A closer look at the hits generates 2 new hypotheses

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.

Blog Post 9 fig

 

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].

Old spider web. Shallow DOF.
© Bolotov | Stock Free Images

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:

  1. 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.
  2. 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.
  3. 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.
  4. 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.
  5. 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.
  6. 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.
  7. 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.
  8. 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.
  9. 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.
  10. 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.
  11. 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.
  12. 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
  13. 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.
  14. 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.
  15. 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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