Loss of the N-terminal methyltransferase NRMT1 increases sensitivity to DNA damage and promotes mammary oncogenesis.

Though discovered over four decades ago, the function of N-terminal methylation has mostly remained a mystery. Our discovery of the first mammalian N-terminal methyltransferase, NRMT1, has led to the discovery of many new functions for N-terminal methylation, including regulation of DNA/protein interactions, accurate mitotic division, and nucleotide excision repair (NER). Here we test whether NRMT1 is also important for DNA double-strand break (DSB) repair, and given its previously known roles in cell cycle regulation and the DNA damage response, assay if NRMT1 is acting as a tumor suppressor. We find that NRMT1 knockdown significantly enhances the sensitivity of breast cancer cell lines to both etoposide treatment and γ-irradiation, as well as, increases proliferation rate, invasive potential, anchorage-independent growth, xenograft tumor size, and tamoxifen sensitivity. Interestingly, this positions NRMT1 as a tumor suppressor protein involved in multiple DNA repair pathways, and indicates, similar to BRCA1 and BRCA2, its loss may result in tumors with enhanced sensitivity to diverse DNA damaging chemotherapeutics.

NRMT1 knockout mice exhibit phenotypes associated with impaired DNA repair and premature aging.

Though defective genome maintenance and DNA repair have long been known to promote phenotypes of premature aging, the role protein methylation plays in these processes is only now emerging. We have recently identified the first N-terminal methyltransferase, NRMT1, which regulates protein-DNA interactions and is necessary for both accurate mitotic division and nucleotide excision repair. To demonstrate if complete loss of NRMT1 subsequently resulted in developmental or aging phenotypes, we constructed the first NRMT1 knockout (Nrmt1(-/-)) mouse. The majority of these mice die shortly after birth. However, the ones that survive, exhibit decreased body size, female-specific infertility, kyphosis, decreased mitochondrial function, and early-onset liver degeneration; phenotypes characteristic of other mouse models deficient in DNA repair. The livers from Nrmt1(-/-) mice produce less reactive oxygen species (ROS) than wild type controls, and Nrmt1(-/-) mouse embryonic fibroblasts show a decreased capacity for handling oxidative damage. This indicates that decreased mitochondrial function may benefit Nrmt1(-/-) mice and protect them from excess internal ROS and subsequent DNA damage. These studies position the NRMT1 knockout mouse as a useful new system for studying the effects of genomic instability and defective DNA damage repair on organismal and tissue-specific aging.

Machine oil inhibits the osseointegration of orthopaedic implants by impairing osteoblast attachment and spreading.

The most important factor contributing to short-term and long-term success of cementless total joint arthroplasties is osseointegration. Osseointegration leads to a direct structural and functional connection between living bone and the surface of an implant. Surface contaminants may remain on orthopaedic implants after sterilization procedures and impair osseointegration. For example, specific lots of hip replacement Sulzer Inter-OP(TM) acetabular shells that were associated with impaired osseointegration and early failure rates were found to be contaminated with both bacterial debris and machine oil residues. However, the effect of machine oil on implant integration is unknown. Therefore, the goal of this study was to determine if machine oil inhibits the osseointegration of orthopaedic implants. To test this hypothesis in vivo we used our murine model of osseointegration where titanium alloy implants are implanted into a unicortical pilot hole in the mid-diaphysis of the femur. We found that machine oil inhibited bone-to-implant contact and biomechanical pullout measures. Machine oil on titanium alloy discs inhibited early stages of MC3T3-E1 osteogenesis in vitro such as attachment and spreading. Inhibition of osteoblast attachment and spreading occurred in both areas with and without detectable oil. Osteoblast growth was in turn inhibited on discs with machine oil due to both a decrease in proliferation and an increase in cell death. Later stages of osteogenic differentiation and mineralization on titanium alloy discs were also inhibited. Thus, machine oil can inhibit osseointegration through cell autonomous effects on osteoblast cells. These results support routine testing by manufacturers of machine oil residues on orthopaedic implants.

NRMT2 is an N-terminal monomethylase that primes for its homologue NRMT1.

NRMT (N-terminal regulator of chromatin condensation 1 methyltransferase) was the first eukaryotic methyltransferase identified to specifically methylate the free α-amino group of proteins. Since the discovery of this N-terminal methyltransferase, many new substrates have been identified and the modification itself has been shown to regulate DNA-protein interactions. Sequence analysis predicts one close human homologue of NRMT, METTL11B (methyltransferase-like protein 11B, now renamed NRMT2). We show in the present paper for the first time that NRMT2 also has N-terminal methylation activity and recognizes the same N-terminal consensus sequences as NRMT (now NRMT1). Both enzymes have similar tissue expression and cellular localization patterns. However, enzyme assays and MS experiments indicate that they differ in their specific catalytic functions. Although NRMT1 is a distributive methyltransferase that can mono-, di- and tri-methylate its substrates, NRMT2 is primarily a monomethylase. Concurrent expression of NRMT1 and NRMT2 accelerates the production of trimethylation, and we propose that NRMT2 activates NRMT1 by priming its substrates for trimethylation.

Adherent lipopolysaccharide inhibits the osseointegration of orthopedic implants by impairing osteoblast differentiation.

Osseointegration is the process by which an orthopedic implant makes direct bone-to-implant contact and is crucial for the long-term function of the implant. Surface contaminants, such as bacterial debris and manufacturing residues, may remain on orthopedic implants after sterilization and impair osseointegration. For example, specific lots of implants that were associated with impaired osseointegration and high failure rates were discovered to have contaminants including bacterial debris. Therefore, the goals of this study were to determine if bacterial debris exists on sterile orthopedic implants and if adherent bacterial debris inhibits the osseointegration of orthopedic implants. We found that debris containing lipopolysaccharide (LPS) from Gram-negative bacteria exists on both sterile craniofacial implants and wrist implants. Levels of bacterial debris vary not only between different lots of implants but within an individual lot. Using our murine model of osseointegration, we found that ultrapure LPS adherent to the implants inhibited bone-to-implant contact and biomechanical pullout measures. Analysis of osseointegration in knock-out mice demonstrated that adherent LPS inhibited osseointegration by signaling through its primary receptor, Toll-like receptor 4, and not by signaling through Toll-like receptor 2. Ultrapure LPS adherent to titanium alloy discs had no detectable effect on early stages of MC3T3-E1 osteogenesis in vitro such as attachment, spreading or growth. However, later stages of osteogenic differentiation and mineralization were inhibited by adherent LPS. Thus, LPS may inhibit osseointegration in part through cell autonomous effects on osteoblasts. These results highlight bacterial debris as a type of surface contaminant that can impair the osseointegration of orthopedic implants.

Surface contaminants inhibit osseointegration in a novel murine model.

Surface contaminants, such as bacterial debris and manufacturing residues, may remain on orthopedic implants after sterilization procedures and affect osseointegration. The goals of this study were to develop a murine model of osseointegration in order to determine whether removing surface contaminants enhances osseointegration. To develop the murine model, titanium alloy implants were implanted into a unicortical pilot hole in the mid-diaphysis of the femur and osseointegration was measured over a five week time course. Histology, backscatter scanning electron microscopy and X-ray energy dispersive spectroscopy showed areas of bone in intimate physical contact with the implant, confirming osseointegration. Histomorphometric quantification of bone-to-implant contact and peri-implant bone and biomechanical pullout quantification of ultimate force, stiffness and work to failure increased significantly over time, also demonstrating successful osseointegration. We also found that a rigorous cleaning procedure significantly enhances bone-to-implant contact and biomechanical pullout measures by two-fold compared with implants that were autoclaved, as recommended by the manufacturer. The most likely interpretation of these results is that surface contaminants inhibit osseointegration. The results of this study justify the need for the development of better detection and removal techniques for contaminants on orthopedic implants and other medical devices.