PARP10 promotes cellular proliferation and tumorigenesis by alleviating replication stress.

During carcinogenesis, cells are exposed to increased replication stress due to replication fork arrest at sites of DNA lesions and difficult to replicate genomic regions. Efficient fork restart and DNA repair are important for cancer cell proliferation. We previously showed that the ADP-ribosyltransferase PARP10 interacts with the replication protein proliferating cell nuclear antigen and promotes lesion bypass by recruiting specialized, non-replicative DNA polymerases. Here, we show that PARP10 is overexpressed in a large proportion of human tumors. To understand the role of PARP10 in cellular transformation, we inactivated PARP10 in HeLa cancer cells by CRISPR/Cas9-mediated gene knockout, and overexpressed it in non-transformed RPE-1 cells. We found that PARP10 promotes cellular proliferation, and its overexpression alleviates cellular sensitivity to replication stress and fosters the restart of stalled replication forks. Importantly, mouse xenograft studies showed that loss of PARP10 reduces the tumorigenesis activity of HeLa cells, while its overexpression results in tumor formation by non-transformed RPE-1 cells. Our findings indicate that PARP10 promotes cellular transformation, potentially by alleviating replication stress and suggest that targeting PARP10 may represent a novel therapeutic approach.

Cell-associated heparin-like molecules modulate the ability of LDL to regulate PCSK9 uptake.

Proprotein convertase subtilisin/kexin type 9 (PCSK9) targets the LDL receptor (LDLR) for degradation, increasing plasma LDL and, consequently, cardiovascular risk. Uptake of secreted PCSK9 is required for its effect on the LDLR, and LDL itself inhibits this uptake, though how it does so remains unclear. In this study, we investigated the relationship between LDL, the PCSK9:LDLR interaction, and PCSK9 uptake. We show that LDL inhibits binding of PCSK9 to the LDLR in vitro more impressively than it inhibits PCSK9 uptake in cells. Furthermore, cell-surface heparin-like molecules (HLMs) can partly explain this difference, consistent with heparan sulfate proteoglycans (HSPGs) acting as coreceptors for PCSK9. We also show that HLMs can interact with either PCSK9 or LDL to modulate the inhibitory activity of LDL on PCSK9 uptake, with such inhibition rescued by competition with the entire PCSK9 prodomain, but not its truncated variants. Additionally, we show that the gain-of-function PCSK9 variant, S127R, located in the prodomain near the HSPG binding site, exhibits increased affinity for HLMs, potentially explaining its phenotype. Overall, our findings suggest a model where LDL acts as a negative regulator of PCSK9 function by decreasing its uptake via direct interactions with either the LDLR or HLMs.

Stepwise processing analyses of the single-turnover PCSK9 protease reveal its substrate sequence specificity and link clinical genotype to lipid phenotype.

Proprotein convertase subtilisin/kexin type 9 (PCSK9) down-regulates the low-density lipoprotein (LDL) receptor, elevating LDL cholesterol and accelerating atherosclerotic heart disease, making it a promising cardiovascular drug target. To achieve its maximal effect on the LDL receptor, PCSK9 requires autoproteolysis. After cleavage, PCSK9 retains its prodomain in the active site as a self-inhibitor. Unlike other proprotein convertases, however, this retention is permanent, inhibiting any further protease activity for the remainder of its life cycle. Such inhibition has proven a major challenge toward a complete biochemical characterization of PCSK9's proteolytic function, which could inform therapeutic approaches against its hypercholesterolemic effects. To address this challenge, we employed a cell-based, high-throughput method using a luciferase readout to evaluate the single-turnover PCSK9 proteolytic event. We combined this method with saturation mutagenesis libraries to interrogate the sequence specificities of PCSK9 cleavage and proteolysis-independent secretion. Our results highlight several key differences in sequence identity between these two steps, complement known structural data, and suggest that PCSK9 self-proteolysis is the rate-limiting step of secretion. Additionally, we found that for missense SNPs within PCSK9, alterations in both proteolysis and secretion are common. Last, we show that some SNPs allosterically modulate PCSK9's substrate sequence specificity. Our findings indicate that PCSK9 proteolysis acts as a commonly perturbed but critical switch in controlling lipid homeostasis and provide a new hope for the development of small-molecule PCSK9 inhibitors.

Repurposing Waste Streams: Lessons on Integrating Hospital Food Waste into a Community Garden.

There have been increasing efforts in recent decades to divert institutional food waste into composting programs. As major producers of food waste who must increasingly demonstrate community benefit, hospitals have an incentive to develop such programs. In this article, we explain the emerging opportunity to link hospitals' food services to local community gardens in order to implement robust composting programs. We describe a partnership model at our hospital in central Pennsylvania, share preliminary outcomes establishing feasibility, and offer guidance for future efforts. We also demonstrate that the integration of medical students in such efforts can foster systems thinking in the development of programs to manage hospital waste streams in more ecologically-friendly ways.

PARP10 deficiency manifests by severe developmental delay and DNA repair defect.

DNA repair mechanisms such as nucleotide excision repair (NER) and translesion synthesis (TLS) are dependent on proliferating cell nuclear antigen (PCNA), a DNA polymerase accessory protein. Recently, homozygosity for p.Ser228Ile mutation in the PCNA gene was reported in patients with neurodegeneration and impaired NER. Using exome sequencing, we identified a homozygous deleterious mutation, c.648delAG, in the PARP10 gene, in a patient suffering from severe developmental delay. In agreement, PARP10 protein was absent from the patient cells. We have previously shown that PARP10 is recruited by PCNA to DNA damage sites and is required for DNA damage resistance. The patient cells were significantly more sensitive to hydroxyurea and UV-induced DNA damage than control cells, resulting in increased apoptosis, indicating DNA repair impairment in the patient cells. PARP10 deficiency joins the long list of DNA repair defects associated with neurodegenerative disorders, including ataxia telangiectasia, xeroderma pigmentosum, Cockayne syndrome, and the recently reported PCNA mutation.

A High-Throughput Luciferase Assay to Evaluate Proteolysis of the Single-Turnover Protease PCSK9.

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a single-turnover protease which regulates serum low-density lipoprotein (LDL) levels and, consequently, cardiovascular disease. Although PCSK9 proteolysis is required for its full hypercholesterolemic effect, the evaluation of its proteolytic function is challenging: PCSK9 is only known to cleave itself, undergoes only a single turnover, and after proteolysis, retains its substrate in its active site as an auto-inhibitor. The methods presented here describe an assay which overcomes these challenges. The assay focuses on intermolecular proteolysis in a cell-based context and links successful cleavage to the secreted luciferase activity, which can be easily read out in the conditioned medium. Via sequential steps of mutagenesis, transient transfection, and a luciferase readout, the assay can probe PCSK9 proteolysis under conditions of either genetic or molecular perturbation in a high-throughput manner. This system is well suited for both the biochemical evaluation of clinically discovered missense single-nucleotide polymorphisms (SNPs), as well as for the screening of small-molecule inhibitors of PCSK9 proteolysis.