{"gene":"MACROD1","run_date":"2026-06-10T02:59:50","timeline":{"discoveries":[{"year":2007,"finding":"LRP16 (MACROD1) physically interacts with estrogen receptor alpha (ERα) via its macro domain, in a manner that is estrogen-independent but enhanced by estrogen, and binds specifically to the A/B activation function 1 (AF-1) domain of ERα, enhancing ERα-mediated transcriptional activity. GST pulldown and coimmunoprecipitation confirmed the interaction.","method":"GST pulldown, coimmunoprecipitation, mammalian two-hybrid assay, siRNA knockdown with luciferase reporter and target gene expression","journal":"Endocrine-related cancer","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal GST pulldown + CoIP + functional reporter assays, replicated across multiple cell line models in a single rigorous study","pmids":["17914104"],"is_preprint":false},{"year":2008,"finding":"LRP16 (MACROD1) binds to androgen receptor (AR) via its macro domain and functions as a coactivator to amplify AR transactivation in response to androgen. The single macro domain is sufficient for AR coactivation. RNAi knockdown of LRP16 impairs AR function and attenuates coactivation by ART-27 and SRC-1, and inhibits androgen-stimulated proliferation of LNCaP cells but not AR-negative PC-3 cells.","method":"Co-immunoprecipitation, luciferase reporter assay, RNAi knockdown, domain mapping","journal":"Endocrine-related cancer","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal CoIP, domain mapping, functional reporter assays, rescue experiments with isogenic AR-positive vs. AR-negative cell lines","pmids":["19022849"],"is_preprint":false},{"year":2018,"finding":"MacroD1 (MACROD1) is a mono-ADP-ribose hydrolase localized primarily in mitochondria (not nucleus or cytosol) of skeletal muscle cells. It can efficiently remove ADP-ribose from 5' and 3'-phosphorylated double-stranded DNA adducts in vitro, targeting ester bonds of ADP-ribosylated phosphorylated dsDNA ends.","method":"Subcellular fractionation with endogenous protein detection, mitochondrial enrichment, in vitro ADP-ribose hydrolase enzymatic assay with phosphorylated dsDNA substrates","journal":"Frontiers in microbiology","confidence":"High","confidence_rationale":"Tier 1-2 / Moderate — endogenous protein fractionation plus in vitro enzymatic assay with defined substrates, single lab but orthogonal methods","pmids":["29410655"],"is_preprint":false},{"year":2011,"finding":"LRP16 (MACROD1) integrates into the NF-κB transcriptional complex by associating with the p65 subunit. RNAi knockdown of LRP16 does not affect TNF-α-induced nuclear translocation of NF-κB but blunts the formation/stabilization of the functional NF-κB/p300/CBP transcription complex in the nucleus, impairing NF-κB target gene expression and sensitizing cells to TNF-α-induced apoptosis.","method":"GST pulldown, coimmunoprecipitation, luciferase reporter assay, RNAi knockdown, flow cytometry (Annexin V), target gene expression analysis","journal":"PloS one","confidence":"High","confidence_rationale":"Tier 2 / Moderate — GST pulldown + CoIP + functional reporter + mechanistic dissection of nuclear vs. cytoplasmic events, single lab with multiple orthogonal methods","pmids":["21483817"],"is_preprint":false},{"year":2015,"finding":"LRP16 (MACROD1) constitutively interacts with PARP1 and IKKγ, forming a preassembly complex. This interaction is essential for efficient interactions among PARP1, IKKγ, and PIASy, the DSB-induced SUMOylation and phosphorylation of IKKγ, and subsequent NF-κB activation following DNA double-strand break induction. The regulation is dependent on the DSB sensors Ku70/Ku80.","method":"Coimmunoprecipitation, genetic knockdown with functional NF-κB activation assays, DSB induction experiments","journal":"Nucleic acids research","confidence":"High","confidence_rationale":"Tier 2 / Moderate — reciprocal CoIP showing constitutive complex, functional NF-κB assays post-DSB, epistasis with Ku70/Ku80, single lab with multiple orthogonal methods","pmids":["25735744"],"is_preprint":false},{"year":2009,"finding":"Keratin 18 (K18) physically interacts with LRP16 (MACROD1) and sequesters it in the cytoplasm, reducing nuclear LRP16 availability. This attenuates LRP16-ERα association, ERα-activated transcription, and estrogen-stimulated cell cycle progression. K18 knockdown has the opposite effect, increasing nuclear LRP16 and ERα-mediated signaling.","method":"Yeast two-hybrid screening, GST pulldown, coimmunoprecipitation, fluorescence microscopy (GFP-LRP16 localization), immunoblotting of nuclear/cytoplasmic fractions, BrdU incorporation assay","journal":"BMC cell biology","confidence":"High","confidence_rationale":"Tier 2 / Moderate — yeast two-hybrid + GST pulldown + CoIP + live-cell localization + functional proliferation assay, single lab with multiple orthogonal methods","pmids":["20035625"],"is_preprint":false},{"year":2017,"finding":"LRP16 (MACROD1) selectively interacts with and activates double-stranded RNA-dependent kinase PKR, and acts as a scaffold to assist formation of a ternary complex of PKR and IKKβ, prolonging PAR-dependent NF-κB transactivation induced by DNA-damaging agents and conferring acquired chemoresistance. The small molecule MRS2578 abrogates LRP16 binding to PKR and IKKβ, converting LRP16 into a pro-death molecule.","method":"Co-immunoprecipitation, small molecule inhibitor (MRS2578) competition assay, NF-κB luciferase reporter, xenograft tumor models","journal":"eLife","confidence":"High","confidence_rationale":"Tier 2 / Moderate — reciprocal CoIP demonstrating ternary complex, functional assays with specific inhibitor, in vivo xenograft validation, single lab with multiple orthogonal approaches","pmids":["28820388"],"is_preprint":false},{"year":2020,"finding":"Crystal structure of MacroD1 (MACROD1) in complex with ADP-ribose reveals that the β5-α10-loop acts as a switch loop for substrate recognition and orientation. The conserved Phe272 in this loop orients the distal ribose of ADPR, and a conserved hydrogen-bond network positions catalytic water for ADPR hydrolysis. MacroD1 is recruited to DNA damage sites via recognition of ADP-ribosylation at DNA lesions, and MacroD1-mediated ADPR hydrolysis is essential for DNA damage repair.","method":"X-ray crystallography (MacroD1-ADP-ribose complex), site-directed mutagenesis of catalytic residues, in vitro hydrolase assay, DNA damage recruitment assay (laser-induced damage with fluorescence microscopy)","journal":"DNA repair","confidence":"High","confidence_rationale":"Tier 1 / Moderate — crystal structure with functional mutagenesis validation and in vitro enzymatic assay plus cellular DNA damage recruitment, single lab with multiple orthogonal high-quality methods","pmids":["32683309"],"is_preprint":false},{"year":2020,"finding":"MACROD1 localizes predominantly to mitochondria (especially in skeletal muscle), and loss of MACROD1 causes disruption of mitochondrial morphology. BioID interactome mapping reveals MACROD1-interacting proteins concentrated in mitochondria, suggesting involvement in mitochondrial RNA metabolism.","method":"Monoclonal antibody validation, immunofluorescence localization, mitochondrial morphology assessment upon MACROD1 knockout, BioID proximity labeling interactome mapping","journal":"Scientific reports","confidence":"High","confidence_rationale":"Tier 2 / Strong — validated monoclonal antibodies plus BioID interactome plus KO phenotype, replicated mitochondrial localization across multiple labs/studies","pmids":["32427867"],"is_preprint":false},{"year":2007,"finding":"LRP16 (MACROD1) represses E-cadherin transcription in an ERα-dependent manner, promoting invasive growth of Ishikawa endometrial cancer cells. Chromatin immunoprecipitation showed LRP16 antagonizes ERα binding to the E-cadherin promoter. E-cadherin downregulation requires ERα mediation as estrogen deprivation abolishes the effect.","method":"Transwell invasion assay, promoter-luciferase reporter analysis, chromatin immunoprecipitation (ChIP), siRNA/ectopic expression","journal":"Cell research","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — ChIP showing promoter interaction, functional invasion assay, ERα dependency established by estrogen deprivation, single lab","pmids":["17893710"],"is_preprint":false},{"year":2007,"finding":"Estrogen receptor alpha (ERα) and Sp1 cooperate at GC-rich motifs in the proximal promoter of LRP16 (specifically the -213/-184 bp fragment) to mediate maximal estrogen-induced transcription of LRP16, as demonstrated by gel mobility shift assays showing Sp1 binding enhanced by ERα titer and ChIP confirming ERα/Sp1 interaction at GC-rich sites.","method":"Deletion and mutation analysis of LRP16 promoter-luciferase constructs, Sp1-siRNA, gel mobility shift assay, chromatin immunoprecipitation (ChIP)","journal":"The Journal of steroid biochemistry and molecular biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — EMSA + ChIP + promoter deletion mapping + siRNA, single lab with multiple methods","pmids":["18206366"],"is_preprint":false},{"year":2024,"finding":"Macrod1 (MACROD1) suppresses diabetic cardiomyopathy by inhibiting PARP1 expression, thereby reducing NAD+ consumption and activating the deacetylase SIRT3 to resist oxidative stress. Knockout of Macrod1 worsened glycemic control, cardiac remodeling, and mitochondrial dysfunction; cardiac-specific overexpression partially reversed these effects. In cardiomyocytes, Macrod1 overexpression inhibited PARP1 and restored NAD+ levels to activate SIRT3.","method":"Macrod1 knockout and cardiac-specific overexpression mouse models (HFD/STZ-induced DCM), Western blot (PARP1, SIRT3), NAD+ measurement, mitochondrial function assays, in vitro neonatal cardiomyocyte palmitate model","journal":"Acta pharmacologica Sinica","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic KO and OE with defined molecular readouts (PARP1, NAD+, SIRT3), single lab, multiple orthogonal methods","pmids":["38459256"],"is_preprint":false},{"year":2013,"finding":"LRP16 (MACROD1) acts as a negative regulator of insulin signaling in 3T3-L1 adipocytes by activating the mTOR pathway, which in turn promotes TNF-α secretion, inhibits IRS-1/PI3K/Akt phosphorylation, and reduces PPARγ expression. Rapamycin (mTOR inhibitor) rescues the LRP16-overexpression phenotype, placing mTOR downstream of LRP16 in insulin resistance.","method":"Lentiviral siRNA knockdown and ectopic overexpression of LRP16 in 3T3-L1 cells, glucose uptake assay, Western blot (IRS-1, PI3K, Akt, mTOR phosphorylation), rapamycin epistasis","journal":"Hormone and metabolic research","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — gain- and loss-of-function with rapamycin epistasis placing mTOR in pathway, single lab","pmids":["23389992"],"is_preprint":false},{"year":2018,"finding":"LRP16 (MACROD1) promotes LPS-stimulated inflammatory responses in adipocytes through activation of a Rac1-dependent ERK1/2 (MAPK) signaling pathway. LRP16 overexpression activates ERK1/2 and Rac1; Rac1 knockdown or ERK inhibitor (PD98059) abolishes the stimulatory effect of LRP16 on inflammatory cytokine expression.","method":"LC-MS proteomics, Western blot (ERK1/2, Rac1 phosphorylation), siRNA knockdown of LRP16 and Rac1, pharmacological ERK inhibition (PD98059)","journal":"Cellular physiology and biochemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — pharmacological and genetic epistasis placing Rac1 and ERK downstream of LRP16, single lab with multiple approaches","pmids":["30562745"],"is_preprint":false},{"year":2021,"finding":"Loss of Macrod1 in mice results in a female-specific motor-coordination defect, consistent with its mitochondrial localization and suggesting a role in mitochondria-dependent neuromotor function. Loss of Macrod2 (a paralog) produces a distinct hyperactivity/bradykinesia phenotype.","method":"Macrod1 and Macrod2 knockout mouse models, behavioral battery testing (motor coordination, locomotion assays)","journal":"Cells","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — clean KO with defined behavioral phenotype but no direct molecular mechanism identified, single lab","pmids":["33578760"],"is_preprint":false}],"current_model":"MACROD1 (LRP16) is a mitochondria-enriched mono-ADP-ribose hydrolase that removes ADP-ribose from acidic amino acids and DNA ends via a catalytic mechanism requiring Phe272 and a hydrogen-bond network (crystal structure solved); it also functions as a nuclear coactivator of estrogen receptor α and androgen receptor through its single macro domain, integrates into the NF-κB transcriptional complex via p65 and a preassembled PARP1/IKKγ scaffold to potentiate DSB-induced NF-κB signaling, interacts with and activates PKR to form a PKR–IKKβ ternary complex that confers chemoresistance, and is sequestered in the cytoplasm by keratin 18 to modulate ERα signaling availability; in mitochondria it suppresses PARP1-mediated NAD+ consumption to activate SIRT3-dependent antioxidant signaling."},"narrative":{"mechanistic_narrative":"MACROD1 (LRP16) is a mono-ADP-ribose hydrolase whose single macro domain serves dual roles as an enzyme that reverses ADP-ribosylation and as a protein-interaction module integrating it into nuclear transcription and DNA-damage signaling [PMID:29410655, PMID:32683309, PMID:17914104]. As a catalyst, it removes ADP-ribose from phosphorylated double-stranded DNA ends, and its crystal structure in complex with ADP-ribose defines a β5-α10 switch loop in which Phe272 orients the distal ribose and a conserved hydrogen-bond network positions the catalytic water; this hydrolase activity supports its recruitment to ADP-ribosylated DNA lesions and contributes to DNA damage repair [PMID:29410655, PMID:32683309]. Independently of catalysis, the macro domain binds the AF-1 domain of estrogen receptor α and binds androgen receptor, acting as a transcriptional coactivator that amplifies hormone-driven transcription and, in an ERα-dependent manner, represses E-cadherin to promote invasive growth [PMID:17914104, PMID:19022849, PMID:17893710]. MACROD1 also functions in NF-κB signaling, associating with the p65 subunit to stabilize the nuclear NF-κB/p300/CBP complex and residing in a constitutive PARP1/IKKγ preassembly complex required for DSB-induced IKKγ modification and NF-κB activation; it further scaffolds a PKR–IKKβ ternary complex that prolongs PAR-dependent NF-κB transactivation and confers chemoresistance [PMID:21483817, PMID:25735744, PMID:28820388]. The protein is enriched in mitochondria, particularly in skeletal muscle, where its loss disrupts mitochondrial morphology, and it suppresses PARP1-mediated NAD+ consumption to activate SIRT3-dependent antioxidant signaling protective against diabetic cardiomyopathy [PMID:29410655, PMID:32427867, PMID:38459256]. Its nuclear availability is controlled by keratin 18, which sequesters it in the cytoplasm to limit ERα signaling [PMID:20035625].","teleology":[{"year":2007,"claim":"Established MACROD1 as a hormone-receptor coactivator by showing its macro domain binds the ERα AF-1 domain and amplifies ERα-driven transcription, defining its first molecular function.","evidence":"GST pulldown, CoIP, mammalian two-hybrid, and luciferase reporter with siRNA knockdown across cell lines","pmids":["17914104"],"confidence":"High","gaps":["Did not test whether catalytic/enzymatic activity is involved in coactivation","Structural basis of macro domain–AF-1 contact not resolved"]},{"year":2007,"claim":"Connected MACROD1 coactivation to an oncogenic output by showing it represses E-cadherin transcription in an ERα-dependent manner to promote endometrial cancer invasion.","evidence":"Transwell invasion, promoter-luciferase, and ChIP with siRNA/ectopic expression in Ishikawa cells","pmids":["17893710"],"confidence":"Medium","gaps":["Mechanism by which MACROD1 antagonizes ERα at the E-cadherin promoter unresolved","Single cell-line context"]},{"year":2007,"claim":"Defined a feedforward loop in which estrogen induces MACROD1 expression itself, via ERα/Sp1 cooperation at its proximal promoter.","evidence":"Promoter deletion/mutation luciferase, Sp1-siRNA, EMSA, and ChIP","pmids":["18206366"],"confidence":"Medium","gaps":["In vivo relevance of the autoregulatory loop not tested"]},{"year":2008,"claim":"Generalized the coactivator role beyond ERα by showing the single macro domain is sufficient for androgen receptor coactivation and is required for androgen-driven prostate cancer cell proliferation.","evidence":"CoIP, domain mapping, luciferase reporter, RNAi rescue in AR-positive vs AR-negative cell lines","pmids":["19022849"],"confidence":"High","gaps":["Whether the same domain surface mediates both ERα and AR binding not determined"]},{"year":2009,"claim":"Identified a spatial control mechanism: keratin 18 sequesters MACROD1 in the cytoplasm to limit its nuclear ERα coactivation.","evidence":"Yeast two-hybrid, GST pulldown, CoIP, GFP localization, nuclear/cytoplasmic fractionation, BrdU assay","pmids":["20035625"],"confidence":"High","gaps":["Signals that release MACROD1 from K18 sequestration unknown"]},{"year":2011,"claim":"Extended MACROD1 into NF-κB signaling by showing it associates with p65 to stabilize the nuclear NF-κB/p300/CBP complex rather than affecting nuclear translocation.","evidence":"GST pulldown, CoIP, luciferase reporter, RNAi, Annexin V flow cytometry","pmids":["21483817"],"confidence":"High","gaps":["Whether enzymatic ADP-ribose hydrolysis contributes to complex stabilization not addressed"]},{"year":2013,"claim":"Linked MACROD1 to metabolic signaling by showing it negatively regulates insulin signaling through mTOR-dependent TNF-α secretion and IRS-1/PI3K/Akt suppression.","evidence":"Lentiviral knockdown/overexpression in 3T3-L1 adipocytes, glucose uptake, phospho-Western, rapamycin epistasis","pmids":["23389992"],"confidence":"Medium","gaps":["Direct molecular link between MACROD1 and mTOR activation not defined"]},{"year":2015,"claim":"Defined a constitutive PARP1/IKKγ preassembly platform centered on MACROD1 required for DSB-induced IKKγ SUMOylation/phosphorylation and downstream NF-κB activation.","evidence":"Reciprocal CoIP, knockdown with NF-κB activation assays, DSB induction, Ku70/Ku80 epistasis","pmids":["25735744"],"confidence":"High","gaps":["Whether ADP-ribose hydrolase activity is required for scaffold function untested"]},{"year":2017,"claim":"Showed MACROD1 scaffolds a PKR–IKKβ ternary complex that prolongs PAR-dependent NF-κB transactivation and drives chemoresistance, identifying a druggable interaction.","evidence":"CoIP, MRS2578 competition, NF-κB reporter, xenograft models","pmids":["28820388"],"confidence":"High","gaps":["How PKR activation relates to MACROD1 enzymatic activity unclear"]},{"year":2018,"claim":"Established MACROD1 as a mitochondria-enriched mono-ADP-ribose hydrolase acting on phosphorylated dsDNA ADP-ribose adducts, anchoring its enzymatic identity and localization.","evidence":"Subcellular fractionation of endogenous protein and in vitro hydrolase assay with defined dsDNA substrates","pmids":["29410655"],"confidence":"High","gaps":["Physiological mitochondrial substrates not identified","Reconciliation of mitochondrial localization with nuclear coactivator roles not addressed"]},{"year":2018,"claim":"Placed MACROD1 in adipocyte inflammatory signaling via a Rac1-dependent ERK1/2 pathway promoting cytokine expression.","evidence":"LC-MS proteomics, phospho-Western, siRNA of MACROD1 and Rac1, PD98059 inhibition","pmids":["30562745"],"confidence":"Medium","gaps":["Direct interaction between MACROD1 and Rac1 not demonstrated"]},{"year":2020,"claim":"Solved the catalytic mechanism: the crystal structure with ADP-ribose defined the β5-α10 switch loop, Phe272 substrate orientation, and a hydrogen-bond network positioning catalytic water, with recruitment to ADP-ribosylated DNA lesions required for repair.","evidence":"X-ray crystallography, catalytic-residue mutagenesis, in vitro hydrolase assay, laser-induced DNA damage recruitment","pmids":["32683309"],"confidence":"High","gaps":["In vivo DNA repair contribution relative to paralogs not quantified"]},{"year":2020,"claim":"Confirmed predominant mitochondrial localization with a KO morphology phenotype and a mitochondria-concentrated interactome implicating mitochondrial RNA metabolism.","evidence":"Validated monoclonal antibodies, immunofluorescence, KO morphology assessment, BioID proximity labeling","pmids":["32427867"],"confidence":"High","gaps":["Specific role in mitochondrial RNA metabolism not functionally demonstrated"]},{"year":2021,"claim":"Linked MACROD1 loss to a female-specific motor-coordination defect, providing organismal evidence consistent with its mitochondrial role.","evidence":"Macrod1/Macrod2 knockout mice with behavioral battery testing","pmids":["33578760"],"confidence":"Medium","gaps":["No molecular mechanism linking enzymatic activity to the behavioral phenotype","Basis of sex-specificity unexplained"]},{"year":2024,"claim":"Defined a protective mitochondrial axis in which MACROD1 suppresses PARP1 to preserve NAD+ and activate SIRT3-dependent antioxidant signaling against diabetic cardiomyopathy.","evidence":"Macrod1 KO and cardiac-specific overexpression mouse models, NAD+ measurement, PARP1/SIRT3 Western, mitochondrial function assays","pmids":["38459256"],"confidence":"Medium","gaps":["Whether MACROD1 hydrolase activity drives PARP1 suppression not resolved","Direct mechanism of PARP1 downregulation unclear"]},{"year":null,"claim":"How a single macro domain reconciles its catalytic ADP-ribose hydrolase function with its non-catalytic scaffolding/coactivator roles, and which activity dominates in each subcellular compartment, remains unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No study has tested whether catalytic mutants retain coactivator/scaffold function","Physiological in vivo substrates of the hydrolase not identified","Mechanism partitioning nuclear vs mitochondrial pools beyond K18 sequestration unknown"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0016787","term_label":"hydrolase activity","supporting_discovery_ids":[2,7]},{"term_id":"GO:0140097","term_label":"catalytic activity, acting on DNA","supporting_discovery_ids":[2,7]},{"term_id":"GO:0140110","term_label":"transcription regulator activity","supporting_discovery_ids":[0,1,3]},{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[0,1,4,6]}],"localization":[{"term_id":"GO:0005739","term_label":"mitochondrion","supporting_discovery_ids":[2,8]},{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[0,3,5]},{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[5]}],"pathway":[{"term_id":"R-HSA-73894","term_label":"DNA Repair","supporting_discovery_ids":[7]},{"term_id":"R-HSA-74160","term_label":"Gene expression (Transcription)","supporting_discovery_ids":[0,1,3]},{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[4,6,12,13]},{"term_id":"R-HSA-8953897","term_label":"Cellular responses to stimuli","supporting_discovery_ids":[11]}],"complexes":["NF-κB/p300/CBP transcription complex","PARP1/IKKγ preassembly complex","PKR–IKKβ ternary complex"],"partners":["ESR1","AR","RELA","PARP1","IKBKG","EIF2AK2","IKBKB","KRT18"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q9BQ69","full_name":"ADP-ribose glycohydrolase MACROD1","aliases":["MACRO domain-containing protein 1","O-acetyl-ADP-ribose deacetylase MACROD1","Protein LRP16","[Protein ADP-ribosylaspartate] hydrolase MACROD1","[Protein ADP-ribosylglutamate] hydrolase MACROD1"],"length_aa":325,"mass_kda":35.5,"function":"Removes ADP-ribose from aspartate and glutamate residues in proteins bearing a single ADP-ribose moiety (PubMed:23474712, PubMed:23474714). Inactive towards proteins bearing poly-ADP-ribose (PubMed:23474712, PubMed:23474714). Deacetylates O-acetyl-ADP ribose, a signaling molecule generated by the deacetylation of acetylated lysine residues in histones and other proteins (PubMed:21257746). Plays a role in estrogen signaling (PubMed:17893710, PubMed:17914104, PubMed:19403568). Binds to androgen receptor (AR) and amplifies the transactivation function of AR in response to androgen (PubMed:19022849). May play an important role in carcinogenesis and/or progression of hormone-dependent cancers by feed-forward mechanism that activates ESR1 transactivation (PubMed:17893710, PubMed:17914104). Could be an ESR1 coactivator, providing a positive feedback regulatory loop for ESR1 signal transduction (PubMed:17914104). Could be involved in invasive growth by down-regulating CDH1 in endometrial cancer cells (PubMed:17893710). Enhances ESR1-mediated transcription activity (PubMed:17914104)","subcellular_location":"Nucleus","url":"https://www.uniprot.org/uniprotkb/Q9BQ69/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/MACROD1","classification":"Not Classified","n_dependent_lines":9,"n_total_lines":1208,"dependency_fraction":0.0074503311258278145},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/MACROD1","total_profiled":1310},"omim":[{"mim_id":"614393","title":"O-ACYL-ADP-RIBOSE DEACYLASE 1; OARD1","url":"https://www.omim.org/entry/614393"},{"mim_id":"610400","title":"MONO-ADP RIBOSYLHYDROLASE 1; MACROD1","url":"https://www.omim.org/entry/610400"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Supported","locations":[{"location":"Nucleoplasm","reliability":"Supported"}],"tissue_specificity":"Tissue enhanced","tissue_distribution":"Detected in many","driving_tissues":[{"tissue":"skeletal muscle","ntpm":270.3}],"url":"https://www.proteinatlas.org/search/MACROD1"},"hgnc":{"alias_symbol":["LRP16"],"prev_symbol":[]},"alphafold":{"accession":"Q9BQ69","domains":[{"cath_id":"3.40.220.10","chopping":"106-322","consensus_level":"high","plddt":96.37,"start":106,"end":322}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9BQ69","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q9BQ69-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q9BQ69-F1-predicted_aligned_error_v6.png","plddt_mean":82.25},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=MACROD1","jax_strain_url":"https://www.jax.org/strain/search?query=MACROD1"},"sequence":{"accession":"Q9BQ69","fasta_url":"https://rest.uniprot.org/uniprotkb/Q9BQ69.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q9BQ69/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9BQ69"}},"corpus_meta":[{"pmid":"19022849","id":"PMC_19022849","title":"The single-macro domain protein LRP16 is an essential cofactor of androgen receptor.","date":"2008","source":"Endocrine-related cancer","url":"https://pubmed.ncbi.nlm.nih.gov/19022849","citation_count":51,"is_preprint":false},{"pmid":"29410655","id":"PMC_29410655","title":"MacroD1 Is a Promiscuous ADP-Ribosyl Hydrolase Localized to Mitochondria.","date":"2018","source":"Frontiers in microbiology","url":"https://pubmed.ncbi.nlm.nih.gov/29410655","citation_count":49,"is_preprint":false},{"pmid":"17914104","id":"PMC_17914104","title":"Estrogenically regulated LRP16 interacts with estrogen receptor alpha and enhances the receptor's transcriptional activity.","date":"2007","source":"Endocrine-related cancer","url":"https://pubmed.ncbi.nlm.nih.gov/17914104","citation_count":46,"is_preprint":false},{"pmid":"12790785","id":"PMC_12790785","title":"Up-regulation of LRP16 mRNA by 17beta-estradiol through activation of estrogen receptor alpha (ERalpha), but not ERbeta, and promotion of human breast cancer MCF-7 cell proliferation: a preliminary report.","date":"2003","source":"Endocrine-related cancer","url":"https://pubmed.ncbi.nlm.nih.gov/12790785","citation_count":34,"is_preprint":false},{"pmid":"21483817","id":"PMC_21483817","title":"LRP16 integrates into NF-κB transcriptional complex and is required for its functional activation.","date":"2011","source":"PloS one","url":"https://pubmed.ncbi.nlm.nih.gov/21483817","citation_count":34,"is_preprint":false},{"pmid":"32427867","id":"PMC_32427867","title":"Comparative analysis of MACROD1, MACROD2 and TARG1 expression, localisation and interactome.","date":"2020","source":"Scientific reports","url":"https://pubmed.ncbi.nlm.nih.gov/32427867","citation_count":34,"is_preprint":false},{"pmid":"17893710","id":"PMC_17893710","title":"Induction of the LRP16 gene by estrogen promotes the invasive growth of Ishikawa human endometrial cancer cells through the downregulation of E-cadherin.","date":"2007","source":"Cell 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fused to RUNX1 in monocytic leukemia cell line with t(11;21)(q13;q22).","date":"2007","source":"European journal of haematology","url":"https://pubmed.ncbi.nlm.nih.gov/17532767","citation_count":19,"is_preprint":false},{"pmid":"25735744","id":"PMC_25735744","title":"An LRP16-containing preassembly complex contributes to NF-κB activation induced by DNA double-strand breaks.","date":"2015","source":"Nucleic acids research","url":"https://pubmed.ncbi.nlm.nih.gov/25735744","citation_count":18,"is_preprint":false},{"pmid":"32683309","id":"PMC_32683309","title":"Molecular basis for the MacroD1-mediated hydrolysis of ADP-ribosylation.","date":"2020","source":"DNA repair","url":"https://pubmed.ncbi.nlm.nih.gov/32683309","citation_count":17,"is_preprint":false},{"pmid":"20035625","id":"PMC_20035625","title":"Keratin 18 attenuates estrogen receptor alpha-mediated signaling by sequestering LRP16 in cytoplasm.","date":"2009","source":"BMC cell biology","url":"https://pubmed.ncbi.nlm.nih.gov/20035625","citation_count":17,"is_preprint":false},{"pmid":"38459256","id":"PMC_38459256","title":"Macrod1 suppresses diabetic cardiomyopathy via regulating PARP1-NAD+-SIRT3 pathway.","date":"2024","source":"Acta pharmacologica Sinica","url":"https://pubmed.ncbi.nlm.nih.gov/38459256","citation_count":16,"is_preprint":false},{"pmid":"33578760","id":"PMC_33578760","title":"Behavioural Characterisation of Macrod1 and Macrod2 Knockout Mice.","date":"2021","source":"Cells","url":"https://pubmed.ncbi.nlm.nih.gov/33578760","citation_count":15,"is_preprint":false},{"pmid":"19403568","id":"PMC_19403568","title":"Differential induction of LRP16 by liganded and unliganded estrogen receptor alpha in SKOV3 ovarian carcinoma cells.","date":"2009","source":"The Journal of endocrinology","url":"https://pubmed.ncbi.nlm.nih.gov/19403568","citation_count":13,"is_preprint":false},{"pmid":"30562745","id":"PMC_30562745","title":"MACROD1/LRP16 Enhances LPS-Stimulated Inflammatory Responses by Up-Regulating a Rac1-Dependent Pathway in Adipocytes.","date":"2018","source":"Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology","url":"https://pubmed.ncbi.nlm.nih.gov/30562745","citation_count":12,"is_preprint":false},{"pmid":"18206366","id":"PMC_18206366","title":"GC-rich promoter elements maximally confers estrogen-induced transactivation of LRP16 gene through ERalpha/Sp1 interaction in MCF-7 cells.","date":"2007","source":"The Journal of steroid biochemistry and molecular biology","url":"https://pubmed.ncbi.nlm.nih.gov/18206366","citation_count":12,"is_preprint":false},{"pmid":"16831279","id":"PMC_16831279","title":"[Expression and clinical significance of LRP16 gene in human breast cancer].","date":"2006","source":"Ai zheng = Aizheng = Chinese journal of cancer","url":"https://pubmed.ncbi.nlm.nih.gov/16831279","citation_count":11,"is_preprint":false},{"pmid":"23389992","id":"PMC_23389992","title":"Identification of LRP16 as a negative regulator of insulin action and adipogenesis in 3T3-L1 adipocytes.","date":"2013","source":"Hormone and metabolic research = Hormon- und Stoffwechselforschung = Hormones et metabolisme","url":"https://pubmed.ncbi.nlm.nih.gov/23389992","citation_count":8,"is_preprint":false},{"pmid":"22800886","id":"PMC_22800886","title":"LRP16 gene protects mouse insulinoma MIN6 cells against fatty acid-induced apoptosis through Akt/FoxO1 signaling.","date":"2012","source":"Chinese medical journal","url":"https://pubmed.ncbi.nlm.nih.gov/22800886","citation_count":6,"is_preprint":false},{"pmid":"29551900","id":"PMC_29551900","title":"Leukemia-related protein 16 (LRP16) promotes tumor growth and metastasis in pancreatic cancer.","date":"2018","source":"OncoTargets and therapy","url":"https://pubmed.ncbi.nlm.nih.gov/29551900","citation_count":4,"is_preprint":false},{"pmid":"29748698","id":"PMC_29748698","title":"LRP16 prevents hepatocellular carcinoma progression through regulation of Wnt/β-catenin signaling.","date":"2018","source":"Journal of molecular medicine (Berlin, Germany)","url":"https://pubmed.ncbi.nlm.nih.gov/29748698","citation_count":4,"is_preprint":false},{"pmid":"12578638","id":"PMC_12578638","title":"[The Application of RACE Technique to Clone the Full-Length cDNA of A Novel Leukemia Associated Gene LRP16].","date":"2001","source":"Zhongguo shi yan xue ye xue za zhi","url":"https://pubmed.ncbi.nlm.nih.gov/12578638","citation_count":4,"is_preprint":false},{"pmid":"16584612","id":"PMC_16584612","title":"[Analysis of LRP16 gene promoter activity].","date":"2006","source":"Zhongguo shi yan xue ye xue za zhi","url":"https://pubmed.ncbi.nlm.nih.gov/16584612","citation_count":3,"is_preprint":false},{"pmid":"22528219","id":"PMC_22528219","title":"Reduced expression of the LRP16 gene in mouse insulinoma (MIN6) cells exerts multiple effects on insulin content, proliferation and apoptosis.","date":"2012","source":"Journal of Huazhong University of Science and Technology. Medical sciences = Hua zhong ke ji da xue xue bao. Yi xue Ying De wen ban = Huazhong keji daxue xuebao. Yixue Yingdewen ban","url":"https://pubmed.ncbi.nlm.nih.gov/22528219","citation_count":2,"is_preprint":false},{"pmid":"21756814","id":"PMC_21756814","title":"[LRP16 gene causes insulin resistance in C2-C12 cells by inhibiting the IRS-1 signaling and the transcriptional activity of peroxisome proliferator actived receptor γ].","date":"2011","source":"Zhonghua yi xue za zhi","url":"https://pubmed.ncbi.nlm.nih.gov/21756814","citation_count":2,"is_preprint":false},{"pmid":"40525343","id":"PMC_40525343","title":"[Prokaryotic expression of mouse LRP16, preparation and identification of rabbit anti-mouse LRP16 polyclonal antibody].","date":"2025","source":"Xi bao yu fen zi mian yi xue za zhi = Chinese journal of cellular and molecular immunology","url":"https://pubmed.ncbi.nlm.nih.gov/40525343","citation_count":0,"is_preprint":false},{"pmid":"19840441","id":"PMC_19840441","title":"[Promotive effect of LRP16 gene on proliferation of K562 cells].","date":"2009","source":"Zhongguo shi yan xue ye xue za zhi","url":"https://pubmed.ncbi.nlm.nih.gov/19840441","citation_count":0,"is_preprint":false},{"pmid":null,"id":"bio_10.1101_2025.11.04.686626","title":"Mitochondrial bioenergetic signatures differentiate asymptomatic from symptomatic Alzheimer’s disease","date":"2025-11-06","source":"bioRxiv","url":"https://doi.org/10.1101/2025.11.04.686626","citation_count":0,"is_preprint":true}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":18402,"output_tokens":4243,"usd":0.059425,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":12386,"output_tokens":4687,"usd":0.089552,"stage2_stop_reason":"end_turn"},"total_usd":0.148977,"stage1_batch_id":"msgbatch_01BbTCMyrRPyhtEq6b1MdwCN","stage2_batch_id":"msgbatch_011dEed5bbxYvTAaQWKJevxu","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2007,\n      \"finding\": \"LRP16 (MACROD1) physically interacts with estrogen receptor alpha (ERα) via its macro domain, in a manner that is estrogen-independent but enhanced by estrogen, and binds specifically to the A/B activation function 1 (AF-1) domain of ERα, enhancing ERα-mediated transcriptional activity. GST pulldown and coimmunoprecipitation confirmed the interaction.\",\n      \"method\": \"GST pulldown, coimmunoprecipitation, mammalian two-hybrid assay, siRNA knockdown with luciferase reporter and target gene expression\",\n      \"journal\": \"Endocrine-related cancer\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal GST pulldown + CoIP + functional reporter assays, replicated across multiple cell line models in a single rigorous study\",\n      \"pmids\": [\"17914104\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"LRP16 (MACROD1) binds to androgen receptor (AR) via its macro domain and functions as a coactivator to amplify AR transactivation in response to androgen. The single macro domain is sufficient for AR coactivation. RNAi knockdown of LRP16 impairs AR function and attenuates coactivation by ART-27 and SRC-1, and inhibits androgen-stimulated proliferation of LNCaP cells but not AR-negative PC-3 cells.\",\n      \"method\": \"Co-immunoprecipitation, luciferase reporter assay, RNAi knockdown, domain mapping\",\n      \"journal\": \"Endocrine-related cancer\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal CoIP, domain mapping, functional reporter assays, rescue experiments with isogenic AR-positive vs. AR-negative cell lines\",\n      \"pmids\": [\"19022849\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"MacroD1 (MACROD1) is a mono-ADP-ribose hydrolase localized primarily in mitochondria (not nucleus or cytosol) of skeletal muscle cells. It can efficiently remove ADP-ribose from 5' and 3'-phosphorylated double-stranded DNA adducts in vitro, targeting ester bonds of ADP-ribosylated phosphorylated dsDNA ends.\",\n      \"method\": \"Subcellular fractionation with endogenous protein detection, mitochondrial enrichment, in vitro ADP-ribose hydrolase enzymatic assay with phosphorylated dsDNA substrates\",\n      \"journal\": \"Frontiers in microbiology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Moderate — endogenous protein fractionation plus in vitro enzymatic assay with defined substrates, single lab but orthogonal methods\",\n      \"pmids\": [\"29410655\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"LRP16 (MACROD1) integrates into the NF-κB transcriptional complex by associating with the p65 subunit. RNAi knockdown of LRP16 does not affect TNF-α-induced nuclear translocation of NF-κB but blunts the formation/stabilization of the functional NF-κB/p300/CBP transcription complex in the nucleus, impairing NF-κB target gene expression and sensitizing cells to TNF-α-induced apoptosis.\",\n      \"method\": \"GST pulldown, coimmunoprecipitation, luciferase reporter assay, RNAi knockdown, flow cytometry (Annexin V), target gene expression analysis\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — GST pulldown + CoIP + functional reporter + mechanistic dissection of nuclear vs. cytoplasmic events, single lab with multiple orthogonal methods\",\n      \"pmids\": [\"21483817\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"LRP16 (MACROD1) constitutively interacts with PARP1 and IKKγ, forming a preassembly complex. This interaction is essential for efficient interactions among PARP1, IKKγ, and PIASy, the DSB-induced SUMOylation and phosphorylation of IKKγ, and subsequent NF-κB activation following DNA double-strand break induction. The regulation is dependent on the DSB sensors Ku70/Ku80.\",\n      \"method\": \"Coimmunoprecipitation, genetic knockdown with functional NF-κB activation assays, DSB induction experiments\",\n      \"journal\": \"Nucleic acids research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal CoIP showing constitutive complex, functional NF-κB assays post-DSB, epistasis with Ku70/Ku80, single lab with multiple orthogonal methods\",\n      \"pmids\": [\"25735744\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"Keratin 18 (K18) physically interacts with LRP16 (MACROD1) and sequesters it in the cytoplasm, reducing nuclear LRP16 availability. This attenuates LRP16-ERα association, ERα-activated transcription, and estrogen-stimulated cell cycle progression. K18 knockdown has the opposite effect, increasing nuclear LRP16 and ERα-mediated signaling.\",\n      \"method\": \"Yeast two-hybrid screening, GST pulldown, coimmunoprecipitation, fluorescence microscopy (GFP-LRP16 localization), immunoblotting of nuclear/cytoplasmic fractions, BrdU incorporation assay\",\n      \"journal\": \"BMC cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — yeast two-hybrid + GST pulldown + CoIP + live-cell localization + functional proliferation assay, single lab with multiple orthogonal methods\",\n      \"pmids\": [\"20035625\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"LRP16 (MACROD1) selectively interacts with and activates double-stranded RNA-dependent kinase PKR, and acts as a scaffold to assist formation of a ternary complex of PKR and IKKβ, prolonging PAR-dependent NF-κB transactivation induced by DNA-damaging agents and conferring acquired chemoresistance. The small molecule MRS2578 abrogates LRP16 binding to PKR and IKKβ, converting LRP16 into a pro-death molecule.\",\n      \"method\": \"Co-immunoprecipitation, small molecule inhibitor (MRS2578) competition assay, NF-κB luciferase reporter, xenograft tumor models\",\n      \"journal\": \"eLife\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal CoIP demonstrating ternary complex, functional assays with specific inhibitor, in vivo xenograft validation, single lab with multiple orthogonal approaches\",\n      \"pmids\": [\"28820388\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"Crystal structure of MacroD1 (MACROD1) in complex with ADP-ribose reveals that the β5-α10-loop acts as a switch loop for substrate recognition and orientation. The conserved Phe272 in this loop orients the distal ribose of ADPR, and a conserved hydrogen-bond network positions catalytic water for ADPR hydrolysis. MacroD1 is recruited to DNA damage sites via recognition of ADP-ribosylation at DNA lesions, and MacroD1-mediated ADPR hydrolysis is essential for DNA damage repair.\",\n      \"method\": \"X-ray crystallography (MacroD1-ADP-ribose complex), site-directed mutagenesis of catalytic residues, in vitro hydrolase assay, DNA damage recruitment assay (laser-induced damage with fluorescence microscopy)\",\n      \"journal\": \"DNA repair\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — crystal structure with functional mutagenesis validation and in vitro enzymatic assay plus cellular DNA damage recruitment, single lab with multiple orthogonal high-quality methods\",\n      \"pmids\": [\"32683309\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"MACROD1 localizes predominantly to mitochondria (especially in skeletal muscle), and loss of MACROD1 causes disruption of mitochondrial morphology. BioID interactome mapping reveals MACROD1-interacting proteins concentrated in mitochondria, suggesting involvement in mitochondrial RNA metabolism.\",\n      \"method\": \"Monoclonal antibody validation, immunofluorescence localization, mitochondrial morphology assessment upon MACROD1 knockout, BioID proximity labeling interactome mapping\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — validated monoclonal antibodies plus BioID interactome plus KO phenotype, replicated mitochondrial localization across multiple labs/studies\",\n      \"pmids\": [\"32427867\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"LRP16 (MACROD1) represses E-cadherin transcription in an ERα-dependent manner, promoting invasive growth of Ishikawa endometrial cancer cells. Chromatin immunoprecipitation showed LRP16 antagonizes ERα binding to the E-cadherin promoter. E-cadherin downregulation requires ERα mediation as estrogen deprivation abolishes the effect.\",\n      \"method\": \"Transwell invasion assay, promoter-luciferase reporter analysis, chromatin immunoprecipitation (ChIP), siRNA/ectopic expression\",\n      \"journal\": \"Cell research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — ChIP showing promoter interaction, functional invasion assay, ERα dependency established by estrogen deprivation, single lab\",\n      \"pmids\": [\"17893710\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"Estrogen receptor alpha (ERα) and Sp1 cooperate at GC-rich motifs in the proximal promoter of LRP16 (specifically the -213/-184 bp fragment) to mediate maximal estrogen-induced transcription of LRP16, as demonstrated by gel mobility shift assays showing Sp1 binding enhanced by ERα titer and ChIP confirming ERα/Sp1 interaction at GC-rich sites.\",\n      \"method\": \"Deletion and mutation analysis of LRP16 promoter-luciferase constructs, Sp1-siRNA, gel mobility shift assay, chromatin immunoprecipitation (ChIP)\",\n      \"journal\": \"The Journal of steroid biochemistry and molecular biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — EMSA + ChIP + promoter deletion mapping + siRNA, single lab with multiple methods\",\n      \"pmids\": [\"18206366\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"Macrod1 (MACROD1) suppresses diabetic cardiomyopathy by inhibiting PARP1 expression, thereby reducing NAD+ consumption and activating the deacetylase SIRT3 to resist oxidative stress. Knockout of Macrod1 worsened glycemic control, cardiac remodeling, and mitochondrial dysfunction; cardiac-specific overexpression partially reversed these effects. In cardiomyocytes, Macrod1 overexpression inhibited PARP1 and restored NAD+ levels to activate SIRT3.\",\n      \"method\": \"Macrod1 knockout and cardiac-specific overexpression mouse models (HFD/STZ-induced DCM), Western blot (PARP1, SIRT3), NAD+ measurement, mitochondrial function assays, in vitro neonatal cardiomyocyte palmitate model\",\n      \"journal\": \"Acta pharmacologica Sinica\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic KO and OE with defined molecular readouts (PARP1, NAD+, SIRT3), single lab, multiple orthogonal methods\",\n      \"pmids\": [\"38459256\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"LRP16 (MACROD1) acts as a negative regulator of insulin signaling in 3T3-L1 adipocytes by activating the mTOR pathway, which in turn promotes TNF-α secretion, inhibits IRS-1/PI3K/Akt phosphorylation, and reduces PPARγ expression. Rapamycin (mTOR inhibitor) rescues the LRP16-overexpression phenotype, placing mTOR downstream of LRP16 in insulin resistance.\",\n      \"method\": \"Lentiviral siRNA knockdown and ectopic overexpression of LRP16 in 3T3-L1 cells, glucose uptake assay, Western blot (IRS-1, PI3K, Akt, mTOR phosphorylation), rapamycin epistasis\",\n      \"journal\": \"Hormone and metabolic research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — gain- and loss-of-function with rapamycin epistasis placing mTOR in pathway, single lab\",\n      \"pmids\": [\"23389992\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"LRP16 (MACROD1) promotes LPS-stimulated inflammatory responses in adipocytes through activation of a Rac1-dependent ERK1/2 (MAPK) signaling pathway. LRP16 overexpression activates ERK1/2 and Rac1; Rac1 knockdown or ERK inhibitor (PD98059) abolishes the stimulatory effect of LRP16 on inflammatory cytokine expression.\",\n      \"method\": \"LC-MS proteomics, Western blot (ERK1/2, Rac1 phosphorylation), siRNA knockdown of LRP16 and Rac1, pharmacological ERK inhibition (PD98059)\",\n      \"journal\": \"Cellular physiology and biochemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — pharmacological and genetic epistasis placing Rac1 and ERK downstream of LRP16, single lab with multiple approaches\",\n      \"pmids\": [\"30562745\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"Loss of Macrod1 in mice results in a female-specific motor-coordination defect, consistent with its mitochondrial localization and suggesting a role in mitochondria-dependent neuromotor function. Loss of Macrod2 (a paralog) produces a distinct hyperactivity/bradykinesia phenotype.\",\n      \"method\": \"Macrod1 and Macrod2 knockout mouse models, behavioral battery testing (motor coordination, locomotion assays)\",\n      \"journal\": \"Cells\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — clean KO with defined behavioral phenotype but no direct molecular mechanism identified, single lab\",\n      \"pmids\": [\"33578760\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"MACROD1 (LRP16) is a mitochondria-enriched mono-ADP-ribose hydrolase that removes ADP-ribose from acidic amino acids and DNA ends via a catalytic mechanism requiring Phe272 and a hydrogen-bond network (crystal structure solved); it also functions as a nuclear coactivator of estrogen receptor α and androgen receptor through its single macro domain, integrates into the NF-κB transcriptional complex via p65 and a preassembled PARP1/IKKγ scaffold to potentiate DSB-induced NF-κB signaling, interacts with and activates PKR to form a PKR–IKKβ ternary complex that confers chemoresistance, and is sequestered in the cytoplasm by keratin 18 to modulate ERα signaling availability; in mitochondria it suppresses PARP1-mediated NAD+ consumption to activate SIRT3-dependent antioxidant signaling.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"MACROD1 (LRP16) is a mono-ADP-ribose hydrolase whose single macro domain serves dual roles as an enzyme that reverses ADP-ribosylation and as a protein-interaction module integrating it into nuclear transcription and DNA-damage signaling [#2, #7, #0]. As a catalyst, it removes ADP-ribose from phosphorylated double-stranded DNA ends, and its crystal structure in complex with ADP-ribose defines a β5-α10 switch loop in which Phe272 orients the distal ribose and a conserved hydrogen-bond network positions the catalytic water; this hydrolase activity supports its recruitment to ADP-ribosylated DNA lesions and contributes to DNA damage repair [#2, #7]. Independently of catalysis, the macro domain binds the AF-1 domain of estrogen receptor α and binds androgen receptor, acting as a transcriptional coactivator that amplifies hormone-driven transcription and, in an ERα-dependent manner, represses E-cadherin to promote invasive growth [#0, #1, #9]. MACROD1 also functions in NF-κB signaling, associating with the p65 subunit to stabilize the nuclear NF-κB/p300/CBP complex and residing in a constitutive PARP1/IKKγ preassembly complex required for DSB-induced IKKγ modification and NF-κB activation; it further scaffolds a PKR–IKKβ ternary complex that prolongs PAR-dependent NF-κB transactivation and confers chemoresistance [#3, #4, #6]. The protein is enriched in mitochondria, particularly in skeletal muscle, where its loss disrupts mitochondrial morphology, and it suppresses PARP1-mediated NAD+ consumption to activate SIRT3-dependent antioxidant signaling protective against diabetic cardiomyopathy [#2, #8, #11]. Its nuclear availability is controlled by keratin 18, which sequesters it in the cytoplasm to limit ERα signaling [#5].\",\n  \"teleology\": [\n    {\n      \"year\": 2007,\n      \"claim\": \"Established MACROD1 as a hormone-receptor coactivator by showing its macro domain binds the ERα AF-1 domain and amplifies ERα-driven transcription, defining its first molecular function.\",\n      \"evidence\": \"GST pulldown, CoIP, mammalian two-hybrid, and luciferase reporter with siRNA knockdown across cell lines\",\n      \"pmids\": [\"17914104\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not test whether catalytic/enzymatic activity is involved in coactivation\", \"Structural basis of macro domain–AF-1 contact not resolved\"]\n    },\n    {\n      \"year\": 2007,\n      \"claim\": \"Connected MACROD1 coactivation to an oncogenic output by showing it represses E-cadherin transcription in an ERα-dependent manner to promote endometrial cancer invasion.\",\n      \"evidence\": \"Transwell invasion, promoter-luciferase, and ChIP with siRNA/ectopic expression in Ishikawa cells\",\n      \"pmids\": [\"17893710\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mechanism by which MACROD1 antagonizes ERα at the E-cadherin promoter unresolved\", \"Single cell-line context\"]\n    },\n    {\n      \"year\": 2007,\n      \"claim\": \"Defined a feedforward loop in which estrogen induces MACROD1 expression itself, via ERα/Sp1 cooperation at its proximal promoter.\",\n      \"evidence\": \"Promoter deletion/mutation luciferase, Sp1-siRNA, EMSA, and ChIP\",\n      \"pmids\": [\"18206366\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"In vivo relevance of the autoregulatory loop not tested\"]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"Generalized the coactivator role beyond ERα by showing the single macro domain is sufficient for androgen receptor coactivation and is required for androgen-driven prostate cancer cell proliferation.\",\n      \"evidence\": \"CoIP, domain mapping, luciferase reporter, RNAi rescue in AR-positive vs AR-negative cell lines\",\n      \"pmids\": [\"19022849\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether the same domain surface mediates both ERα and AR binding not determined\"]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"Identified a spatial control mechanism: keratin 18 sequesters MACROD1 in the cytoplasm to limit its nuclear ERα coactivation.\",\n      \"evidence\": \"Yeast two-hybrid, GST pulldown, CoIP, GFP localization, nuclear/cytoplasmic fractionation, BrdU assay\",\n      \"pmids\": [\"20035625\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Signals that release MACROD1 from K18 sequestration unknown\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Extended MACROD1 into NF-κB signaling by showing it associates with p65 to stabilize the nuclear NF-κB/p300/CBP complex rather than affecting nuclear translocation.\",\n      \"evidence\": \"GST pulldown, CoIP, luciferase reporter, RNAi, Annexin V flow cytometry\",\n      \"pmids\": [\"21483817\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether enzymatic ADP-ribose hydrolysis contributes to complex stabilization not addressed\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Linked MACROD1 to metabolic signaling by showing it negatively regulates insulin signaling through mTOR-dependent TNF-α secretion and IRS-1/PI3K/Akt suppression.\",\n      \"evidence\": \"Lentiviral knockdown/overexpression in 3T3-L1 adipocytes, glucose uptake, phospho-Western, rapamycin epistasis\",\n      \"pmids\": [\"23389992\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct molecular link between MACROD1 and mTOR activation not defined\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Defined a constitutive PARP1/IKKγ preassembly platform centered on MACROD1 required for DSB-induced IKKγ SUMOylation/phosphorylation and downstream NF-κB activation.\",\n      \"evidence\": \"Reciprocal CoIP, knockdown with NF-κB activation assays, DSB induction, Ku70/Ku80 epistasis\",\n      \"pmids\": [\"25735744\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether ADP-ribose hydrolase activity is required for scaffold function untested\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Showed MACROD1 scaffolds a PKR–IKKβ ternary complex that prolongs PAR-dependent NF-κB transactivation and drives chemoresistance, identifying a druggable interaction.\",\n      \"evidence\": \"CoIP, MRS2578 competition, NF-κB reporter, xenograft models\",\n      \"pmids\": [\"28820388\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How PKR activation relates to MACROD1 enzymatic activity unclear\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Established MACROD1 as a mitochondria-enriched mono-ADP-ribose hydrolase acting on phosphorylated dsDNA ADP-ribose adducts, anchoring its enzymatic identity and localization.\",\n      \"evidence\": \"Subcellular fractionation of endogenous protein and in vitro hydrolase assay with defined dsDNA substrates\",\n      \"pmids\": [\"29410655\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Physiological mitochondrial substrates not identified\", \"Reconciliation of mitochondrial localization with nuclear coactivator roles not addressed\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Placed MACROD1 in adipocyte inflammatory signaling via a Rac1-dependent ERK1/2 pathway promoting cytokine expression.\",\n      \"evidence\": \"LC-MS proteomics, phospho-Western, siRNA of MACROD1 and Rac1, PD98059 inhibition\",\n      \"pmids\": [\"30562745\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct interaction between MACROD1 and Rac1 not demonstrated\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Solved the catalytic mechanism: the crystal structure with ADP-ribose defined the β5-α10 switch loop, Phe272 substrate orientation, and a hydrogen-bond network positioning catalytic water, with recruitment to ADP-ribosylated DNA lesions required for repair.\",\n      \"evidence\": \"X-ray crystallography, catalytic-residue mutagenesis, in vitro hydrolase assay, laser-induced DNA damage recruitment\",\n      \"pmids\": [\"32683309\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"In vivo DNA repair contribution relative to paralogs not quantified\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Confirmed predominant mitochondrial localization with a KO morphology phenotype and a mitochondria-concentrated interactome implicating mitochondrial RNA metabolism.\",\n      \"evidence\": \"Validated monoclonal antibodies, immunofluorescence, KO morphology assessment, BioID proximity labeling\",\n      \"pmids\": [\"32427867\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Specific role in mitochondrial RNA metabolism not functionally demonstrated\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Linked MACROD1 loss to a female-specific motor-coordination defect, providing organismal evidence consistent with its mitochondrial role.\",\n      \"evidence\": \"Macrod1/Macrod2 knockout mice with behavioral battery testing\",\n      \"pmids\": [\"33578760\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No molecular mechanism linking enzymatic activity to the behavioral phenotype\", \"Basis of sex-specificity unexplained\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Defined a protective mitochondrial axis in which MACROD1 suppresses PARP1 to preserve NAD+ and activate SIRT3-dependent antioxidant signaling against diabetic cardiomyopathy.\",\n      \"evidence\": \"Macrod1 KO and cardiac-specific overexpression mouse models, NAD+ measurement, PARP1/SIRT3 Western, mitochondrial function assays\",\n      \"pmids\": [\"38459256\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether MACROD1 hydrolase activity drives PARP1 suppression not resolved\", \"Direct mechanism of PARP1 downregulation unclear\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How a single macro domain reconciles its catalytic ADP-ribose hydrolase function with its non-catalytic scaffolding/coactivator roles, and which activity dominates in each subcellular compartment, remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No study has tested whether catalytic mutants retain coactivator/scaffold function\", \"Physiological in vivo substrates of the hydrolase not identified\", \"Mechanism partitioning nuclear vs mitochondrial pools beyond K18 sequestration unknown\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0016787\", \"supporting_discovery_ids\": [2, 7]},\n      {\"term_id\": \"GO:0140097\", \"supporting_discovery_ids\": [2, 7]},\n      {\"term_id\": \"GO:0140110\", \"supporting_discovery_ids\": [0, 1, 3]},\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [0, 1, 4, 6]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005739\", \"supporting_discovery_ids\": [2, 8]},\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [0, 3, 5]},\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [5]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-73894\", \"supporting_discovery_ids\": [7]},\n      {\"term_id\": \"R-HSA-74160\", \"supporting_discovery_ids\": [0, 1, 3]},\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [4, 6, 12, 13]},\n      {\"term_id\": \"R-HSA-8953897\", \"supporting_discovery_ids\": [11]}\n    ],\n    \"complexes\": [\n      \"NF-κB/p300/CBP transcription complex\",\n      \"PARP1/IKKγ preassembly complex\",\n      \"PKR–IKKβ ternary complex\"\n    ],\n    \"partners\": [\n      \"ESR1\",\n      \"AR\",\n      \"RELA\",\n      \"PARP1\",\n      \"IKBKG\",\n      \"EIF2AK2\",\n      \"IKBKB\",\n      \"KRT18\"\n    ],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":6,"faith_total":6,"faith_pct":100.0}}