Jeffrey A. Cohlberg

Parkinson's disease is the second most common neurodegenerative disease and results from loss of dopaminergic neurons in the substantia nigra. The aggregation and fibrillation of alpha-synuclein have been implicated as a causative factor in the disease. Glycosaminoglycans (GAGs) are routinely found associated with amyloid deposits in most amyloidosis diseases, and there is evidence to support an active role of GAGs in amyloid fibril formation in some cases. In contrast to the extracellular amyloid deposits, the alpha-synuclein deposits in Lewy body diseases are intracellular, and thus it is less clear whether GAGs may be involved. To determine whether the presence of GAGs does affect the fibrillation of alpha-synuclein, the kinetics of fibril formation were investigated in the presence of a number of GAGs and other charged polymers. Certain GAGs (heparin, heparan sulfate) and other highly sulfated polymers (dextran sulfate) were found to significantly stimulate the formation of alpha-synuclein fibrils. Interestingly, the interaction of GAGs with alpha-synuclein is quite specific, since some GAGs, e.g., keratan sulfate, had negligible effect. Heparin not only increased the rate of fibrillation but also apparently increased the yield of fibrils. The molar ratio of heparin to alpha-synuclein and the incorporation of fluorescein-labeled heparin into the fibrils demonstrate that the heparin is integrated into the fibrils and is not just a catalyst for fibrillation. The apparent dissociation constant for heparin in stimulating alpha-synuclein fibrillation was 0.19 microM, indicating a strong affinity. Similar effects of heparin were observed with the A53T and A30P mutants of alpha-synuclein. Since there is some evidence that Lewy bodies may contain GAGs, these observations may be very relevant in the context of the etiology of Parkinson's disease.

Mutations in the gene encoding Cu-Zn superoxide dismutase (SOD1) are one of the causes of familial amyotrophic lateral sclerosis (FALS). Fibrillar inclusions containing SOD1 and SOD1 inclusions that bind the amyloid-specific dye thioflavin S have been found in neurons of transgenic mice expressing mutant SOD1. Therefore, the formation of amyloid fibrils from human SOD1 was investigated. When agitated at acidic pH in the presence of low concentrations of guanidine or acetonitrile, metalated SOD1 formed fibrillar material which bound both thioflavin T and Congo red and had circular dichroism and infrared spectra characteristic of amyloid. While metalated SOD1 did not form amyloid-like aggregates at neutral pH, either removing metals from SOD1 with its intramolecular disulfide bond intact or reducing the intramolecular disulfide bond of metalated SOD1 was sufficient to promote formation of these aggregates. SOD1 formed amyloid-like aggregates both with and without intermolecular disulfide bonds, depending on the incubation conditions, and a mutant SOD1 lacking free sulfhydryl groups (AS-SOD1) formed amyloid-like aggregates at neutral pH under reducing conditions. ALS mutations enhanced the ability of disulfide-reduced SOD1 to form amyloid-like aggregates, and apo-AS-SOD1 formed amyloid-like aggregates at pH 7 only when an ALS mutation was also present. These results indicate that some mutations related to ALS promote formation of amyloid-like aggregates by facilitating the loss of metals and/or by making the intramolecular disulfide bond more susceptible to reduction, thus allowing the conversion of SOD1 to a form that aggregates to form resembling amyloid. Furthermore, the occurrence of amyloid-like aggregates per se does not depend on forming intermolecular disulfide bonds, and multiple forms of such aggregates can be produced from SOD1.

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTStructure and arrangement of the regulatory subunits in aspartate transcarbamylaseJeffrey A. Cohlberg, Vincent P. Pigiet, Jr., and H. K. SchachmanCite this: Biochemistry 1972, 11, 18, 3396–3411Publication Date (Print):August 1, 1972Publication History Published online1 May 2002Published inissue 1 August 1972https://pubs.acs.org/doi/10.1021/bi00768a013https://doi.org/10.1021/bi00768a013research-articleACS PublicationsRequest reuse permissionsArticle Views73Altmetric-Citations106LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InRedditEmail Other access optionsGet e-Alertsclose Get e-Alerts

Mutations in the gene encoding human copper-zinc superoxide dismutase (SOD1) cause a dominant form of the progressive neurodegenerative disease amyotrophic lateral sclerosis. Transgenic mice expressing the human G85R SOD1 variant develop paralytic symptoms concomitant with the appearance of SOD1-enriched proteinaceous inclusions in their neural tissues. The process(es) through which misfolding or aggregation of G85R SOD1 induces motor neuron toxicity is not understood. Here we present structures of the human G85R SOD1 variant determined by single crystal x-ray diffraction. Alterations in structure of the metal-binding loop elements relative to the wild type enzyme suggest a molecular basis for the metal ion deficiency of the G85R SOD1 protein observed in the central nervous system of transgenic mice and in purified recombinant G85R SOD1. These findings support the notion that metal-deficient and/or disulfide-reduced mutant SOD1 species contribute to toxicity in SOD1-linked amyotrophic lateral sclerosis. Mutations in the gene encoding human copper-zinc superoxide dismutase (SOD1) cause a dominant form of the progressive neurodegenerative disease amyotrophic lateral sclerosis. Transgenic mice expressing the human G85R SOD1 variant develop paralytic symptoms concomitant with the appearance of SOD1-enriched proteinaceous inclusions in their neural tissues. The process(es) through which misfolding or aggregation of G85R SOD1 induces motor neuron toxicity is not understood. Here we present structures of the human G85R SOD1 variant determined by single crystal x-ray diffraction. Alterations in structure of the metal-binding loop elements relative to the wild type enzyme suggest a molecular basis for the metal ion deficiency of the G85R SOD1 protein observed in the central nervous system of transgenic mice and in purified recombinant G85R SOD1. These findings support the notion that metal-deficient and/or disulfide-reduced mutant SOD1 species contribute to toxicity in SOD1-linked amyotrophic lateral sclerosis. Superoxide ( O2.¯) is a reactive byproduct of mitochondrial respiration and fatty acid oxidation that can damage critical cellular components. To protect against such such copper-zinc superoxide dismutase (SOD1) copper-zinc superoxide amyotrophic lateral for SOD1. copper-zinc superoxide amyotrophic lateral for SOD1. and that to superoxide to molecular and metal SOD1 and of the protein in and and against the superoxide by tissues. of SOD1 can the by in the human SOD1 gene that to of the neurodegenerative disease amyotrophic lateral in transgenic mice that SOD1 motor neuron through the of a and not a of inclusions in SOD1 observed in transgenic and in that SOD1-linked to protein misfolding or aggregation the molecular SOD1 toxicity to motor and to the observed inclusions or of motor neuron the of the the human SOD1 variant in which is for in transgenic G85R SOD1 progressive motor neuron in the of inclusions in with the of paralytic symptoms in mice and in the disease the disease inclusions in SOD1 to in the motor in motor neuron with a that the G85R metal ion and the relative to wild type recombinant G85R SOD1 observed to metal-deficient a basis relative to the wild type enzyme in the system in mice that of the G85R SOD1 protein in the of is with and we present structures of the human G85R variant determined by single crystal x-ray diffraction. The structures suggest a molecular basis for in metal and a which the metal deficiency and SOD1 toxicity can and encoding mutant G85R human SOD1 by and the which the of the protein the of G85R SOD1 in the of that the the of protein by the the the that the not and a a to the protein the to to the which in and the in the G85R SOD1 in with of in and in the and and G85R protein in a of in the in the x-ray the and a x-ray with system The and The and in structures determined molecular with the SOD1 structure the The the in of the and in the of the a of to a to or and and in for and The for structures and The a of the and the with and The a of the and the with and the and to in the of and in the G85R SOD1 in with of in and and in the and G85R SOD1 in a of with a in the and of the structures determined with the wild type human SOD1 the The the in of the and in the of the the to a to or and for structures and The a of the and with and The a of the and with and and to in the of and metal-binding 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molecular structure a ion G85R G85R and with in a The metal is to through the that G85R SOD1 the and to the by a and a of SOD1 and in G85R in of the human G85R SOD1 to in the with that the human G85R SOD1 variant and the G85R SOD1 to to and the by a of G85R SOD1 to the of mutant to the of SOD1 and the with of G85R and or of ion in the and that G85R SOD1 the to their G85R for the of the G85R to the which of and and the of with the of of the wild type wild that the with of the wild type in the of the loop the in and the of with the the of and the of a with the and the loop to the loop the is to in and disulfide-reduced SOD1 The loop and which in the is to the in to which and in the form of the is the and with the of a in the loop and for to a a the loop to the is to a that can protein by or in the of or a loop or of the of to The loop to the to and the loop to The of the that can with or the of the of the that the of G85R that or and in loop that and loop and loop observed in SOD1 molecular and in the of the of in the can loop and the wild type of a of the of the by the and to in the protein the a to the in and to the of to and the of the wild type the of is to the of of the and and the of to the the of of the loop the of the and the and is critical to by the and against for The SOD1 mutant is to G85R and in that metal in to the and in is the the of the or the of the metal-binding through with and that to the the by the of the of to the to critical to the of the and loop and is not that loop elements in G85R in G85R and with in a G85R SOD1 not observed to in wild type SOD1 structure determined to is observed to through and in the crystal structure of the SOD1 which is that to metal-binding in the is the observed in is to metal such and in and and to a in in a of that cause human neurodegenerative and for a G85R and for SOD1 that the the of the concomitant with ion to the SOD1 protein The of to the is to a for of SOD1 by and is to that G85R SOD1 and not to their to The that is to the in that in to the protein to to or The of G85R SOD1 for a of G85R SOD1 that is not to with through is that the G85R with the in the of SOD1 by which in a disulfide-reduced of with the of and in the central nervous system of transgenic mice expressing the G85R SOD1 and G85R SOD1 in the and loop elements in human SOD1 is to in of of of the which can in to that in the of and that the disulfide-reduced wild type SOD1 protein a and disulfide-reduced SOD1 to to for and aggregation a and in and and a SOD1 in oxidation of SOD1 aggregation and the of the with the of to protect against aggregation of the mutant to the SOD1 to the against and to protect against aggregation in that the SOD1 and the of SOD1 in the proteinaceous inclusions of transgenic mice expressing the SOD1 and in the SOD1 protein motor that the disulfide-reduced wild type SOD1 protein a the SOD1 such and in that is in with the of human G85R in by and that G85R SOD1 protein in central nervous enzyme and in the central nervous system relative to and of disulfide-reduced SOD1 in the SOD1 in transgenic that not the in and a with a with the that the mice with a of with for mice expressing SOD1 the that in the neural of the mice and that the of to the of disulfide-reduced and not SOD1 to support the notion that the disulfide-reduced SOD1 species that the in SOD1-linked the of toxicity the the G85R SOD1 variant suggest a molecular basis for the metal ion deficiency observed for protein in in and in metal deficiency is to for the G85R SOD1 protein to a disulfide-reduced species that is the in SOD1-linked and a of the to by Superoxide ( O2.¯) is a reactive byproduct of mitochondrial respiration and fatty acid oxidation that can damage critical cellular components. To protect against such such copper-zinc superoxide dismutase (SOD1) copper-zinc superoxide amyotrophic lateral for SOD1. copper-zinc superoxide amyotrophic lateral for SOD1. and that to superoxide to molecular and metal SOD1 and of the protein in and and against the superoxide by tissues. of SOD1 can the by in the human SOD1 gene that to of the neurodegenerative disease amyotrophic lateral in transgenic mice that SOD1 motor neuron through the of a and not a of inclusions in SOD1 observed in transgenic and in that SOD1-linked to protein misfolding or aggregation the molecular SOD1 toxicity to motor and to the observed inclusions or of motor neuron the of the the human SOD1 variant in which is for in transgenic G85R SOD1 progressive motor neuron in the of inclusions in with the of paralytic symptoms in mice and in the disease the disease inclusions in SOD1 to in the motor in motor neuron with a that the G85R metal ion and the relative to wild type recombinant G85R SOD1 observed to metal-deficient a basis relative to the wild type enzyme in the system in mice that of the G85R SOD1 protein in the of is with and Here we present structures of the human G85R variant determined by single crystal x-ray diffraction. The structures suggest a molecular basis for in metal and a which the metal deficiency and SOD1 toxicity can and encoding mutant G85R human SOD1 by and the which the of the protein the of G85R SOD1 in the of that the the of protein by the the the that the not and a a to the protein the to to the which in and the in the G85R SOD1 in with of in and in the and and G85R protein in a of in the in the x-ray the and a x-ray with system The and The and in structures determined molecular with the SOD1 structure the The the in of the and in the of the a of to a to or and and in for and The for structures and The a of the and the with and The a of the and the with and the and to in the of and in the G85R SOD1 in with of in and and in the and G85R SOD1 in a of with a in the and of the structures determined with the wild type human SOD1 the The the in of the and in the of the the to a to or and for structures and The a of the and with and The a of the and with and and to in the of and metal-binding of and of and to by of the The of the metal with of the and/or acid and with the observed in structures and the ion G85R SOD1 is a a of in and in The The is the SOD1 to through a and a of The ion and in These the crystal the The ion in the is a the the of the in the to the in the with a and the The wild type enzyme is in and the G85R SOD1 mutant is in The to in the G85R structure is a and encoding mutant G85R human SOD1 by and the which the of the protein the of G85R SOD1 in the of that the the of protein by the the the that the not and a a to the protein the to to the which in and the in the G85R SOD1 in with of in and in the and and G85R protein in a of in the in the x-ray the and a x-ray with system The and The and in The structures determined molecular with the SOD1 structure the The the in of the and in the of the a of to a to or and and in for and The for structures and The a of the and the with and The a of the and the with and the and to in the of and in the G85R SOD1 in with 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SOD1 and of structure and of the G85R SOD1 in to of structure and of structure a of metal in the in the G85R a of and to the These in metal by G85R SOD1 cause in their which in by the crystal form in which in and that the in the and loop elements that the to structures and the the x-ray for structure the for structure structure is metal-deficient relative to structure in metal-binding metal ion a of metal is observed in wild type human SOD1 G85R observed the G85R the of the wild type The observed of the to the G85R with the metal in in for a of the molecular structure a ion G85R G85R and with in a The metal is to through the that G85R SOD1 the and to the by a and a of SOD1 of G85R SOD1 and a of the human wild type SOD1 and and loop elements the to form the of the and in metal The the of the The the a by the metal-binding The loop acid that the to the the of loop is to the through a and The loop and the of the and the of the SOD1 x-ray crystal structures of the human G85R mutant determined to the and for or to the protein in crystal by of to the and The of is by a metal ion and the metal to in and of the The of to the not a to the metal the x-ray the and G85R a The in the of metal in of G85R SOD1 and of structure and of the G85R SOD1 in to of structure and of structure a of metal in the in the G85R a of and to the These in metal by G85R SOD1 cause in their which in by the crystal form in which in and that the in the and loop elements that the The to structures and the the x-ray for structure the for structure structure is metal-deficient relative to structure in metal-binding metal ion a of metal is observed in wild type human SOD1 The G85R observed the G85R the of the wild type The observed of the to the G85R with the metal in in for a of the molecular structure a ion G85R G85R and with in a The metal is to through the that G85R SOD1 the and to the by a and a of SOD1 and in G85R in of the human G85R SOD1 to in the with that the human G85R SOD1 variant and the G85R SOD1 to to and the by a of G85R SOD1 to the of mutant to the of SOD1 and the with of G85R and or of ion in the and that G85R SOD1 the to their G85R for the of the G85R to the which of and and the of with the of of the wild type wild that the with of the wild type in the of the loop the in and the of with the the of and the of a with the and the loop to the loop the is to in and disulfide-reduced SOD1 The loop and which in the is to the in to which and in the form of the is the and with the of a in the loop and for to a a the loop to the is to a that can protein by or in the of or a loop or of the of to The loop to the to and the loop to The of the that can with or the of the of the that the of G85R that or and in loop that and loop and loop observed in SOD1 molecular and in the of the of in the can loop and the wild type of a of the of the by the and to in the protein the a to the in and to the of to and the of the wild type the of is to the of of the and and the of to the the of of the loop the of the and the and is critical to by the and against for The SOD1 mutant is to G85R and in that metal in to the and in is the the of the or the of the metal-binding through with and that to the the by the of the of to the to critical to the of the and loop and is not that loop elements in G85R in G85R and with in a G85R SOD1 not observed to in wild type SOD1 structure determined to is observed to through and in the crystal structure of the SOD1 which is that to metal-binding in the is the observed in is to metal such and in and and to a in in a of that cause human neurodegenerative and for a G85R and for SOD1 that the the of the concomitant with ion to the SOD1 protein The of to the is to a for of SOD1 by and is to that G85R SOD1 and not to their to The that is to the in that in to the protein to to or The of G85R SOD1 for a of G85R SOD1 that is not to with through is that the G85R with the in the of SOD1 by which in a disulfide-reduced of with the of and in the central nervous system of transgenic mice expressing the G85R SOD1 and G85R SOD1 in the and loop elements in human SOD1 is to in of of of the which can in to that in the of and that the disulfide-reduced wild type SOD1 protein a and disulfide-reduced SOD1 to to for and aggregation a and in and and a SOD1 in oxidation of SOD1 aggregation and the of the with the of to protect against aggregation of the mutant to the SOD1 to the against and to protect against aggregation in that the SOD1 and the of SOD1 in the proteinaceous inclusions of transgenic mice expressing the SOD1 and in the SOD1 protein motor that the disulfide-reduced wild type SOD1 protein a the SOD1 such and in that is in with the of human G85R in by and that G85R SOD1 protein in central nervous enzyme and in the central nervous system relative to and of disulfide-reduced SOD1 in the SOD1 in transgenic that not the in and a with a with the that the mice with a of with for mice expressing SOD1 the that in the neural of the mice and that the of to the of disulfide-reduced and not SOD1 to support the notion that the disulfide-reduced SOD1 species that the in SOD1-linked the of toxicity the the G85R SOD1 variant suggest a molecular basis for the metal ion deficiency observed for protein in in and in metal deficiency is to for the G85R SOD1 protein to a disulfide-reduced species that is the in SOD1-linked and a of the to by and in G85R in of the human G85R SOD1 to in the with that the human G85R SOD1 variant and the G85R SOD1 to to and the by a of G85R SOD1 to the of mutant to the of SOD1 and the with of G85R and or of ion in the and that G85R SOD1 the to their The G85R for the of the G85R to the which of and and the of with the of of the wild type wild that the with of the wild type in the of the loop the in and the of with the the of and the of a with the and the loop to the The loop the is to in and disulfide-reduced SOD1 The loop and which in the is to the in to which and in the form of the is the and with the of a in the loop and for to a a the loop to the is to a that can protein by or in the of or a loop or of the of to The loop to the to and the loop to The of the that can with or the of the of the that the of G85R that or and in loop that and loop and loop observed in SOD1 molecular and in the of the of in the can loop and the wild type of a of the of the by the and to in the protein the a to the in and to the of to and the of the wild type the of is to the of of the and and the of to the the of of the loop the of the and the and is critical to by the and against for The SOD1 mutant is to G85R and in that metal in to the and in is the the of the or the of the metal-binding through with and that to the the by the of the of to the to critical to the of the and loop and is not that loop elements in G85R SOD1. in G85R and with in a G85R SOD1 not observed to in wild type SOD1 structure determined to is observed to through and in the crystal structure of the SOD1 which is that to metal-binding in the is the observed in is to metal such and in and and to a in in a of that cause human neurodegenerative and for a The G85R and for SOD1 that the the of the concomitant with ion to the SOD1 protein The of to the is to a for of SOD1 by and is to that G85R SOD1 and not to their to The that is to the in that in to the protein to to or The of G85R SOD1 for a of G85R SOD1 that is not to with through is that the G85R with the in the of SOD1 by which in a disulfide-reduced of with the of and in the central nervous system of transgenic mice expressing the G85R SOD1 and G85R SOD1 in the and loop elements in human SOD1 is to in of of of the which can in to that in the of and that the disulfide-reduced wild type SOD1 protein a and disulfide-reduced SOD1 to to for and aggregation a and in and and a SOD1 in oxidation of SOD1 aggregation and the of the with the of to protect against aggregation of the mutant to the SOD1 to the against and to protect against aggregation in that the SOD1 and the of SOD1 in the proteinaceous inclusions of transgenic mice expressing the SOD1 and in the SOD1 protein motor that the disulfide-reduced wild type SOD1 protein a the SOD1 such and in that is in with the of human G85R in by and that G85R SOD1 protein in central nervous enzyme and in the central nervous system relative to and of disulfide-reduced SOD1 in tissues. the SOD1 in transgenic that not the in and a with a with the that the mice with a of with for mice expressing SOD1 the that in the neural of the mice and that the of to the of disulfide-reduced and not SOD1 to support the notion that the disulfide-reduced SOD1 species that the in SOD1-linked the of toxicity the the G85R SOD1 variant suggest a molecular basis for the metal ion deficiency observed for protein in in and in metal deficiency is to for the G85R SOD1 protein to a disulfide-reduced species that is the in SOD1-linked and a of the to by for the by The of and the is to for and to in the and for support of and the of