Oligomerization mediated by the D2 domain of DTX3L is critical for DTX3L-PARP9 reading function of mono-ADP-ribosylated androgen receptor.

Deltex proteins are a family of E3 ubiquitin ligases that encode C-terminal RING and DTC domains that mediate interactions with E2 ubiquitin-conjugating enzymes and recognize ubiquitination substrates. DTX3L is unique among the Deltex proteins based on its N-terminal domain architecture. The N-terminal D1 and D2 domains of DTX3L mediate homo-oligomerization, and the D3 domain interacts with PARP9, a protein that contains tandem macrodomains with ADP-ribose reader function. While DTX3L and PARP9 are known to heterodimerize, and assemble into a high molecular weight oligomeric complex, the nature of the oligomeric structure, including whether this contributes to the ADP-ribose reader function is unknown. Here, we report a crystal structure of the DTX3L N-terminal D2 domain and show that it forms a tetramer with, conveniently, D2 symmetry. We identified two interfaces in the structure: a major, conserved interface with a surface of 973 Å2 and a smaller one of 415 Å2. Using native mass spectrometry, we observed molecular species that correspond to monomers, dimers and tetramers of the D2 domain. Reconstitution of DTX3L knockout cells with a D1-D2 deletion mutant showed the domain is dispensable for DTX3L-PARP9 heterodimer formation, but necessary to assemble an oligomeric complex with efficient reader function for ADP-ribosylated androgen receptor. Our results suggest that homo-oligomerization of DTX3L is important for the DTX3L-PARP9 complex to read mono-ADP-ribosylation on a ligand-regulated transcription factor.

Preparation of screening assays for ADP-ribosyl readers and erasers using the GAP-tag as a binding probe.

Here, we describe a protocol to set up a screening assay for ADP-ribosyl binding proteins including proteins that possess O-glycosidase or N-glycosidase activities. The FRET-based assay measures the interaction of any ADP-ribosyl binding protein fused to CFP with a cysteine-ADP-ribosylated GAP-tag fused to YFP. Recombinant PtxS1 and PARP2 are used to mono-ADP-ribosylate and poly-ADP-ribosylate the GAP-tag. The protocol does not require specialized compounds or substrates, making it accessible and easy to adapt in any laboratory or for other proteins of interest. For complete details on the use and execution of this profile, please refer to Sowa et al. (2021).

Reconstitution of the DTX3L-PARP9 complex reveals determinants for high-affinity heterodimerization and multimeric assembly.

Ubiquitination and ADP-ribosylation are post-translational modifications that play major roles in pathways including the DNA damage response and viral infection. The enzymes responsible for these modifications are therefore potential targets for therapeutic intervention. DTX3L is an E3 Ubiquitin ligase that forms a heterodimer with PARP9. In addition to its ubiquitin ligase activity, DTX3L-PARP9 also acts as an ADP-ribosyl transferase for Gly76 on the C-terminus of ubiquitin. NAD+-dependent ADP-ribosylation of ubiquitin by DTX3L-PARP9 prevents ubiquitin from conjugating to protein substrates. To gain insight into how DTX3L-PARP9 generates these post-translational modifications, we produced recombinant forms of DTX3L and PARP9 and studied their physical interactions. We show the DTX3L D3 domain (230-510) mediates the interaction with PARP9 with nanomolar affinity and an apparent 1 : 1 stoichiometry. We also show that DTX3L and PARP9 assemble into a higher molecular weight oligomer, and that this is mediated by the DTX3L N-terminal region (1-200). Lastly, we show that ADP-ribosylation of ubiquitin at Gly76 is reversible in vitro by several Macrodomain-type hydrolases. Our study provides a framework to understand how DTX3L-PARP9 mediates ADP-ribosylation and ubiquitination through both intra- and inter-subunit interactions.

A molecular toolbox for ADP-ribosyl binding proteins.

Proteins interacting with ADP-ribosyl groups are often involved in disease-related pathways or viral infections, making them attractive drug targets. We present a robust and accessible assay applicable to both hydrolyzing or non-hydrolyzing binders of mono- and poly-ADP-ribosyl groups. This technology relies on a C-terminal tag based on a Gi protein alpha subunit peptide (GAP), which allows for site-specific introduction of cysteine-linked mono- and poly-ADP-ribosyl groups or analogs. By fusing the GAP-tag and ADP-ribosyl binders to fluorescent proteins, we generate robust FRET partners and confirm the interaction with 22 known ADP-ribosyl binders. The applicability for high-throughput screening of inhibitors is demonstrated with the SARS-CoV-2 nsp3 macrodomain, for which we identify suramin as a moderate-affinity yet non-specific inhibitor. High-affinity ADP-ribosyl binders fused to nanoluciferase complement this technology, enabling simple blot-based detection of ADP-ribosylated proteins. All these tools can be produced in Escherichia coli and will help in ADP-ribosylation research and drug discovery.

Activity-Based Screening Assay for Mono-ADP-Ribosylhydrolases.

ADP-ribosylation is a post-translational modification involved in the regulation of many vital cellular processes. This posttranslational modification is carried out by ADP-ribosyltransferases converting ß-NAD+ into nicotinamide and a protein-linked ADP-ribosyl group or a chain of PAR. The reverse reaction, release of ADP-ribose from the acceptor molecule, is catalyzed by ADP-ribosylhydrolases. Several hydrolases contain a macrodomain fold, and activities of human macrodomain protein modules vary from reading or erasing mono- and poly-ADP-ribosylation. Macrodomains have been linked to diseases such as cancer, making them potential drug targets. Discovery of inhibitors requires robust biochemical tools mostly lacking for hydrolases, and here we describe an inhibitor screening assay against mono-ADP-ribosylhydrolyzing enzymes. The activity-based assay uses an α-NAD+, anomer of ß-NAD+, which is accepted as a substrate by MacroD1, MacroD2, and ARH3 due to its resemblance to the protein-linked ADP-ribose. The amount of α-NAD+ present after hydrolysis is measured by chemically converting it on a microtiter plate to a fluorescent compound. We optimized the assay for MacroD2 and performed a proof-of-concept compound screening. Three compounds were identified as screening hits with micromolar potency. However, further characterization of the compounds identified them as protein destabilizers, excluding further follow-up studies. Validation and screening demonstrated the usability of the in vitro assay for MacroD2, and we also demonstrate the applicability of the assay as a tool for other human ADP-ribosylhydrolases.

NHLRC2 variants identified in patients with fibrosis, neurodegeneration, and cerebral angiomatosis (FINCA): characterisation of a novel cerebropulmonary disease.

A novel multi-organ disease that is fatal in early childhood was identified in three patients from two non-consanguineous families. These children were born asymptomatic but at the age of 2 months they manifested progressive multi-organ symptoms resembling no previously known disease. The main clinical features included progressive cerebropulmonary symptoms, malabsorption, progressive growth failure, recurrent infections, chronic haemolytic anaemia and transient liver dysfunction. In the affected children, neuropathology revealed increased angiomatosis-like leptomeningeal, cortical and superficial white matter vascularisation and congestion, vacuolar degeneration and myelin loss in white matter, as well as neuronal degeneration. Interstitial fibrosis and previously undescribed granuloma-like lesions were observed in the lungs. Hepatomegaly, steatosis and collagen accumulation were detected in the liver. A whole-exome sequencing of the two unrelated families with the affected children revealed the transmission of two heterozygous variants in the NHL repeat-containing protein 2 (NHLRC2); an amino acid substitution p.Asp148Tyr and a frameshift 2-bp deletion p.Arg201GlyfsTer6. NHLRC2 is highly conserved and expressed in multiple organs and its function is unknown. It contains a thioredoxin-like domain; however, an insulin turbidity assay on human recombinant NHLRC2 showed no thioredoxin activity. In patient-derived fibroblasts, NHLRC2 levels were low, and only p.Asp148Tyr was expressed. Therefore, the allele with the frameshift deletion is likely non-functional. Development of the Nhlrc2 null mouse strain stalled before the morula stage. Morpholino knockdown of nhlrc2 in zebrafish embryos affected the integrity of cells in the midbrain region. This is the first description of a fatal, early-onset disease; we have named it FINCA disease based on the combination of pathological features that include fibrosis, neurodegeneration, and cerebral angiomatosis.

A novel stress-induced EDEM variant regulating endoplasmic reticulum-associated glycoprotein degradation.

Proteins expressed in the endoplasmic reticulum (ER) are subjected to a tight quality control. Persistent association with ER-resident molecular chaperones prevents exit of misfolded or incompletely assembled polypeptides from the ER and forward transport along the secretory line. ER-associated degradation (ERAD) is in place to avoid ER constipation. Folding-incompetent products have to be identified to interrupt futile folding attempts and then targeted for unfolding and dislocation into the cytosol for proteasome-mediated destruction. These processes are better understood for N-glycosylated proteins that represent the majority of polypeptides expressed in the ER. EDEM, a mannosidase-like chaperone, regulates the extraction of misfolded glycoproteins from the calnexin cycle. Here we identify and characterize EDEM2, a novel, stress-regulated mannosidase-like protein that operates in the ER lumen. We show that transcriptional up-regulation of EDEM2 depends on the ER stress-activated transcription factor Xbp1, that EDEM2 up-regulation selectively accelerates ERAD of terminally misfolded glycoproteins by facilitating their extraction from the calnexin cycle, and that the previously characterized homolog EDEM is also a soluble protein of the ER lumen in HEK293 cells.

Molecular characterization of the principal substrate binding site of the ubiquitous folding catalyst protein disulfide isomerase.

Disulfide bond formation in the endoplasmic reticulum of eukaryotes is catalyzed by the ubiquitously expressed enzyme protein disulfide isomerase (PDI). The effectiveness of PDI as a catalyst of native disulfide bond formation in folding polypeptides depends on the ability to catalyze disulfide-dithiol exchange, to bind non-native proteins, and to trigger conformational changes in the bound substrate, allowing access to buried cysteine residues. It is known that the b' domain of PDI provides the principal peptide binding site of PDI and that this domain is critical for catalysis of isomerization but not oxidation reactions in protein substrates. Here we use homology modeling to define more precisely the boundaries of the b' domain and show the existence of an intradomain linker between the b' and a' domains. We have expressed the recombinant b' domain thus defined; the stability and conformational properties of the recombinant product confirm the validity of the domain boundaries. We have modeled the tertiary structure of the b' domain and identified the primary substrate binding site within it. Mutations within this site, expressed both in the isolated domain and in full-length PDI, greatly reduce the binding affinity for small peptide substrates, with the greatest effect being I272W, a mutation that appears to have no structural effect.

Functional characterization of ERp18, a new endoplasmic reticulum-located thioredoxin superfamily member.

Native disulfide bond formation in the endoplasmic reticulum is a critical process in the maturation of many secreted and outer membrane proteins. Although a large number of proteins have been implicated in this process, it is clear that our current understanding is far from complete. Here we describe the functional characterization of a new 18-kDa protein (ERp18) related to protein-disulfide isomerase. We show that ERp18 is located in the endoplasmic reticulum and that it contains a single catalytic domain with an unusual CGAC active site motif and a probable insertion between beta3 and alpha3 of the thioredoxin fold. From circular dichroism and NMR measurements, ERp18 is well structured and undergoes only a minor conformational change upon dithioldisulfide exchange in the active site. Guanidinium chloride denaturation curves indicate that the reduced form of the protein is more stable than the oxidized form, suggesting that it is involved in disulfide bond formation. Furthermore, in vitro ERp18 possesses significant peptide thiol-disulfide oxidase activity, which is dependent on the presence of both active site cysteine residues. This activity differs from that of the human PDI family in that under standard assay conditions it is limited by substrate oxidation and not by enzyme reoxidation. A putative physiological role for Erp18 in native disulfide bond formation is discussed.