Papers - Furukawa, Yoshiaki

Cysteine residues in Cu,Zn-superoxide dismutase are essential to toxicity in Caenorhabditis elegans model of amyotrophic lateral sclerosis

Ogawa, Mariko, Shidara, Hisashi, Oka, Kotaro, Kurosawa, Masaru, Nukina, Nobuyuki, Furukawa, Yoshiaki

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 463 ( 4 ) 1196 - 1202 2015.08

Research paper (scientific journal), Joint Work, Accepted,  ISSN  0006-291X

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Conformational Disorder of the Most Immature Cu, Zn-Superoxide Dismutase Leading to Amyotrophic Lateral Sclerosis

Furukawa, Yoshiaki, Anzai, Itsuki, Akiyama, Shuji, Imai, Mizue, Cruz, Fatima Joy C., Saio, Tomohide, Nagasawa, Kenichi, Nomura, Takao, Ishimori, Koichiro

JOURNAL OF BIOLOGICAL CHEMISTRY 291 ( 8 ) 4144 - 4155 2016.02

Research paper (scientific journal), Joint Work, Accepted,  ISSN  0021-9258

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A molecular mechanism realizing sequence-specific recognition of nucleic acids by TDP-43

Furukawa, Yoshiaki, Suzuki, Yoh, Fukuoka, Mami, Nagasawa, Kenichi, Nakagome, Kenta, Shimizu, Hideaki, Mukaiyama, Atsushi, Akiyama, Shuji

SCIENTIFIC REPORTS 6   4144 - 4155 2016.02

Research paper (scientific journal), Joint Work, Accepted,  ISSN  2045-2322

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Oxidative denaturation of Cu/Zn-superoxide dismutase associated with neurodegenerative diseases

Yoshida M., Muraki N., Tajiri M., Hengphasatporn K., Sue K., Kubo T., Akashi S., Shigeta Y., Kambe T., Furukawa Y.

Protein Science 34 ( 11 )  2025.11

ISSN  09618368

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Misfolding of mutant Cu/Zn-superoxide dismutase (SOD1) is a well-established pathological feature of familial amyotrophic lateral sclerosis (ALS). While amino acid substitutions in mutant SOD1 destabilize its structure and promote misfolding, oxidation has also been implicated in the pathological alterations of wild-type SOD1, particularly in neurodegenerative diseases including sporadic ALS. However, the impact of oxidation on SOD1 folding remains to be fully elucidated. Here, we demonstrate that Cys111 is primarily oxidized to sulfonic acid upon exposure of apo-SOD1 to hydrogen peroxide, as confirmed by the quantitation of thiol groups and mass spectrometry. Molecular dynamics simulations showed that sulfonylation of Cys111 disrupts the dimer interface and promotes monomerization. This monomeric form then facilitates the subsequent oxidation of buried Cys6, leading to structural disruption, as evidenced by circular dichroism spectroscopy and loss of thiol groups. SOD1 denaturation triggered by Cys111 oxidation became evident when zinc binding was impaired due to pathological mutations and/or under zinc-deficient conditions. Given that increased oxidative stress is frequently associated with many neurodegenerative diseases, modulating Cys111 oxidation may offer a potential strategy for maintaining SOD1 structural stability and preventing its pathological misfolding.

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Cu/Zn-superoxide dismutase naturally fused with a β-propeller lactonase in Deinococcus radiodurans.

Yoshiaki Furukawa, Masamichi Megata, Atsuko Shintani, Kaori Sue, Tomohiro Morohoshi, Masato Akutsu, Norifumi Muraki

The Journal of biological chemistry 301 ( 8 ) 110499 - 110499 2025.08

ISSN  00219258

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Cu/Zn-superoxide dismutase (Cu/Zn-SOD) is an antioxidant enzyme widely present across species; however, the structural diversity and physiological roles of Cu/Zn-SOD are yet to be fully uncovered. Here, we show a unique type of Cu/Zn-SOD from Deinococcus radiodurans (DrSOD) with an additional β-propeller domain. Our structural analysis on DrSOD revealed a typical bacterial Cu/Zn-SOD domain, binding both a copper and zinc ion, alongside a six-bladed β-propeller domain coordinating a calcium ion. DrSOD was indeed expressed in D. radiodurans, but its deletion did not lead to any noticeable changes in resistance to DNA-damaging stresses, a characteristic trait of D. radiodurans. Despite this, the Cu/Zn-SOD domain retained superoxide dismutase activity, and the β-propeller domain was found to exhibit a lactonase activity specifically for hydrolyzing 2-coumaranone. Taken together, while the precise physiological role of DrSOD needs to be further investigated, our findings here reveal a unique multi-functional enzyme architecture, expanding the known structural diversity of Cu/Zn-SODs.

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Metal-Responsive Up-Regulation of Bifunctional Disulfides for Suppressing Protein Misfolding and Promoting Oxidative Folding.

Keita Mori, Tsubura Kuramochi, Motonori Matsusaki, Yuki Hashiguchi, Masaki Okumura, Tomohide Saio, Yoshiaki Furukawa, Kenta Arai, Takahiro Muraoka

Angewandte Chemie (International ed. in English) 64 ( 36 ) e202502187 2025

ISSN  14337851

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The stress-responsive up-regulation process is a sophisticated biological response to maintain cellular homeostasis. In intracellular anti-oxidant systems, the expression level of oxidoreductases is up-regulated under oxidative stress, mitigating oxidative damage on biomolecules and enhancing protein folding capacity. Herein, inspired by the biological system, we developed a synthetic folding promotor whose reactivity is up-regulated under stress conditions. We conjugated two metal-binding 1,4,7,11-tetraazacyclotetradecane (cyclam) ligands and a redox-active disulfide to obtain cyclam-SS, whose reactivity can be enhanced under metal-induced stress. Metal coordination increased the redox potential of cyclam-SS, activating it as an oxidant. While CuII ions severely hampered the oxidative folding of substrate polypeptides, cyclam-SS exhibited bifunctional folding-promoting properties, i) suppressing CuII-mediated misfolding and aggregation, and ii) harnessing CuII to enhance oxidative folding. Cyclam-SS was also useful for disulfide-bond formation to promote oxidative folding of pharmaceutical and pathological proteins, as demonstrated with proinsulin and superoxide dismutase 1 (SOD1). Furthermore, cyclam-SS protected cultured cells from copper-induced stress. Thus, we demonstrated the induction of the stress-responsive up-regulation process by a bifunctional folding promotor controlling the folding status of biologically important proteins under metal-induced stress. The strategy of "stress-responsive up-regulation" could aid the development of novel synthetic materials for treating intracellular stress and related disorders.

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Disulfide-mediated oligomerization of mutant Cu/Zn-superoxide dismutase associated with canine degenerative myelopathy.

Yuki Shino, Norifumi Muraki, Yui Kobatake, Hiroaki Kamishina, Ryuichi Kato, Yoshiaki Furukawa

Protein science : a publication of the Protein Society 33 ( 12 ) e5210 2024.12

ISSN  09618368

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A homozygous E40K mutation in the gene coding canine Cu/Zn-superoxide dismutase (cSOD1) causes degenerative myelopathy (DM) in dogs. A pathological hallmark of DM with the cSOD1 mutation is the aggregation of mutant cSOD1 proteins in neurons. The amino acid substitution E40K disrupts a salt bridge between Glu40 and Lys91 and is considered to destabilize the native state of cSOD1; however, the mechanism by which mutant cSOD1 aggregates remains unclear. Here, we show that mutant cSOD1 losing a copper and zinc ion forms oligomers crosslinked via disulfide bonds. The E40K substitution was found to result in the increased solvent exposure of the Cys7 side chain, which then attacked the disulfide bond (Cys57-Cys146) in cSOD1 to form disulfide-linked oligomers. We also successfully prevented the Cys7 exposure and thus the oligomerization of mutant cSOD1 by a fragment antibody that specifically recognizes the region around the mutation site. The fragment antibody covered the β-plug region, reinforcing the interactions compromised by the E40K substitution and thus contributing to the maintenance of the structural integrity of the β-barrel core of cSOD1. Taken together, we propose that the Cys7 exposure in cSOD1 upon the salt bridge disruption plays a central role in the aggregation mechanism of DM-associated mutant cSOD1.

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Engineering a monobody specific to monomeric Cu/Zn-superoxide dismutase associated with amyotrophic lateral sclerosis.

Hiroshi Amesaka, Mizuho Hara, Yuki Sakai, Atsuko Shintani, Kaori Sue, Tomoyuki Yamanaka, Shun-Ichi Tanaka, Yoshiaki Furukawa

Protein science : a publication of the Protein Society 33 ( 4 ) e4961 2024.04

ISSN  09618368

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Misfolding of mutant Cu/Zn-superoxide dismutase (SOD1) has been implicated in familial form of amyotrophic lateral sclerosis (ALS). A natively folded SOD1 forms a tight homodimer, and the dimer dissociation has been proposed to trigger the oligomerization/aggregation of SOD1. Besides increasing demand for probes allowing the detection of monomerized forms of SOD1 in various applications, the development of probes has been limited to conventional antibodies. Here, we have developed Mb(S4) monobody, a small synthetic binding protein based on the fibronectin type III scaffold, that recognizes a monomeric but not dimeric form of SOD1 by performing combinatorial library selections using phage and yeast-surface display methods. Although Mb(S4) was characterized by its excellent selectivity to the monomeric conformation of SOD1, the monomeric SOD1/Mb(S4) complex was not so stable (apparent Kd ~ μM) as to be detected in conventional pull-down experiments. Instead, the complex of Mb(S4) with monomeric but not dimeric SOD1 was successfully trapped by proximity-enabled chemical crosslinking even when reacted in the cell lysates. We thus anticipate that Mb(S4) binding followed by chemical crosslinking would be a useful strategy for in vitro and also ex vivo detection of the monomeric SOD1 proteins.

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Characterization of a novel cysteine-less Cu/Zn-superoxide dismutase in Paenibacillus lautus missing a conserved disulfide bond.

Yoshiaki Furukawa, Atsuko Shintani, Shuhei Narikiyo, Kaori Sue, Masato Akutsu, Norifumi Muraki

The Journal of biological chemistry 299 ( 8 ) 105040 - 105040 2023.08

ISSN  00219258

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Cu/Zn-superoxide dismutase (CuZnSOD) is an enzyme that binds a copper and zinc ion and also forms an intramolecular disulfide bond. Together with the copper ion as the active site, the disulfide bond is completely conserved among these proteins; indeed, the disulfide bond plays critical roles in maintaining the catalytically competent conformation of CuZnSOD. Here, we found that a CuZnSOD protein in Paenibacillus lautus (PaSOD) has no Cys residue but exhibits a significant level of enzyme activity. The crystal structure of PaSOD revealed hydrophobic and hydrogen-bonding interactions in substitution for the disulfide bond of the other CuZnSOD proteins. Also notably, we determined that PaSOD forms a homodimer through an additional domain with a novel fold at the N terminus. While the advantages of lacking Cys residues and adopting a novel dimer configuration remain obscure, PaSOD does not require a disulfide-introducing/correcting system for maturation and could also avoid misfolding caused by aberrant thiol oxidations under an oxidative environment.

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Intrinsic structural vulnerability in the hydrophobic core induces species-specific aggregation of canine SOD1 with degenerative myelopathy-linked E40K mutation.

Kei Hashimoto, Seiji Watanabe, Masato Akutsu, Norifumi Muraki, Hiroaki Kamishina, Yoshiaki Furukawa, Koji Yamanaka

The Journal of biological chemistry 299 ( 6 ) 104798 - 104798 2023.06

ISSN  00219258

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Canine degenerative myelopathy (DM), a fatal neurodegenerative disease in dogs, shares clinical and genetic features with amyotrophic lateral sclerosis (ALS), a human motor neuron disease. Mutations in the SOD1 gene encoding Cu/Zn superoxide dismutase (SOD1) cause canine DM and a subset of inherited human ALS. The most frequent DM causative mutation is homozygous E40K mutation which induces the aggregation of canine SOD1 but not of human SOD1. However, the mechanism through which canine E40K mutation induces species-specific aggregation of SOD1 remains unknown. By screening human/canine chimeric SOD1s, we identified that the humanized mutation of the 117th residue (M117L), encoded by exon 4, significantly reduced aggregation propensity of canine SOD1E40K. Conversely, introducing a mutation of leucine 117 to methionine, a residue homologous to canine, promoted E40K-dependent aggregation in human SOD1. M117L mutation improved protein stability and reduced cytotoxicity of canine SOD1E40K. Furthermore, crystal structural analysis of canine SOD1 proteins revealed that M117L increased the packing within the hydrophobic core of the β-barrel structure, contributing to the increased protein stability. Our findings indicate that the structural vulnerability derived intrinsically from Met 117 in the hydrophobic core of the β-barrel structure induces E40K-dependent species-specific aggregation in canine SOD1.

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Zinc-mediated interaction of copper chaperones through their heavy-metal associated domains

Furukawa Y., Matsumoto K., Nakagome K., Shintani A., Sue K.

Journal of Trace Elements in Medicine and Biology 75 2023.01

ISSN  0946672X

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Background: A copper chaperone CCS is a multi-domain protein that supplies a copper ion to Cu/Zn-superoxide dismutase (SOD1). Among the domains of CCS, the N-terminal domain (CCS^dI) belongs to a heavy metal-associated (HMA) domain, in which a Cys-x-x-Cys (CxxC) motif binds a heavy metal ion. It has hence been expected that the HMA domain in CCS has a role in the metal trafficking; however, the CxxC motif in the domain is dispensable for supplying a copper ion to SOD1, leaving an open question on roles of CCS^dI in CCS. Methods: To evaluate protein-protein interactions of CCS through CCS^dI, yeast two-hybrid assay, a pull-down assay using recombinant proteins, and the analysis with fluorescence resonance energy transfer were performed. Results: We found that CCS specifically interacted with another copper chaperone HAH1, a HMA domain protein, through CCS^dI. The interaction between CCS^dI and HAH1 was not involved in the copper supply from CCS to SOD1 but was mediated by a zinc ion ligated with Cys residues of the CxxC motifs in CCS^dI and HAH1. Conclusion: While physiological significance of the interaction between copper chaperones awaits further investigation, we propose that CCS^dI would have a role in the metal-mediated interaction with other proteins including heterologous copper chaperones.

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Quantum-dot-labeled synuclein seed assay identifies drugs modulating the experimental prion-like transmission.

Yukio Imamura, Ayami Okuzumi, Saki Yoshinaga, Akiko Hiyama, Yoshiaki Furukawa, Tomohiro Miyasaka, Nobutaka Hattori, Nobuyuki Nukina

Communications biology 5 ( 1 ) 636 - 636 2022.12

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Synucleinopathies are neurodegenerative disorders including Parkinson disease (PD), dementia with Lewy body (DLB), and multiple system atrophy (MSA) that involve deposits of the protein alpha-synuclein (α-syn) in the brain. The inoculation of α-syn aggregates derived from synucleinopathy or preformed fibrils (PFF) formed in vitro induces misfolding and deposition of endogenous α-syn. This is referred to as prion-like transmission, and the mechanism is still unknown. In this study, we label α-syn PFF with quantum dots and visualize their movement directly in acute slices of brain tissue inoculated with α-syn PFF seeds. Using this system, we find that the trafficking of α-syn seeds is dependent on fast axonal transport and the seed spreading is dependent on endocytosis and neuronal activity. We also observe pharmacological effects on α-syn seed spreading; clinically available drugs including riluzole are effective in reducing the spread of α-syn seeds and this effect is also observed in vivo. Our quantum-dot-labeled α-syn seed assay system combined with in vivo transmission experiment reveals an early phase of transmission, in which uptake and spreading of seeds occur depending on neuronal activity, and a later phase, in which seeds induce the propagation of endogenous misfolded α-syn.

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FUS regulates RAN translation through modulating the G-quadruplex structure of GGGGCC repeat RNA inC9orf72-linked ALS/FTD

Yuzo Fujino, Morio Ueyama, Taro Ishiguro, Daisaku Ozawa, Toshihiko Sugiki, Hayato Ito, Asako Murata, Akira Ishiguro, Tania F. Gendron, Kohji Mori, Eiichi Tokuda, Tomoya Taminato, Takuya Konno, Akihide Koyama, Yuya Kawabe, Toshihide Takeuchi, Yoshiaki Furukawa, Toshimichi Fujiwara, Manabu Ikeda, Toshiki Mizuno, Hideki Mochizuki, Hidehiro Mizusawa, Keiji Wada, Kinya Ishikawa, Osamu Onodera, Kazuhiko Nakatani, Hideki Taguchi, Leonard Petrucelli, Yoshitaka Nagai

(eLife Sciences Publications, Ltd)   2022.11

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Abnormal expansions of GGGGCC repeat sequence in the noncoding region of the C9orf72 gene is the most common cause of familial amyotrophic lateral sclerosis and frontotemporal dementia (C9-ALS/FTD). The expanded repeat sequence is translated into dipeptide repeat proteins (DPRs) by noncanonical repeat-associated non-AUG (RAN) translation. Since DPRs play central roles in the pathogenesis of C9-ALS/FTD, we here investigate the regulatory mechanisms of RAN translation, focusing on the effects of RNA-binding proteins (RBPs) targeting GGGGCC repeat RNAs. Using C9-ALS/FTD model flies, we demonstrated that the ALS/FTD-linked RBP FUS suppresses RAN translation and neurodegeneration in an RNA-binding activity-dependent manner. Moreover, we found that FUS directly binds to and modulates the G-quadruplex structure of GGGGCC repeat RNA as an RNA chaperone, resulting in the suppression of RAN translation in vitro. These results reveal a previously unrecognized regulatory mechanism of RAN translation by G-quadruplex-targeting RBPs, providing therapeutic insights for C9-ALS/FTD and other repeat expansion diseases.

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A rapid and simple electrochemical detection of the free drug concentration in human serum using boron-doped diamond electrodes

Moriyama H., Ogata G., Nashimoto H., Sawamura S., Furukawa Y., Hibino H., Kusuhara H., Einaga Y.

Analyst 147 ( 20 ) 4442 - 4449 2022.09

ISSN  00032654

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Monitoring drug concentration in blood and reflecting this in the dosage are crucial for safe and effective drug treatment. Most drug assays are based on total concentrations of bound and unbound proteins in the serum, although only the unbound concentration causes beneficial and adverse events. Monitoring the unbound concentration alone is expected to provide a means for further optimisation of drug treatment. However, unbound concentration monitoring has not been routinely used for drug treatment due to the long analysis time and the high cost of conventional methods. Here, we have developed a rapid electrochemical method to determine the unbound concentration in ultrafiltered human serum using boron-doped diamond (BDD) electrodes. When the anticancer drug doxorubicin was used as the test drug, the catalytic doxorubicin-mediated reduction of dissolved oxygen provided a sensitive electrochemical signal, with a detection limit of 0.14 nM. In contrast, the sensitivity of glassy carbon (GC) was inferior under the same conditions due to interference from the dissolved oxygen reduction current. The signal background ratio (S/B) of BDD and GC was 11.5 (10 nM doxorubicin) and 1.1 (50 nM), respectively. The results show that a fast measurement time within ten seconds is possible in the clinical concentration range. Additionally, in the ultrafiltered human serum, the obtained values of unbound doxorubicin concentration showed good agreement with those quantified by conventional liquid chromatography-mass spectrometry. This approach has the potential for application in clinical settings where rapid and simple analysis methods would be beneficial.

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Novel oxindole compounds inhibit the aggregation of amyloidogenic proteins associated with neurodegenerative diseases.

Shintaro Kimura, Hiroaki Kamishina, Yoko Hirata, Kyoji Furuta, Yoshiaki Furukawa, Osamu Yamato, Sadatoshi Maeda, Yuji O Kamatari

Biochimica et biophysica acta. General subjects (Biochimica et Biophysica Acta - General Subjects)  1866 ( 5 ) 130114 - 130114 2022.05

ISSN  03044165

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Amyloidogenic proteins form aggregates in cells, thereby leading to neurodegenerative disorders, including Alzheimer's and prion's disease, amyotrophic lateral sclerosis (ALS) in humans, and degenerative myelopathy (DM) and cognitive dysfunction in dogs. Hence, many small-molecule compounds have been screened to examine their inhibitory effects on amyloidogenic protein aggregation. However, no effective drug suitable for transition to clinical use has been found. Here we examined several novel oxindole compounds (GIF compounds) for their inhibitory effects on aggregate formation of the canine mutant superoxide dismutase 1 (cSOD1 E40K), a causative mutation resulting in DM, using Thioflavin-T fluorescence. Most GIF compounds inhibited the aggregation of cSOD1 E40K. Among the compounds, GIF-0854-r and GIF-0890-r were most effective. Their inhibitory effects were also observed in cSOD1 E40K-transfected cells. Additionally, GIF-0890-r effectively inhibited the aggregate formation of human SOD1 G93A, a causative mutation of ALS. GIF-0827-r and GIF-0856-r also effectively inhibited aggregate formation of human prion protein (hPrP). Subsequently, the correlation between their inhibitory effects on cSOD1 and hPrP aggregation was shown, indicating GIF compounds inhibited the aggregate formation of multiple amyloidogenic proteins. Conclusively, the novel oxindole compounds (GIF-0827-r, GIF-0854-r, GIF-0856-r, and GIF-0890-r) are proposed as useful therapeutic candidates for amyloidogenic neurodegenerative disorders.

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Metal distribution in Cu/Zn-superoxide dismutase revealed by native mass spectrometry.

Michiko Tajiri, Hiroto Aoki, Atsuko Shintani, Kaori Sue, Satoko Akashi, Yoshiaki Furukawa

Free radical biology & medicine (Free Radical Biology and Medicine)  183   60 - 68 2022.04

ISSN  08915849

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Cu/Zn-superoxide dismutase (SOD1) is a homodimer with two identical subunits, each of which binds a copper and zinc ion in the native state. In contrast to such a text book case, SOD1 proteins purified in vitro or even in vivo have been often reported to bind a non-stoichiometric amount of the metal ions. Nonetheless, it is difficult to probe how those metal ions are distributed in the two identical subunits. By utilizing native mass spectrometry, we showed here that addition of a sub-stoichiometric copper/zinc ion to SOD1 led to the formation of a homodimer with a stochastic combination of the subunits binding 0, 1, and even 2 metal ions. We also found that the homodimer was able to bind four copper or four zinc ions, implying the binding of a copper and zinc ion at the canonical zinc and copper site, respectively. Such ambiguity in the metal quota and selectivity could be avoided when an intra-subunit disulfide bond in SOD1 was reduced before addition of the metal ions. Apo-SOD1 in the disulfide-reduced state was monomeric and was found to bind only one zinc ion per monomer. By binding a zinc ion, the disulfide-reduced SOD1 became conformationally compact and acquired the ability to dimerize. Based upon the results in vitro, we describe the pathway in vivo enabling SOD1 to bind copper and zinc ions with high accuracy in their quota and selectivity. A failure of correct metallation in SOD1 will also be discussed in relation to amyotrophic lateral sclerosis.

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A pathological link between dysregulated copper binding in Cu/Zn-superoxide dismutase and amyotrophic lateral sclerosis

Furukawa Y.

Journal of Clinical Biochemistry and Nutrition 71 ( 2 ) 73 - 77 2022

ISSN  09120009

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Mutations in the gene coding Cu/Zn-superoxide dismutase (SOD1) are linked to a familial form of amyotrophic lateral sclerosis (ALS), and its pathological hallmark includes abnormal accumulation of mutant SOD1 proteins in spinal motorneurons. Mutant SOD1 proteins are considered to be susceptible to misfolding, resulting in the accumulation as oligomers/aggregates. While it remains obscure how and why SOD1 becomes misfolded under pathological conditions in vivo, the failure to bind a copper and zinc ion in SOD1 in vitro leads to the significant destabilization of its natively folded structure. Therefore, genetic and pharmacological attempts to promote the metal binding in mutant SOD1 could serve as an effective treatment of ALS. Here, I briefly review the copper and zinc binding process of SOD1 in vivo and discuss a copper chaperone for SOD1 as a potential target for developing ALS therapeutics.

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Stagnation of glymphatic interstitial fluid flow and delay in waste clearance in the SOD1-G93A mouse model of ALS.

Mikako Hirose, Mito Asano, Saori Watanabe-Matsumoto, Koji Yamanaka, Yoichiro Abe, Masato Yasui, Eiichi Tokuda, Yoshiaki Furukawa, Hidemi Misawa

Neuroscience research (Neuroscience Research)  171   74 - 82 2021.10

Accepted,  ISSN  01680102

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Overexpression and mislocalization of aquaporin-4 (AQP4) in the SOD1G93A mouse model of amyotrophic lateral sclerosis (ALS) have previously been reported. However, how alterations of AQP4 affect interstitial bulk flow in the brain and spinal cord, the so-called glymphatic system, is unclear. Here, we report an enhanced accumulation of disease-associated SOD1 species including SOD1 oligomers in SOD1G93A;AQP4-/- mice compared with SOD1G93A mice during ALS disease progression, as analyzed by sandwich ELISA. By directly injecting SOD1 oligomers into the spinal cord parenchyma, we observed a significantly larger delay in clearance of biotinylated or fluorescent-labeled SOD1 oligomers in AQP4-/- mice than in wild-type mice. Furthermore, when we injected the fluorescent-labeled tracer protein ovalbumin into the cisterna magna and analyzed the tracer distribution in the cervical spinal cord, approximately 35 % processing ability was found to be reduced in SOD1G93A mice compared to wild-type mice. These results suggest that the glymphatic system is abnormal and that waste clearance is delayed in SOD1G93A mice.

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A dual role of cysteine residues in the maturation of prokaryotic Cu/Zn-superoxide dismutase.

Yoshiaki Furukawa, Atsuko Shintani, Teppei Kokubo

Metallomics : integrated biometal science (Metallomics)  13 ( 9 )  2021.09

Accepted,  ISSN  17565901

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Bacterial Cu/Zn-superoxide dismutase (SodC) is an enzyme catalyzing the disproportionation of superoxide radicals, to which the binding of copper and zinc ions and the formation of an intramolecular disulfide bond are essential. We previously showed that Escherichia coli SodC (SodC) was prone to spontaneous degradation in vivo in an immature form prior to the introduction of the disulfide bond. The post-translational maintenance involving the metal binding and the disulfide formation would thus control the stability as well as the enzymatic function of SodC; however, a mechanism of the SodC maturation remains obscure. Here, we show that the disulfide-reduced SodC can secure a copper ion as well as a zinc ion through the thiolate groups. Furthermore, the disulfide-reduced SodC was found to bind cuprous and cupric ions more tightly than SodC with the disulfide bond. The thiolate groups ligating the copper ion were then autooxidized to form the intramolecular disulfide bond, leading to the production of enzymatically active SodC. Based upon the experiments in vitro, therefore, we propose a mechanism for the activation of SodC, in which the conserved Cys residues play a dual role: the acquisition of a copper ion for the enzymatic activity and the formation of the disulfide bond for the structural stabilization.

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Good and bad of Cu/Zn-superoxide dismutase controlled by metal ions and disulfide bonds

Furukawa Y.

Chemistry Letters 50 ( 2 ) 331 - 341 2021

ISSN  03667022

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Cu/Zn-superoxide dismutase (SOD1) is a metalloenzyme that catalyzes the disproportionation of superoxide. This review summarizes intracellular processes for metal binding and disulfide formation in SOD1, both of which are essential to stabilization of the protein structure as well as its enzymatic function. Also, failure of those processes as a possible cause of a neurodegenerative disease through protein misfolding will be described.

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