investigated to bridge this gap. A wide variety of bacteria isolated from soil or activated sludge, including Proteobacterium Y-134, Sphingomonas sp. JEM-1, Nocardia nova, Rhodococcus sp. RHA1, and Pseduomonas aeruginosa, utilized 7KC as a sole carbon and energy source, resulting in its mineralization. Nocardia nova, which is known to produce biosurfactants, was the fastest degrader. This study supports the notion that microbial catabolic enzymes could be exploited to control 7KC levels in potential biotechnological applications for agricultural, environmental, or medical use.

  • Rittmann BE, Schloendorn J. Engineering away lysosomal junk: medical bioremediation. Rejuvenation Res 2007;10(3):359-365. PubMed: 17708688. Categories: LysoSENS

    Engineering away lysosomal junk: medical bioremediation.

    Rejuvenation Res 2007;10(3):359-365.

    Engineering away lysosomal junk: medical bioremediation.

    Rittmann BE, Schloendorn J.

    Abstract

    Abstract:

    Atherosclerosis, macular degeneration, and neurodegenerative diseases such as Alzheimer's disease, are associated with the intracellular accumulation of substances that impair cellular function and viability. Reversing this accumulation may be a valuable therapy, but the accumulating substances resist normal cellular catabolism. On the other hand, these substances are naturally degraded in the soil and water by microorganisms. Thus, we propose the concept of "medical bioremediation," which derives from the successful field of in situ environmental bioremediation of petroleum hydrocarbons. In environmental bioremediation, communities of microorganisms mineralize hydrophobic organics using a series of enzymes. In medical bioremediation, we hope to utilize one or several microbial enzymes to degrade the intracellular accumulators enough that they can be cleared from the affected cells. Here, we present preliminary, but promising results for the bacterial biodegradation of 7-ketocholesterol, the main accumulator of foam cells associated with atherosclerosis. In particular, we report on the isolation of several Nocardia strains able to biodegrade 7-ketocholesterol and as an ester of 7-ketocholoesterol. We also outline key intermediates in the biodegradation pathway, a key step towards identifying the key enzymes that may lead to a therapy.

  • de Grey ADNJ, Alvarez PJJ, Brady RO, Cuervo AM, Jerome WG, McCarty PL, Nixon RA, Rittmann BE, Sparrow JR. Medical bioremediation: prospects for the application of microbial catabolic diversity to aging and several major age-related diseases. Ageing Res Rev. 2005 Aug;4(3):315-38. PubMed: 16040282. Categories: LysoSENS

    Medical bioremediation: prospects for the application of microbial catabolic diversity to aging and several major age-related diseases.

    Ageing Res Rev. 2005 Aug;4(3):315-38.

    Medical bioremediation: prospects for the application of microbial catabolic diversity to aging and several major age-related diseases.

    de Grey ADNJ, Alvarez PJJ, Brady RO, Cuervo AM, Jerome WG, McCarty PL, Nixon RA, Rittmann BE, Sparrow JR.

    Abstract

    Abstract:

    Several major diseases of old age, including atherosclerosis, macular degeneration and neurodegenerative diseases are associated with the intracellular accumulation of substances that impair cellular function and viability. Moreover, the accumulation of lipofuscin, a substance that may have similarly deleterious effects, is one of the most universal markers of aging in postmitotic cells. Reversing this accumulation may thus be valuable, but has proven challenging, doubtless because substances resistant to cellular catabolism are inherently hard to degrade. We suggest a radically new approach: augmenting humans' natural catabolic machinery with microbial enzymes. Many recalcitrant organic molecules are naturally degraded in the soil. Since the soil in certain environments - graveyards, for example - is enriched in human remains but does not accumulate these substances, it presumably harbours microbes that degrade them. The enzymes responsible could be identified and engineered to metabolise these substances in vivo. Here, we survey a range of such substances, their putative roles in age-related diseases and the possible benefits of their removal. We discuss how microbes capable of degrading them can be isolated, characterised and their relevant enzymes engineered for this purpose and ways to avoid potential side-effects.

  • de Grey ADNJ. Bioremediation meets biomedicine: therapeutic translation of microbial catabolism to the lysosome. Trends Biotechnol 2002;20(11):452-455. PubMed: 12413818. Categories: LysoSENS

    Bioremediation meets biomedicine: therapeutic translation of microbial catabolism to the lysosome.

    Trends Biotechnol 2002;20(11):452-455.

    Bioremediation meets biomedicine: therapeutic translation of microbial catabolism to the lysosome.

    de Grey ADNJ.

    Abstract

    Abstract:

    Lysosomal degradation of damaged macromolecules is imperfect: many cell types accumulate lysosomal aggregates with age. Some such deposits are known, or are strongly suspected, to cause age-related disorders such as atherosclerosis and neurodegeration. It is possible that they also influence the rate of aging in general. Lysosomal degradation involves extensive cooperation between the participating enzymes: each generates a substrate for others until breakdown of the target material to recyclable units (such as amino acids) is complete. Hence, the age-related accumulation of lysosomal aggregates might be markedly retarded, or even reversed, by introducing just a few bacterial or fungal enzymes -'xenohydrolases' - that can degrade molecules that our natural machinery cannot. This article examines the feasibility and biomedical potential of such lysosomal enhancement as an approach to retarding or treating age-related physiological decline and disease.