Blog post by Dr. Tamara Maiuri
Forgive me readers for I have been busy, it has been 3 months since my last blog post. During this time, we published a very exciting story identifying a lead compound and how we think it works in HD. Also during this time, I have gone through the list of huntingtin interactors identified in my HDSA-funded project to find drug targets that are relevant to the process of DNA repair. We now have some interesting results!
As explained in my last post, the original aim to identify huntingtin interactors from human cells was not technically feasible and mouse cells were used instead. Several hundred protein-protein interactions were detected, then the list was further refined by only considering high and medium confidence hits. We now have a list of 314 proteins that interact with huntingtin reproducibly across biological replicates. See the end of this post for the lists of ROS-dependent interactors (Tables 1-3), proteins that interacted with huntingtin only in untreated cells (Table 4), and proteins found to be modified by poly ADP-ribose (PARylated proteins; Table 5). These tables have also been deposited to Zenodo.
Indeed, the most notable finding of the ROS-dependent interactome analysis was the high degree of overlap with datasets of “PARylated” proteins. PAR is of interest to us because it is generated in response to DNA damage, and acts to recruit DNA repair proteins to damage sites.
This result led us to pursue two hypotheses with the aim of identifying drug targets relevant to DNA repair:
Huntingtin is a PAR-binding protein: Huntingtin may use PAR binding to interact with PARylated proteins. If so, this is likely to be the mechanism by which huntingtin interacts with chromatin and assembles DNA repair factors in its role as a scaffold, and this may be dysregulated in HD.
PAR signaling is dysregulated in HD: Regardless of whether huntingtin physically binds PAR, it is still possible that we have uncovered a previously unexplored aspect of HD pathology: there is strong genetic evidence that DNA repair genes influence disease [1-3], huntingtin participates in the process of DNA repair [4], and elevated levels of damaged DNA are common to HD tissues and models [4-8].

In response to DNA damage, PAR chains are the first scaffold generated for DNA repair factor assembly: a sticky web bound by proteins involved in DNA repair. So it’s possible that in HD, excess DNA damage accumulates due to sub-optimal huntingtin function in the repair process, leading to Poly ADP-Ribose Polymerase (PARP) hyperactivation and elevated PAR levels.
High PAR levels could not only interfere with huntingtin function if it binds PAR, but also cause an “energy crisis” in high-energy-consuming neurons [9-11]. In fact, energy deficits have been frequently observed across HD models and in HD patients [12]. Similarly, PARP hyperactivation is linked to cerebellar ataxia [13], and PARP inhibition has been reported to improve phenotypes in a HD mouse model, although by a different mechanism [14,15].
In the coming blog posts, I will share preliminary data that suggests huntingtin can bind PAR in a test tube, and that PAR is at least partially responsible for huntingtin recruitment to chromatin. We have also detected increased amounts of PAR and of chromatin-bound huntingtin in cells from an HD patient. Stay tuned!
References:
- Genetic Modifiers of Huntington’s Disease (GeM-HD) Consortium. Identification of Genetic Factors that Modify Clinical Onset of Huntington’s Disease. Cell. 2015;162: 516–526.
- Bettencourt C, Hensman-Moss D, Flower M, Wiethoff S, Brice A, Goizet C, et al. DNA repair pathways underlie a common genetic mechanism modulating onset in polyglutamine diseases. Ann Neurol. 2016;79: 983–990.
- Moss DJH, Pardiñas AF, Langbehn D, Lo K, Leavitt BR, Roos R, et al. Identification of genetic variants associated with Huntington’s disease progression: a genome-wide association study. Lancet Neurol. 2017;16: 701–711.
- Maiuri T, Mocle AJ, Hung CL, Xia J, van Roon-Mom WMC, Truant R. Huntingtin is a scaffolding protein in the ATM oxidative DNA damage response complex. Hum Mol Genet. 2017;26: 395–406.
- Bogdanov MB, Andreassen OA, Dedeoglu A, Ferrante RJ, Beal MF. Increased oxidative damage to DNA in a transgenic mouse model of Huntington’s disease. J Neurochem. 2001;79: 1246–1249.
- Kovtun IV, Liu Y, Bjoras M, Klungland A, Wilson SH, McMurray CT. OGG1 initiates age-dependent CAG trinucleotide expansion in somatic cells. Nature. 2007;447: 447–452.
- Enokido Y, Tamura T, Ito H, Arumughan A, Komuro A, Shiwaku H, et al. Mutant huntingtin impairs Ku70-mediated DNA repair. J Cell Biol. 2010;189: 425–443.
- Askeland G, Dosoudilova Z, Rodinova M, Klempir J, Liskova I, Kuśnierczyk A, et al. Increased nuclear DNA damage precedes mitochondrial dysfunction in peripheral blood mononuclear cells from Huntington’s disease patients. Sci Rep. 2018;8: 9817.
- Morales J, Li L, Fattah FJ, Dong Y, Bey EA, Patel M, et al. Review of poly (ADP-ribose) polymerase (PARP) mechanisms of action and rationale for targeting in cancer and other diseases. Crit Rev Eukaryot Gene Expr. 2014;24: 15–28.
- Andrabi SA, Umanah GKE, Chang C, Stevens DA, Karuppagounder SS, Gagné J-P, et al. Poly(ADP-ribose) polymerase-dependent energy depletion occurs through inhibition of glycolysis. Proc Natl Acad Sci U S A. 2014;111: 10209–10214.
- Fouquerel E, Goellner EM, Yu Z, Gagné J-P, Barbi de Moura M, Feinstein T, et al. ARTD1/PARP1 negatively regulates glycolysis by inhibiting hexokinase 1 independent of NAD+ depletion. Cell Rep. 2014;8: 1819–1831.
- Dickey AS, La Spada AR. Therapy development in Huntington disease: From current strategies to emerging opportunities. Am J Med Genet A. 2017; doi:10.1002/ajmg.a.38494
- Hoch NC, Hanzlikova H, Rulten SL, Tétreault M, Komulainen E, Ju L, et al. XRCC1 mutation is associated with PARP1 hyperactivation and cerebellar ataxia. Nature. 2017;541: 87–91.
- Cardinale A, Paldino E, Giampà C, Bernardi G, Fusco FR. PARP-1 Inhibition Is Neuroprotective in the R6/2 Mouse Model of Huntington’s Disease. PLoS One. 2015;10: e0134482.
- Paldino E, Cardinale A, D’Angelo V, Sauve I, Giampà C, Fusco FR. Selective Sparing of Striatal Interneurons after Poly (ADP-Ribose) Polymerase 1 Inhibition in the R6/2 Mouse Model of Huntington’s Disease. Front Neuroanat. 2017;11: 61.
Table 1: Hit Proteins unique to H2O2-treated cells | ||||
Hit Protein (human homolog) | Molecular Function | Biological Function | Interactions (UniprotKB, BioGrid) | Disease Connections |
TPT1 | metal ion binding; protein binding; RNA binding
|
calcium binding and microtubule stabilization
|
TP53, XRCC5, XRCCC6, HSPB1, EF1D, PCH2, RHEB, EF1D-2, T22D1, AT1A1 | Charcot Marie tooth, Distal hereditary motor neuropathy type II, several cancers |
NDUFV1 | metal ion binding; nucleotide binding; protein binding | ATP synthesis | ||
SLIRP | RNA-binding | RNA-binding protein that acts as a nuclear receptor corepressor | PNMA1, C102B, MTUS2, K1C40, LPPRC | Leigh Syndrome French Canadian Type |
HINT2 | nucleotide binding
|
steroid biosynthesis, apoptosis | 33 interactors, including APP, TOP3A | |
ACAD9 | nucleotide binding | mitochondrial complex I assembly | 79 interactors, including many mitochondrial proteins | |
PMPCB | metal ion binding | cleaves presequences (transit peptides) from mitochondrial protein precursors | 77 interactors, including many mitochondrial proteins | |
IDH3G | metal ion binding nucleotide binding | tricarboxylic acid cycle | 28 interactors, including KPNA2, HNRNPK, PQBP1 | |
OAT | protein binding | amino acid biosynthesis | 44 interactors, including SOD1, SOD2, PARK7, HDAC5, SIRT7, CDK2, FBXO6, FUS | Parkinson’s disease |
ACOT10 | hydrolase activity | acyl-CoA metabolic process | ||
GCAT | acetyltransferase activity | amino acid metabolism | 12 interactors, including ATXN3, MDM2, FBXO6 | Spinocerebellar ataxia 3 |
DKC1 | protein binding; RNA binding | ribosome biogenesis, telomere maintenance | RUVB1, HMBX1, NAF1 | Hoyeraal Hreidaarsson syndrome |
CHST2 | nucleotide binding | carbohydrate metabolism | ||
HIBADH | nucleotide binding | amino acid catabolism | 10 interactors, including BRCA1, SOD1 | |
PPIF | protein binding | protein folding, mitochondrial permeability | TP53, CKLF5, ABI2, BANP | Li-Fraumeni syndrome, several cancers |
MRPS33 | mitochondrial translation | 36 interactors, including several RNA-binding proteins | ||
HAT1 | histone acetyltransferase activity | DNA packaging | RBBP4, H4, VPR, REL, MEOX2, BACD2, ITF2 | Pitt-Hopkins syndrome |
PPM1G | metal ion binding; protein binding; phosphatase activity | cell cycle arrest | TERF1, XRCC5, XRCC6, TAT, YBOX1 | |
VAT1 | metal ion binding | negative regulation of mitochondrial fusion | 25 interactors, including TP53, H2AFX, SOD1, PARK2, CDK2 |
Table 2: Hit Proteins unique to MMS-treated cells | ||||
Hit Protein (human homolog) | Molecular Function | Biological Function | Interactions (UniprotKB, BioGrid) | Disease Connections |
TXNDC17 | antioxidant activity | redox reactions | RUFY1, TINF2, EXOS8 | Dyskeratosis congenita, Pontocerebellar hypoplasia |
MRPS21 | RNA binding structural molecule activity | mitochondrial translation | 38 interactors, including mitochondrial translation proteins, RNA-binding proteins | |
HMGA1 | DNA binding; protein binding | base excision repair, nucleosome disassembly | ORC6, ANM6 | Meier-Gorlin syndrome |
MRPS26 | RNA binding | DNA damage response, mitochondrial translation | 69 interactors, including mitochondrial translation proteins, RNA binding proteins | |
PEBP1 | enzyme regulator activity;
nucleotide binding; protein binding; RNA binding |
MAPK cascase | 41 interactrs, including SOD1, SOD2, RAF1, LOX15, CFL1, PABPC3 | |
CDC37 | enzyme regulator activity;
protein binding |
co-chaperone, mitophagy | 246 interactions, including IKKA, IKKB, APOE, PSN1, LRKK2 | Alzheimer’s disease, frontotemporal dementia, amyotrophic lateral sclerosis, Parkinson’s disease |
TXNL1 | antioxidant activity | redox homeostasis | 64 interactors, including proteasomal proteins | |
NARS | nucleotide binding | tRNA synthesis | 67 interactors, including ATXN1, HDAC2, XRCC6, XRCC5 | Spinocerebellar ataxia 1 |
FDPS | metal ion binding;
RNA binding |
cholesterol biosynthesis | 50 interactors, including ATXN 1, G6PD, CREB3, PABPC1, PSME4 | Spinocerebellar ataxia 1 |
PSME4 | enzyme regulator activity;
protein binding |
DNA repair, histone degradation | 45 interactors, including proteasomal proteins, FDPS |
Table 3: Hit Proteins common to H2O2- and MMS-treated cells | ||||
Hit Protein (human homolog) | Molecular Function | Biological Function | Interactions (UniprotKB, BioGrid) | Disease Connections |
FAM135A | hydrolase activity | lipid metabolism | 10 interactors, including EZR, RAB5A, RAB9A | |
CAVIN1 | protein binding;
RNA binding |
caveolae formation, transcription | 115 interactors, including T10IP, CAV1, CAVN2, CAVN3, RCOR1, RAB5A, RAB7A, RAB5C | Neurodegeneration syndrome |
Table 4: Hit Proteins specific to untreated cells | ||||
Hit Protein (human homolog) | Molecular Function | Biological Function | Interactions (UniprotKB, BioGrid) | Disease Connections |
TCEB1 | protein binding | translation | 154 interactors, including APE1, FUS, SQSTM1, POU5F1, OTUB1, TOP2A, CENPC | |
RPS14 | RNA binding;
structural molecule activity |
translation | 254 interactors, including MDM2 and many ribosomal proteins | |
BRK1 | protein binding | actin and microtubule organization | 25 interactors, including BRAP, PFDN1 | Hermansky-Pudlak syndrome |
RPL24 | protein binding;
RNA binding; structural molecule activity |
translation | 165 interactors, including many ribosomal proteins | |
RPL15 | protein binding;
RNA binding; structural molecule activity |
translation | 197 interactors, including many ribosomal proteins | |
RAD23B | DNA binding;
protein binding |
DNA damage recognition, nucleotide excision repair | 125 interactors, including HMGB1, MLH1, XPC, ATXN3, POU5F1, ERCC3, PUF60, G6PD, EWSR1, BRCA1 | Xeroderma pigmentosum, Spinocerebellar ataxia 3, Verheij syndrome |
NACA | DNA binding;
protein binding |
protein transport, transcription | 55 interactors, including EWSR1, SOD1, BRCA1, PARK2, MDM2, H2AFX, APLP1 | Parkinson’s disease |
CAND1 | protein binding | ubiquitin conjugation | 708 interactors, including many cullins | X-linked syndromic mental retardation |
PAICS | nucleotide binding;
protein binding |
purine biosynthesis | 94 interactors, including BRCA1, FUS, 53BP1 | |
EWSR1 | metal ion binding;
protein binding; RNA binding |
transcription | 650 interactors, including FUS, ATPF2, PABPN1, SOD1 | Ewing sarcoma, Mitochondrial complex V deficiency nuclear 1, Amyotrophic lateral sclerosis, Frontotemporal dementia |
TPM3 | protein binding;
structural molecule activity |
actin filament organization | 133 interactors, including TP53, PARK7, PARK2, BRCA1 | Dystonia, Parkinson’s disease |
FHL1 | metal ion binding;
protein binding |
differentiation | 35 interactors, including EWSR1 | Emery-Dreifuss muscular dystrophy |
HNRNPK | DNA binding;
protein binding; RNA binding |
transcriptional regulation of TP53 DNA damage response | 281 interactors, including TP53, MDM2, PABPC1, FUS, XRCC6, BRCA1, HMGB1, TOP1, PARK2 | Amyotrophic lateral sclerosis, Li Fraumeni syndrome, Parkinson’s disease |
RPL31 | protein binding;
RNA binding; structural molecule activity |
translation | 159 interactors, including BRCA1, EWSR1, APP, TP53 | Diamond-Blackfan anemia |
YBX1 | DNA binding;
protein binding; RNA binding |
transcription, mRNA processing | 290 interactors, including TP53, BRCA1, FUS, PCNA, H2AFX, APE1, EWSR1 | |
LMNA | protein binding;
structural molecule activity |
nuclear assembly, chromatin organization | 645 interactors, including FUS, PARP1, H2AFX | Emery-Dreifuss muscular dystrophy, Charcot Marie tooth disease, Hutchinson-Gilford progeria syndrome, LMNA-related congenital muscular dystrophy |
SNRPC | metal ion binding;
protein binding; RNA binding |
mRNA splicing | 90 interactors, including EWSR1, BARD1 | |
FABP5 | transporter activity | fatty acid transport | 40 interactors, including SOD1, POU5F1 | |
RPL23A | protein binding;
RNA binding; structural molecule activity |
translation | 239 interactors, including TP53, BRCA1, MDM2, PARK2, many ribosomal proteins | |
UBA1 | nucleotide binding;
protein binding; RNA binding
|
ubiquitin conjugation, response to DNA damage | 147 interactors, including FUS, BRCA1, SOD1, PABPC1 | X-linked spinal muscular atrophy, Giant axonal neuropathy |
RPL27 | RNA binding;
structural molecule activity |
translation | 157 interactors, including TP53, BRCA1, PABPC1, many ribosomal proteins | |
RPL7 | DNA binding;
protein binding; RNA binding; structural molecule activity
|
translation | 230 interactors, including TP53, BRCA1, PABPC1, many ribosomal proteins | |
H3F3C | DNA binding;
protein binding |
nucleosome assembly | 24 interactors, including FAN1, DNAJC11 | |
RPL36 | RNA binding;
structural molecule activity |
translation | 138 interactors, including BRCA1, many ribosomal proteins |
Table 5: PARylated proteins | |||||||||
ABI1 | DBD1 | EWSR1 | HMGA1 | KRT1 | PAICS | RPL15 | RPL6 | RPS19 | TUBA4A |
ACTB | DDX3X | FLNA | HMGB1 | KRT10 | PCBP1 | RPL18 | RPL7 | RPS2 | TUBB6 |
AHCY | DEK | FOXP1 | HMGB2 | LMNA | PCBP2 | RPL22 | RPL7A | RPS25 | TUFM |
ATP5A1 | DKC1 | FSCN1 | HNRNPA1 | LONP1 | PCNA | RPL23 | RPLP0 | RPS27A | |
CALR | DNAJA1 | FUS | HNRNPAB | MTHFD1L | PIP5K1A | RPL23A | RPLP1 | RPS3 | |
CAND1 | EEF1A1 | GAPDH | HNRNPD | MYL12B | PRTF | RPL24 | RPS10 | RPS3A | |
CCT3 | EEF1G | GLUD1 | HNRNPK | MYL6 | RAD23B | RPL27 | RPS11 | RPS6 | |
CCT4 | EEF2 | GSTP1 | HSP90AA1 | NACA | RPL11 | RPL27A | RPS12 | RPS8 | |
CCT8 | EIF2S1 | HADHA | HSPH1 | NCKAP1 | RPL 12 | RPL3 | RPS14 | RPSA | |
CFL1 | EIF4A1 | HIST1H2A | KPNA2 | NCL | RPL13 | RPL31 | RPS15A | SFPQ | |
CYFIP1 | ENO1 | HIST4H4 | KPNB1 | PA2G4 | RPL14 | RPL4 | RPS18 | TCP1 |
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