Catching mono- and poly-ADP-ribose readers with synthetic ADP-ribose baits.

ADP-ribosylation is an essential post-translational modification that comes in two varieties: mono-ADP-ribosylation (MAR) and poly-ADP-ribosylation (PAR). Modular interaction domains that read MAR and PAR modifications are critical for interpreting the language of ADP-ribosylation. Kliza et al. (2021) use a chemical biology approach to identify distinct MAR and PAR readers.

Interplay between compartmentalized NAD+ synthesis and consumption: a focus on the PARP family.

Nicotinamide adenine dinucleotide (NAD+) is an essential cofactor for redox enzymes, but also moonlights as a substrate for signaling enzymes. When used as a substrate by signaling enzymes, it is consumed, necessitating the recycling of NAD+ consumption products (i.e., nicotinamide) via a salvage pathway in order to maintain NAD+ homeostasis. A major family of NAD+ consumers in mammalian cells are poly-ADP-ribose-polymerases (PARPs). PARPs comprise a family of 17 enzymes in humans, 16 of which catalyze the transfer of ADP-ribose from NAD+ to macromolecular targets (namely, proteins, but also DNA and RNA). Because PARPs and the NAD+ biosynthetic enzymes are subcellularly localized, an emerging concept is that the activity of PARPs and other NAD+ consumers are regulated in a compartmentalized manner. In this review, I discuss NAD+ metabolism, how different subcellular pools of NAD+ are established and regulated, and how free NAD+ levels can control signaling by PARPs and redox metabolism.

Chemical genetic methodologies for identifying protein substrates of PARPs.

Poly-ADP-ribose-polymerases (PARPs) are a family of 17 enzymes that regulate a diverse range of cellular processes in mammalian cells. PARPs catalyze the transfer of ADP-ribose from NAD+ to target molecules, most prominently amino acids on protein substrates, in a process known as ADP-ribosylation. Identifying the direct protein substrates of individual PARP family members is an essential first step for elucidating the mechanism by which PARPs regulate a particular pathway in cells. Two distinct chemical genetic (CG) strategies have been developed for identifying the direct protein substrates of individual PARP family members. In this review, we discuss the design principles behind these two strategies and how target identification has provided novel insight into the cellular function of individual PARPs and PARP-mediated ADP-ribosylation.

Proximity-dependent mapping of the HCMV US28 interactome identifies RhoGEF signaling as a requirement for efficient viral reactivation.

Human cytomegalovirus (HCMV) encodes multiple putative G protein-coupled receptors (GPCRs). US28 functions as a viral chemokine receptor and is expressed during both latent and lytic phases of virus infection. US28 actively promotes cellular migration, transformation, and plays a major role in mediating viral latency and reactivation; however, knowledge about the interaction partners involved in these processes is still incomplete. Herein, we utilized a proximity-dependent biotinylating enzyme (TurboID) to characterize the US28 interactome when expressed in isolation, and during both latent (CD34+ hematopoietic progenitor cells) and lytic (fibroblasts) HCMV infection. Our analyses indicate that the US28 signalosome converges with RhoA and EGFR signal transduction pathways, sharing multiple mediators that are major actors in processes such as cellular proliferation and differentiation. Integral members of the US28 signaling complex were validated in functional assays by immunoblot and small-molecule inhibitors. Importantly, we identified RhoGEFs as key US28 signaling intermediaries. In vitro latency and reactivation assays utilizing primary CD34+ hematopoietic progenitor cells (HPCs) treated with the small-molecule inhibitors Rhosin or Y16 indicated that US28 -RhoGEF interactions are required for efficient viral reactivation. These findings were recapitulated in vivo using a humanized mouse model where inhibition of RhoGEFs resulted in a failure of the virus to reactivate. Together, our data identifies multiple new proteins in the US28 interactome that play major roles in viral latency and reactivation, highlights the utility of proximity-sensor labeling to characterize protein interactomes, and provides insight into targets for the development of novel anti-HCMV therapeutics.

Aging is associated with maladaptive episodic memory-guided social decision-making.

Older adults are frequent targets and victims of financial fraud. They may be especially susceptible to revictimization because of age-related changes in both episodic memory and social motivation. Here we examined these factors in a context where adaptive social decision-making requires intact associative memory for previous social interactions. Older adults made more maladaptive episodic memory-guided social decisions but not only because of poorer associative memory. Older adults were biased toward remembering people as being fair, while young adults were biased toward remembering people as being unfair. Holding memory constant, older adults engaged more with people that were familiar (regardless of the nature of the previous interaction), whereas young adults were prone to avoiding others that they remembered as being unfair. Finally, older adults were more influenced by facial appearances, choosing to interact with social partners that looked more generous, even though those perceptions were inconsistent with prior experience.

Emergence of the natural history of Myhre syndrome: 47 patients evaluated in the Massachusetts General Hospital Myhre Syndrome Clinic (2016-2023).

Myhre syndrome is an increasingly diagnosed ultrarare condition caused by recurrent germline autosomal dominant de novo variants in SMAD4. Detailed multispecialty evaluations performed at the Massachusetts General Hospital (MGH) Myhre Syndrome Clinic (2016-2023) and by collaborating specialists have facilitated deep phenotyping, genotyping and natural history analysis. Of 47 patients (four previously reported), most (81%) patients returned to MGH at least once. For patients followed for at least 5 years, symptom progression was observed in all. 55% were female and 9% were older than 18 years at diagnosis. Pathogenic variants in SMAD4 involved protein residues p.Ile500Val (49%), p.Ile500Thr (11%), p.Ile500Leu (2%), and p.Arg496Cys (38%). Individuals with the SMAD4 variant p.Arg496Cys were less likely to have hearing loss, growth restriction, and aortic hypoplasia than the other variant groups. Those with the p.Ile500Thr variant had moderate/severe aortic hypoplasia in three patients (60%), however, the small number (n = 5) prevented statistical comparison with the other variants. Two deaths reported in this cohort involved complex cardiovascular disease and airway stenosis, respectively. We provide a foundation for ongoing natural history studies and emphasize the need for evidence-based guidelines in anticipation of disease-specific therapies.

Reward enhancement of item-location associative memory spreads to similar items within a category.

The experience of a reward appears to enhance memory for recent prior events, adaptively making that information more available to guide future decision-making. Here, we tested whether reward enhances memory for associative item-location information and also whether the effect of reward spreads to other categorically-related but unrewarded items. Participants earned either points (Experiment 1) or money (Experiment 2) through a time-estimation reward task, during which stimuli-location pairings around a 2D-ring were shown followed by either high-value or low-value rewards. All stimuli were then tested for location memory or recognition (yes/no), immediately and after a 24-hour delay. Across both experiments (combined analysis), there was a robust improvement in location memory following high-value rewards, even though evidence supporting this effect was reliable in Experiment 2 but not in Experiment 1. The memory-enhancing effect of reward was observed on both the immediate and delayed location-memory tests. Reward-enhanced memory for both directly rewarded stimuli and categorically related stimuli that were not directly rewarded. No reliable effect of reward value on yes/no recognition-memory performance was observed in either experiment. We hypothesise that reward enhances the consolidation of recent experience and conceptually related memories to make these more available for future decisions.

Retrospective assessment of the impact of an education specialist on the care of children with hearing loss.

OBJECTIVE: To assess the impact of an education specialist in a multidisciplinary pediatric hearing loss clinic. STUDY DESIGN: Retrospective review and cross-sectional survey. SETTING: Single tertiary care center. METHODS: Consultations held between an education specialist and families of pediatric deaf or hard of hearing (DHH) children within a two-year period were reviewed. Reasons for referral and services provided to each patient and family who subsequently worked with the educational specialist were assessed. Parents of patients who had previously worked with the education specialist were invited to complete a survey evaluating their experience. RESULTS: 102 patients were referred to the educational specialist in a two-year period. Most common reasons for referral included need for special education plans to accommodate their hearing deficit (32) or family request to support for revisions to such plans (37). 14 patient families completed our survey. 76.9 % of respondents confirmed that the education specialist recommended resources they had not been introduced to before. Given a scale of 1 ("completely dissatisfied") and 10 being "completely satisfied," the average rating of the 14 respondents was 9.0. CONCLUSION: The role of an education specialist in a pediatric hearing loss clinic is to optimize patient and family access to resources that could benefit their DHH child's academic development over time. Future studies should prospectively investigate the impact of education specialist services on the educational progress of DHH patients compared to outcomes without these supports.

Mechanisms governing PARP expression, localization, and activity in cells.

Poly-(ADP)-ribose polymerases (PARPs) are a family of 17 enzymes in humans that have diverse roles in cell physiology including DNA damage repair, transcription, innate immunity, and regulation of signaling pathways. The modular domain architecture of PARPs gives rise to this functional diversity. PARPs catalyze the transfer of ADP-ribose from nicotinamide adenine dinucleotide (NAD+) to targets-proteins and poly-nucleic acids. This enigmatic post-translational modification comes in two varieties: the transfer of a single unit of ADP-ribose, known as mono-ADP-ribosylation (MARylation) or the transfer of multiple units of ADP-ribose, known as poly-ADP-ribosylation (PARylation). Emerging data shows that PARPs are regulated at multiple levels to control when and where PARP-mediated M/PARylation occurs in cells. In this review, we will discuss the latest knowledge regarding the regulation of PARPs in cells: from transcription and protein stability to subcellular localization and modulation of catalytic activity.

Coronavirus infection and PARP expression dysregulate the NAD metabolome: An actionable component of innate immunity.

Poly(ADP-ribose) polymerase (PARP) superfamily members covalently link either a single ADP-ribose (ADPR) or a chain of ADPR units to proteins using NAD as the source of ADPR. Although the well-known poly(ADP-ribosylating) (PARylating) PARPs primarily function in the DNA damage response, many noncanonical mono(ADP-ribosylating) (MARylating) PARPs are associated with cellular antiviral responses. We recently demonstrated robust up-regulation of several PARPs following infection with murine hepatitis virus (MHV), a model coronavirus. Here we show that SARS-CoV-2 infection strikingly up-regulates MARylating PARPs and induces the expression of genes encoding enzymes for salvage NAD synthesis from nicotinamide (NAM) and nicotinamide riboside (NR), while down-regulating other NAD biosynthetic pathways. We show that overexpression of PARP10 is sufficient to depress cellular NAD and that the activities of the transcriptionally induced enzymes PARP7, PARP10, PARP12 and PARP14 are limited by cellular NAD and can be enhanced by pharmacological activation of NAD synthesis. We further demonstrate that infection with MHV induces a severe attack on host cell NAD+ and NADP+ Finally, we show that NAMPT activation, NAM, and NR dramatically decrease the replication of an MHV that is sensitive to PARP activity. These data suggest that the antiviral activities of noncanonical PARP isozyme activities are limited by the availability of NAD and that nutritional and pharmacological interventions to enhance NAD levels may boost innate immunity to coronaviruses.

Chemical Proteomics Approach for Profiling the NAD Interactome.

Nicotinamide adenine dinucleotide (NAD+) is a multifunctional molecule. Beyond redox metabolism, NAD+ has an equally important function as a substrate for post-translational modification enzymes, the largest family being the poly-ADP-ribose polymerases (PARPs, 17 family members in humans). The recent surprising discoveries of noncanonical NAD (NAD+/NADH)-binding proteins suggests that the NAD interactome is likely larger than previously thought; yet, broadly useful chemical tools for profiling and discovering NAD-binding proteins do not exist. Here, we describe the design, synthesis, and validation of clickable, photoaffinity labeling (PAL) probes, 2- and 6-ad-BAD, for interrogating the NAD interactome. We found that 2-ad-BAD efficiently labels PARPs in a UV-dependent manner. Chemical proteomics experiments with 2- and 6-ad-BAD identified known and unknown NAD+/NADH-binding proteins. Together, our study shows the utility of 2- and 6-ad-BAD as clickable PAL NAD probes.

Reversible ADP-ribosylation of RNA.

ADP-ribosylation is a reversible chemical modification catalysed by ADP-ribosyltransferases such as PARPs that utilize nicotinamide adenine dinucleotide (NAD+) as a cofactor to transfer monomer or polymers of ADP-ribose nucleotide onto macromolecular targets such as proteins and DNA. ADP-ribosylation plays an important role in several biological processes such as DNA repair, transcription, chromatin remodelling, host-virus interactions, cellular stress response and many more. Using biochemical methods we identify RNA as a novel target of reversible mono-ADP-ribosylation. We demonstrate that the human PARPs - PARP10, PARP11 and PARP15 as well as a highly diverged PARP homologue TRPT1, ADP-ribosylate phosphorylated ends of RNA. We further reveal that ADP-ribosylation of RNA mediated by PARP10 and TRPT1 can be efficiently reversed by several cellular ADP-ribosylhydrolases (PARG, TARG1, MACROD1, MACROD2 and ARH3), as well as by MACROD-like hydrolases from VEEV and SARS viruses. Finally, we show that TRPT1 and MACROD homologues in bacteria possess activities equivalent to the human proteins. Our data suggest that RNA ADP-ribosylation may represent a widespread and physiologically relevant form of reversible ADP-ribosylation signalling.

ADP-ribosyl-binding and hydrolase activities of the alphavirus nsP3 macrodomain are critical for initiation of virus replication.

Alphaviruses are plus-strand RNA viruses that cause encephalitis, rash, and arthritis. The nonstructural protein (nsP) precursor polyprotein is translated from genomic RNA and processed into four nsPs. nsP3 has a highly conserved macrodomain (MD) that binds ADP-ribose (ADPr), which can be conjugated to protein as a posttranslational modification involving transfer of ADPr from NAD+ by poly ADPr polymerases (PARPs). The nsP3MD also removes ADPr from mono ADP-ribosylated (MARylated) substrates. To determine which aspects of alphavirus replication require nsP3MD ADPr-binding and/or hydrolysis function, we studied NSC34 neuronal cells infected with chikungunya virus (CHIKV). Infection induced ADP-ribosylation of cellular proteins without increasing PARP expression, and inhibition of MARylation decreased virus replication. CHIKV with a G32S mutation that reduced ADPr-binding and hydrolase activities was less efficient than WT CHIKV in establishing infection and in producing nsPs, dsRNA, viral RNA, and infectious virus. CHIKV with a Y114A mutation that increased ADPr binding but reduced hydrolase activity, established infection like WT CHIKV, rapidly induced nsP translation, and shut off host protein synthesis with reduced amplification of dsRNA. To assess replicase function independent of virus infection, a transreplicase system was used. Mutant nsP3MDs D10A, G32E, and G112E with no binding or hydrolase activity had no replicase activity, G32S had little, and Y114A was intermediate to WT. Therefore, ADP ribosylation of proteins and nsP3MD ADPr binding are necessary for initiation of alphavirus replication, while hydrolase activity facilitates amplification of replication complexes. These observations are consistent with observed nsP3MD conservation and limited tolerance for mutation.

Pharmacological bypass of NAD+ salvage pathway protects neurons from chemotherapy-induced degeneration.

Axon degeneration, a hallmark of chemotherapy-induced peripheral neuropathy (CIPN), is thought to be caused by a loss of the essential metabolite nicotinamide adenine dinucleotide (NAD+) via the prodegenerative protein SARM1. Some studies challenge this notion, however, and suggest that an aberrant increase in a direct precursor of NAD+, nicotinamide mononucleotide (NMN), rather than loss of NAD+, is responsible. In support of this idea, blocking NMN accumulation in neurons by expressing a bacterial NMN deamidase protected axons from degeneration. We hypothesized that protection could similarly be achieved by reducing NMN production pharmacologically. To achieve this, we took advantage of an alternative pathway for NAD+ generation that goes through the intermediate nicotinic acid mononucleotide (NAMN), rather than NMN. We discovered that nicotinic acid riboside (NAR), a precursor of NAMN, administered in combination with FK866, an inhibitor of the enzyme nicotinamide phosphoribosyltransferase that produces NMN, protected dorsal root ganglion (DRG) axons against vincristine-induced degeneration as well as NMN deamidase. Introducing a different bacterial enzyme that converts NAMN to NMN reversed this protection. Collectively, our data indicate that maintaining NAD+ is not sufficient to protect DRG neurons from vincristine-induced axon degeneration, and elevating NMN, by itself, is not sufficient to cause degeneration. Nonetheless, the combination of FK866 and NAR, which bypasses NMN formation, may provide a therapeutic strategy for neuroprotection.

Small-Molecule Inhibitors of PARPs: From Tools for Investigating ADP-Ribosylation to Therapeutics.

Over the last 60 years, poly-ADP-ribose polymerases (PARPs, 17 family members in humans) have emerged as important regulators of physiology and disease. Small-molecule inhibitors have been essential tools for unraveling PARP function, and recently the first PARP inhibitors have been approved for the treatment of various human cancers. However, inhibitors have only been developed for a few PARPs and in vitro profiling has revealed that many of these exhibit polypharmacology across the PARP family. In this review, we discuss the history, development, and current state of the field, highlighting the limitations and opportunities for PARP inhibitor development.

Insights into the biogenesis, function, and regulation of ADP-ribosylation.

ADP-ribosylation-the transfer of ADP-ribose (ADPr) from NAD+ onto target molecules-is catalyzed by members of the ADP-ribosyltransferase (ART) superfamily of proteins, found in all kingdoms of life. Modification of amino acids in protein targets by ADPr regulates critical cellular pathways in eukaryotes and underlies the pathogenicity of certain bacteria. Several members of the ART superfamily are highly relevant for disease; these include the poly(ADP-ribose) polymerases (PARPs), recently shown to be important cancer targets, and the bacterial toxins diphtheria toxin and cholera toxin, long known to be responsible for the symptoms of diphtheria and cholera that result in morbidity. In this Review, we discuss the functions of amino acid ADPr modifications and the ART proteins that make them, the nature of the chemical linkage between ADPr and its targets and how this impacts function and stability, and the way that ARTs select specific amino acids in targets to modify.

Impact of cochlear abnormalities on hearing outcomes for children with cochlear implants.

OBJECTIVE: Evaluate the impact of cochlear anomalies on hearing outcomes for pediatric patients with cochlear implants. STUDY DESIGN: Retrospective chart review. SETTING: Tertiary care center. SUBJECTS AND METHODS: Charts were retrospectively reviewed for cases where pediatric cochlear implant surgery was performed between 2002 and 2018 at a single, tertiary care institution. Patients were divided into groups based on the presence or absence of radiological cochlear abnormalities, which were further classified as low or high risk anomalies. Hearing outcomes were evaluated by measuring pure tone averages and word recognition scores preoperatively, 3 and 12 months postoperatively, in addition to the most recent test results. RESULTS: There were 154 ears implanted in our cohort of 100 patients. 107 ears had normal cochlear anatomy, 31 had low risk, and 16 had high risk abnormalities. The most common modality of preoperative imaging was CT scan. Postoperative mean pure tone average (PTA) was significantly higher in patients with inner ear anomalies compared to those with normal anatomy. No significant difference in PTA was noted between low versus high risk patients. <50% of patients had word recognition scores available within the first year following surgery. CONCLUSION: Abnormalities of the inner ear significantly influenced hearing outcomes over time following cochlear implant surgery when compared to pediatric patients with normal anatomy. Obtaining hearing testing can be difficult in very young children and therefore future studies are warranted to further investigate the impact that cochlear abnormalities may have on hearing outcomes following cochlear implant surgery.

Separate Memory-Enhancing Effects of Reward and Strategic Encoding.

Memory encoding for important information can be enhanced both by reward anticipation and by intentional strategies. These effects are hypothesized to depend on distinct neural mechanisms, yet prior work has provided only limited evidence for their separability. We aimed to determine whether reward-driven and strategic mechanisms for prioritizing important information are separable, even if they may also interact. We examined the joint operation of both mechanisms using fMRI measures of brain activity. Participants learned abstract visual images in a value-directed recognition paradigm. On each trial, two novel images were presented simultaneously in different screen quadrants, one arbitrarily designated as high point value and one as low value. Immediately after each block of 16 study trials, the corresponding point rewards could be obtained in a test of item recognition and spatial location memory. During encoding trials leading to successful subsequent memory, especially of high-value images, increased activity was observed in dorsal frontoparietal and lateral occipitotemporal cortex. Furthermore, activity in a network associated with reward was higher during encoding when any image, of high or low value, was subsequently remembered. Functional connectivity between right medial temporal lobe and right ventral tegmental area, measured via psychophysiological interaction, was also greater during successful encoding regardless of value. Strategic control of memory, as indexed by successful prioritization of the high-value image, affected activity in dorsal posterior parietal cortex as well as connectivity between this area and right lateral temporal cortex. These results demonstrate that memory can be strengthened by separate neurocognitive mechanisms for strategic control versus reward-based enhancement of processing.

Assessing the Relative Contribution of Resident Versus Facility Characteristics Associated With Antipsychotic Medication Receipt Among Nursing Facility Residents.

OBJECTIVE: In 2012 Centers for Medicare and Medicaid Services (CMS) launched a multifaceted initiative aimed at reducing the unnecessary use of antipsychotic medications in nursing facilities due to evidence these medications are associated with little or uncertain benefit and substantial risk. Yet, little is known about whether efforts to reduce antipsychotic medication should be focused on residents with targeted characteristics, or on nursing facility regulation (eg, staffing levels). Our objective was to identify the relative contribution of resident and facility characteristics to potentially inappropriate antipsychotic use. METHODS: We examined 1,156,875 long stay residents in 14,699 US nursing facilities in 2014 and predicted resident antipsychotic use controlling sequentially for resident and facility characteristics and calculated the incremental variation explained. RESULTS: We found significant variability in unadjusted rates of potentially inappropriate antipsychotic use among nursing facilities (mean=18.0%; interquartile range: 11.3%-23.7%; SD: 11.1). Regression results indicated that 93% of the explained variation in antipsychotic use was attributed to resident characteristics and 7% was attributed to facility-level factors. At the facility level, worker hours per resident day was not significantly associated with antipsychotic use. Simulations indicated that applying the effect sizes achieved by the best performing facilities to the existing case mix across all nursing facilities would result in no more than a 1 percentage point change in population-level antipsychotic use. CONCLUSIONS: Efforts to reduce antipsychotic use may have greater impact by developing new clinical strategies to address specific diagnoses rather than regulations related to facility-level attributes.