Hexokinase I N-terminal based peptide prevents the VDAC1-SOD1 G93A interaction and re-establishes ALS cell viability

Superoxide Dismutase 1 mutants associate with 20–25% of familial Amyotrophic Lateral Sclerosis (ALS) cases, producing toxic aggregates on mitochondria, notably in spinal cord. The Voltage Dependent Anion Channel isoform 1 (VDAC1) in the outer mitochondrial membrane is a docking site for SOD1 G93A mutant in ALS mice and the physiological receptor of Hexokinase I (HK1), which is poorly expressed in mouse spinal cord. Our results demonstrate that HK1 competes with SOD1 G93A for binding VDAC1, suggesting that in ALS spinal cord the available HK1-binding sites could be used by SOD1 mutants for docking mitochondria, producing thus organelle dysfunction. We tested this model by studying the action of a HK1-N-terminal based peptide (NHK1). This NHK1 peptide specifically interacts with VDAC1, inhibits the SOD1 G93A binding to mitochondria and restores the viability of ALS model NSC34 cells. Altogether, our results suggest that NHK1 peptide could be developed as a therapeutic tool in ALS, predicting an effective role also in other proteinopathies.


Cloning of recombinant proteins
The sequence encoding the human VDAC1 was inserted into the pET-21a expression vector (Novagen) in frame with 6xHis tag at C-terminal domain, as previously reported 1 . The sequence encoding the human SOD1 wild type (SOD1 WT) was amplified by in stock plasmid using a specific couple of primers (SOD1 fw and rev) and cloned into the pET-52b expression vector (Novagen) in frame with Strep-tag at its Nterminal domain using SanDI/SacI sites. To generate the construct for the ALS-linked SOD1 G93A, the QuikChange II site-direct mutagenesis kit (Agilent) was used in combination with a specific couple of mutagenic primers (SOD1 mutG93A fw and rev). Primer sequences are listed in Supplementary Table   1. All sequences were verified by sequencing.

Expression, purification and refolding of VDAC1 proteins
E. coli BL21 (DE3) cells were transformed with the pET constructs harboring VDAC1 sequences. The 6xHis-tagged VDAC1 proteins were induced, purified and refolded as previously reported 1 . The protein purity was verified in 12% SDS-PAGE followed by Comassie staining. Purified samples were stored at -20°C until further use.

Expression, purification and activity assay of SOD1 proteins
The pET vectors containing SOD1 constructs were used for E. coli BL21 (DE3) transformation. Protein expression was achieved by 1 mM isopropyl-β-D-thiogalactopyranoside (IPTG) (Sigma) induction at 3 18°C over night. Cell lysis and purification of Strep-tagged proteins by affinity chromatography using Strep-Tactin Superflow Plus Resin (Qiagen) were performed under native conditions, according to manufacturer's protocol. Metal loading and storage was performed as reported 2 . The protein purity was verified by 12% SDS-PAGE and Comassie staining. Activity of SOD1 proteins was assayed in gel with the Riboflavin/NitroBlue Tetrazolium (RF/NBT) assay as reported 3 .

Plasmids and cell transfection
Sequence encoding for human HK1 was amplified from in stock plasmid using a specific couple of primers (HK1 fw and rev, see Supporting Table 1  After PBS washing, cells were exposed for 1h at RT to the secondary anti-rabbit antibody AlexaFluor 488. Coverslips were mounted with the ProLong Gold antifade mounting medium (Invitrogen) and 5 examined by fluorescence microscopy. Co-localization with mitochondria was obtained by merging the signal HK1 or HA respectively with the signal from Mito-Traker Red (Molecular Probes) or the fluorescent reporter mtDsRED.

Fluorescence microscopy
A Leica DMI 6000B epifluorescence inverted microscope with Adaptive Focus Control was used. This system is outfitted with a controllable X-cite mercury lamp and an extensive collection of filter cubes  Table S1. List of the primer sequences used in this work.