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

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.

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

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.

FUS regulates RAN translation through modulating the G-quadruplex structure of GGGGCC repeat RNA in C9orf72-linked ALS/FTD.

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.

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

Canine degenerative myelopathy (DM), a fatal neurodegenerative disease in dogs, shares clinical and genetic features with amyotrophic lateral sclerosis, 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 amyotrophic lateral sclerosis. 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 ß-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 ß-barrel structure induces E40K-dependent species-specific aggregation in canine SOD1.

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

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 (CCSdI) 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 CCSdI in CCS. METHODS: To evaluate protein-protein interactions of CCS through CCSdI, 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 CCSdI. The interaction between CCSdI 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 CCSdI and HAH1. CONCLUSION: While physiological significance of the interaction between copper chaperones awaits further investigation, we propose that CCSdI would have a role in the metal-mediated interaction with other proteins including heterologous copper chaperones.

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

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.

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

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.

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

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.

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

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.

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

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.

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

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.

The identified clinical features of Parkinson's disease in homo-, heterozygous and digenic variants of PINK1.

To investigate the prevalence and genotype-phenotype correlations of phosphatase and tensin homolog induced putative kinase 1 (PINK1) variants in Parkinson's disease (PD) patients, we analyzed 1700 patients (842 familial PD and 858 sporadic PD patients from Japanese origin). We screened the entire exon and exon-intron boundaries of PINK1 using Sanger sequencing and target sequencing by Ion torrent system. We identified 30 patients with heterozygous variants, 3 with homozygous variants, and 3 with digenic variants of PINK1-PRKN. Patients with homozygous variants presented a significantly younger age at onset than those with heterozygous variants. The allele frequency of heterozygous variants in patients with age at onset at 50 years and younger with familial PD and sporadic PD showed no differences. [123I]meta-iodobenzylguanidine (MIBG) myocardial scintigraphy indicated that half of patients harboring PINK1 heterozygous variants showed a decreased heart to mediastinum ratio (12/23). Our findings emphasize the importance of PINK1 variants for the onset of PD in patients with age at onset at 50 years and younger and the broad spectrum of clinical symptoms in patients with PINK1 variants.

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

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.

Does wild-type Cu/Zn-superoxide dismutase have pathogenic roles in amyotrophic lateral sclerosis?

Amyotrophic lateral sclerosis (ALS) is characterized by adult-onset progressive degeneration of upper and lower motor neurons. Increasing numbers of genes are found to be associated with ALS; among those, the first identified gene, SOD1 coding a Cu/Zn-superoxide dismutase protein (SOD1), has been regarded as the gold standard in the research on a pathomechanism of ALS. Abnormal accumulation of misfolded SOD1 in affected spinal motor neurons has been established as a pathological hallmark of ALS caused by mutations in SOD1 (SOD1-ALS). Nonetheless, involvement of wild-type SOD1 remains quite controversial in the pathology of ALS with no SOD1 mutations (non-SOD1 ALS), which occupies more than 90% of total ALS cases. In vitro studies have revealed post-translationally controlled misfolding and aggregation of wild-type as well as of mutant SOD1 proteins; therefore, SOD1 proteins could be a therapeutic target not only in SOD1-ALS but also in more prevailing cases, non-SOD1 ALS. In order to search for evidence on misfolding and aggregation of wild-type SOD1 in vivo, we reviewed pathological studies using mouse models and patients and then summarized arguments for and against possible involvement of wild-type SOD1 in non-SOD1 ALS as well as in SOD1-ALS.

Serotonin transporter protein in autopsied brain of chronic users of cocaine.

RATIONALE: The long-held speculation that the brain serotonin system mediates some behavioral effects of the psychostimulant cocaine is supported in part by the high affinity of cocaine for the serotonin transporter (SERT) and by reports that the serotonin transporter (SERT), estimated by SERT binding, is increased in brain of human chronic cocaine users. Excessive SERT activity and consequent synaptic serotonin deficiency might cause a behavioral (e.g., mood) abnormality in chronic users of the drug. OBJECTIVE AND METHODS: Previous studies focused on changes in SERT binding, which might not necessarily reflect changes in SERT protein. Therefore, we compared levels of SERT protein, using a quantitative Western blot procedure, in autopsied brain (striatum, cerebral cortices) of chronic human cocaine users (n = 9), who all tested positive for the drug/metabolite in brain, to those in control subjects (n = 15) and, as a separate drug of abuse group, in chronic heroin users (n = 11). RESULTS: We found no significant difference in protein levels of SERT or the serotonin synthesizing enzyme tryptophan hydroxylase-2 among the control and drug abuse groups. In the cocaine users, no significant correlations were observed between SERT and brain levels of cocaine plus metabolites, or with levels of serotonin or its metabolite 5-hydroxyindoleacetic acid. CONCLUSION: Our postmortem data suggest that a robust increase in striatal/cerebral cortical SERT protein is not a common characteristic of chronic, human cocaine users.

Concentration, distribution, and influence of aging on the 18 kDa translocator protein in human brain: Implications for brain imaging studies.

Positron emission tomography (PET) imaging of the translocator protein (TSPO) is widely used as a biomarker of microglial activation. However, TSPO protein concentration in human brain has not been optimally quantified nor has its regional distribution been compared to TSPO binding. We determined TSPO protein concentration, change with age, and regional distribution by quantitative immunoblotting in autopsied human brain. Brain TSPO protein concentration (>0.1 ng/µg protein) was higher than those reported by in vitro binding assays by at least 2 to 70 fold. TSPO protein distributed widely in both gray and white matter regions, with distribution in major gray matter areas ranked generally similar to that of PET binding in second-generation radiotracer studies. TSPO protein concentration in frontal cortex was high at birth, declined precipitously during the first three months, and increased modestly during adulthood/senescence (10%/decade; vs. 30% for comparison astrocytic marker GFAP). As expected, TSPO protein levels were significantly increased (+114%) in degenerating putamen in multiple system atrophy, providing further circumstantial support for TSPO as a gliosis marker. Overall, findings show some similarities between TSPO protein and PET binding characteristics in the human brain but also suggest that part of the TSPO protein pool might be less available for radioligand binding.