Experimental Investigation on the Impact of Graphite Electrodes Grain Size on Technological Parameters and Surface Texture of Hastelloy C-22 after Electrical Discharge Machining with Negative Polarity.

Electrical discharge machining (EDM) is a rapidly evolving method in modern industry that manufactures highly complex components. The physical properties of a tool electrode material are significant factors in determining the effectiveness of the process, as well as the characteristics of the machined surfaces. The current trend of implementing graphite tool electrodes in manufacturing processes is observed. Innovative material engineering solutions enable graphite production with miniaturized grain size. However, the correlation between the graphite electrode grain size and the mechanism of the process removal in the EDM is a challenge for its widespread implementation in the industry. This research introduces a new method to evaluate the impact of the graphite electrode grain size and machining parameters on the material removal effectiveness, relative tool wear rate, and surface roughness (Ra) of Hastelloy C-22 following EDM with negative polarity. The study utilized new graphite materials with a grain size of 1 µm (POCO AF-5) and 10 µm (POCO EDM-180). An assessment of the impact of the EDM process parameters on the technological parameters and the development of the surface roughness was carried out. Electrical discharge machining with fine-grained graphite electrodes increases process efficiency and reduces tool wear. Graphite grains detached from the tool electrode affect the stability of electrical discharges and the efficiency of the process. Based on the experimental results, mathematical models were developed, enabling the prediction of machining effects to advance state-of-the-art manufacturing processes. The obtained mathematical models can be implemented in modern industrial EDM machines as guidelines for selecting adequate machining parameters depending on the desired process efficiency, tool wear rate, and surface roughness for advanced materials.

Finishing Additively Manufactured Ti6Al4V Alloy with Low-Energy Electrical Discharges.

Additive manufacturing has garnered significant interest in various industries due to its flexibility and capability to produce parts with complex shapes. However, issues related to surface quality, such as roughness and microstructural defects, necessitate the use of post-processing techniques to achieve the desired properties. Ti6Al4V alloy, produced additively, was finished using low-energy discharges, and the new surface integrity properties resulting from the induced heat energy were investigated. To further understand the influence of discharge energy on the formation of the new layer, roughness parameters and power spectral density were used to characterize the surface topography. SEM and EDS analyses were performed to examine the morphology and microstructural defects such as microcracks. The results indicate that the heat energy induced by the discharge improved the properties of the surface. SEM analysis revealed that the new layer was characterized by a reduction in defects such as unmelted particles, the balling effect, and microcracks. At the lowest investigated discharge energy of E = 0.21 mJ, surface roughness, Sa, was reduced by about 69%, which is equal to about 2 µm, accompanied by a significant decrease in microcracks. EDS analysis indicated that the diffusion of copper and zinc from the electrode to the top surface was related to the discharge energy. Furthermore, prediction models of the influence of wire electrical discharge polishing parameters, including discharge energy, wire speed, and time interval, on the surface roughness and material removal rate (MRR) were developed using the response surface methodology.

Effects of Wire Electrical Discharge Finishing Cuts on the Surface Integrity of Additively Manufactured Ti6Al4V Alloy.

The Selective laser melting (SLM) technology of recent years allows for building complex-shaped parts with difficult-to-cut materials such as Ti6Al4V alloy. Nevertheless, the surface integrity after SLM is characterized by surface roughness and defects in the microstructure. The use of additional finishing technology, such as machining, laser polishing, or mechanical polishing, is used to achieve desired surface properties. In this study, improving SLM Ti6Al4V alloy surface integrity using wire electrical discharge machining (WEDM) is proposed. The influence of finishing WEDM cuts and the discharge energy on the surface roughness parameters Sa, Svk, Spk, and Sk and the composition of the recast layer were investigated. The proposed finishing technology allows for significant improvement of the surface roughness by up to 88% (from Sa = 6.74 µm to Sa = 0.8 µm). Furthermore, the SEM analyses of surface morphology indicate improving surface integrity properties by removing the balling effect, unmelted particles, and the presence of microcracks. EDS analysis of the recast layer indicated a significant influence of discharge energy and the polarization of the electrode on its composition and thickness. Depending on the used discharge energy and the number of finishing cuts, changes in the composition of the material in the range of 2 to 10 µm were observed.

Effects of a New Type of Grinding Wheel with Multi-Granular Abrasive Grains on Surface Topography Properties after Grinding of Inconel 625.

Finishing operations are one of the most challenging tasks during a manufacturing process, and are responsible for achieving dimensional accuracy of the manufactured parts and the desired surface topography properties. One of the most advanced finishing technologies is grinding. However, typical grinding processes have limitations in the acquired surface topography properties, especially in finishing difficult to cut materials such as Inconel 625. To overcome this limitation, a new type of grinding wheel is proposed. The tool is made up of grains of different sizes, which results in less damage to the work surface and an enhancement in the manufacturing process. In this article, the results of an experimental study of the surface grinding process of Inconel 625 with single-granular and multi-granular wheels are presented. The influence of various input parameters on the roughness parameter (Sa) and surface topography was investigated. Statistical models of the grinding process were developed based on our research. Studies showed that with an increase in the cutting speed, the surface roughness values of the machined samples decreased (Sa = 0.9 µm for a Vc of 33 m/s for a multigranular wheel). Observation of the grinding process showed an unfavorable effect of a low grinding wheel speed on the machined surface. For both conventional and multigranular wheels, the highest value for the Sa parameter was obtained for Vc = 13 m/s. Regarding the surface topography, the observed surfaces did not show defects over large areas in the cases of both wheels. However, a smaller portion of single traces of active abrasive grains was observed in the case of the multi-granular wheel, indicating that this tool performs better finishing operations.

Evaluation of Prediction Models of the Microwire EDM Process of Inconel 718 Using ANN and RSM Methods.

Precise machining of micro parts from difficult-to-cut materials requires using advanced technology such as wire electrical discharge machining (WEDM). In order to enhance the productivity of micro WEDM, the key role is understanding the influence of process parameters on the surface topography and the material's removal rate (MRR). Furthermore, effective models which allow us to predict the influence of the parameters of micro-WEDM on the qualitative effects of the process are required. This paper influences the discharge energy, time interval, and wire speed on the surface topography's properties, namely Sa, Sk, Spk, Svk, and MRR, after micro-WEDM of Inconel 718 were described. Developed RSM and ANN model of the micro-WEDM process, showing that the discharge energy had the main influence (over 70%) on the surface topography's parameters. However, for MRR, the time interval was also significant. Furthermore, a reduction in wire speed can lead to a decrease in the cost process and have a positive influence on the environment and sustainability of the process. Evaluation of developed prediction models of micro-WEDM of Inconel 718 indicates that ANN had a lower value for the relative error compared with the RSM models and did not exceed 4%.

Experimental Investigation of Technological Indicators and Surface Roughness of Hastelloy C-22 after Electrical Discharge Machining Using POCO Graphite Electrodes.

Modern industry is focused on looking for new and effective technologies to manufacture complex shapes from alloys based on nickel and chromium. One of the materials widely used in the chemical and aerospace industry is Hastelloy C-22. This material is difficult to machine by conventional methods, and in many cases, unconventional methods are used to manufacture it, such as electrical discharge machining (EDM). In the EDM process, the material is removed by electrical discharges between a workpiece and a tool electrode. The physical and mechanical properties of the tool electrodes have a direct impact on the process efficiency, machining accuracy, and surface roughness. Currently, there has been a significant increase in the use of graphite as a material for tool electrodes due to the low purchase cost of the raw material, good machinability, and high sublimation temperature. In this work, an experimental investigation of the influence of the grain size of the graphite tool electrode on material removal rate (MRR), tool wear rate (TWR), and surface roughness (Ra) of Hastelloy C-22 was carried out. Two POCO graphite tool electrodes with a grain size of 1 µm (AF-5) and 10 µm (S-180) were used. Based on the experimental studies, empirical models describing the influence of machining parameters on technological indicators and the condition of the surface texture were determined. The research indicates that graphite with a larger grain provides higher process efficiency with high relative wear of the tool electrode. The lowest surface roughness was obtained for graphite with a smaller grain size (AF-5). The analysis of the machining parameters proves that the discharge current and pulse duration are the main factors determining the MRR and Ra values for both AF-5 and S-180 graphite. The time interval is the dominant parameter with regard to the relative wear of the graphite electrode.

Experimental Investigation and Optimization of Rough EDM of High-Thermal-Conductivity Tool Steel with a Thin-Walled Electrode.

The industrial application of electrical discharge machining (EDM) for manufacturing injection molding, in many cases, requires forming depth cavities with high length-to-width ratios, which is quite challenging. During slot EDM with thin-walled electrodes, short-circuits and arcing discharges occur, as a result of low efficiency in removing debris and bubble gas from the gap. Furthermore, unstable discharges can cause increases in tool wear and shape deviation of the machined parts. In order to characterize the influence of the type of electrode material and EDM parameters on the deep slot machining of high-thermal-conductivity tool steel (HTCS), experimental studies were conducted. An analytical and experimental investigation is carried out on the influence of EDM parameters on discharge current and pulse-on-time on the tool wear (TW), surface roughness (Ra), slot width (S)-dimension of the cavity, and material removal rate (MRR). The analyses of the EDS spectrum of the electrode indicate the occurrence of the additional carbon layer on the electrode. Carbon deposition on the anode surface can provide an additional thermal barrier that reduces electrode wear in the case of the copper electrode but for graphite electrodes, uneven deposition of carbon on the electrode leads to unstable discharges and leads to increase tool wear. The response surface methodology (RSM) was used to build empirical models of the influence of the discharge current I and pulse-on-time ton on Ra, S, TW, and MRR. Analysis of variance (ANOVA) was used to establish the statistical significance parameters. The calculated contribution indicated that the discharge current had the most influence (over 70%) on the Ra, S, TW, and MRR, followed by the discharge time. Multicriteria optimization with Derringer's function was then used to minimize the surface roughness, slot width, and TW, while maximizing MRR. A validation test confirms that the maximal error between the predicted and obtained values did not exceed 7%.

Selective depletion of radiolabeled HER2-specific antibody for contrast improvement during PET.

The prolonged in vivo persistence of antibodies results in high background and poor contrast during their use as molecular imaging agents for positron emission tomography (PET). We have recently described a class of engineered Fc fusion proteins that selectively deplete antigen-specific antibodies without affecting the levels of antibodies of other specificities. Here, we demonstrate that these Fc fusions (called Seldegs, for selective degradation) can be used to clear circulating, radiolabeled HER2-specific antibody during diagnostic imaging of HER2-positive tumors in mice. The analyses show that Seldegs have considerable promise for the reduction of whole-body exposure to radiolabel and improvement of contrast during PET.

Investigation of the Influence of Reduced Graphene Oxide Flakes in the Dielectric on Surface Characteristics and Material Removal Rate in EDM.

Electrical discharge machining (EDM) is an advanced technology used to manufacture difficult-to-cut conductive materials. However, the surface layer properties after EDM require additional finishing operations in many cases. Therefore, new methods implemented in EDM are being developed to improve surface characteristics and the material removal rate. This paper presents new research about improving the surface integrity of 55NiCrMoV7 tool steel by using reduced graphene oxide (RGO) flakes in the dielectric. The main goal of the research was to investigate the influence of RGO flakes in the dielectric on electrical discharge propagation and heat dissipation in the gap. The investigation of the influence of discharge current I and pulse time ton during EDM with RGO flakes in the dielectric was carried out using response surface methodology. Furthermore, the surface texture properties and metallographic structure after EDM with RGO in the dielectric and conventional EDM were investigated and described. The obtained results indicate that using RGO flakes in the dielectric leads to a decreased surface roughness and recast layer thickness with an increased material removal rate (MRR). The presence of RGO flakes in the dielectric reduced the breakdown voltage and allowed several discharges to occur during one pulse. The dispersion of the discharge caused a decrease in the energy delivered to the workpiece. In terms of the finishing EDM parameters, there was a 460% reduction in roughness Ra with a uniform distribution of the recast layer on the surface, and a slight increase in MRR (12%) was obtained.

Investigation of the Influence of Reduced Graphene Oxide Flakes in the Dielectric on Surface Characteristics and Material Removal Rate in EDM.

Electrical discharge machining (EDM) is an advanced technology used to manufacture difficult-to-cut conductive materials. However, the surface layer properties after EDM require additional finishing operations in many cases. Therefore, new methods implemented in EDM are being developed to improve surface characteristics and the material removal rate. This paper presents new research about improving the surface integrity of 55NiCrMoV7 tool steel by using reduced graphene oxide (RGO) flakes in the dielectric. The main goal of the research was to investigate the influence of RGO flakes in the dielectric on electrical discharge propagation and heat dissipation in the gap. The investigation of the influence of discharge current I and pulse time ton during EDM with RGO flakes in the dielectric was carried out using response surface methodology. Furthermore, the surface texture properties and metallographic structure after EDM with RGO in the dielectric and conventional EDM were investigated and described. The obtained results indicate that using RGO flakes in the dielectric leads to a decreased surface roughness and recast layer thickness with an increased material removal rate (MRR). The presence of RGO flakes in the dielectric reduced the breakdown voltage and allowed several discharges to occur during one pulse. The dispersion of the discharge caused a decrease in the energy delivered to the workpiece. In terms of the finishing EDM parameters, there was a 460% reduction in roughness Ra with a uniform distribution of the recast layer on the surface, and a slight increase in MRR (12%) was obtained.

The Effects of Reduced Graphene Oxide Flakes in the Dielectric on Electrical Discharge Machining.

Electrical discharge machining (EDM) is a nonconventional technology that is frequently used in manufacturing for difficult-to-cut conductive materials. Drawbacks to using EDM include the resulting surface roughness and integrity. One of the recent innovations for improving surface integrity with EDM is the use of a powder mixed dielectric. The aim of this study is to analyze the influence of having reduced graphene oxide (RGO) in the dielectric on the ionization of the plasma channel and the dispersion of electrical discharges. The main goal is to improve the surface integrity of the tool steel 55NiCrMoV7 during finishing machining. To achieve this goal, an experimental investigation was carried out to establish the smallest possible values of discharge current and pulse time at which it is possible to initiate an electric discharge, which causes material removal. Next, the effect of the direction of the electric discharges (electrode polarity) and the concentration (percentage) of RGO in the dielectric on surface integrity was investigated. The results of this experiment indicate that during EDM with RGO, the discharges are dispersed on the RGO flakes. This leads to a multiplication of the discharges during a single pulse, and this strongly affects the surface integrity. The obtained results indicate that it is possible to reduce surface roughness and thickness of the recast layer by approximately 2.5 times compared with conventional EDM.

Multi-Response Optimization of Electrical Discharge Machining Using the Desirability Function.

Electrical discharge machining (EDM) is a modern technology that is widely used in the production of difficult to cut conductive materials. The basic problem of EDM is the stochastic nature of electrical discharges. The optimal selection of machining parameters to achieve micron surface roughness and the recast layer with the maximal possible value of the material removal rate (MRR) is quite challenging. In this paper, we performed an analytical and experimental investigation of the influence of the EDM parameters: Surface integrity and MRR. Response surface methodology (RSM) was used to build empirical models on the influence of the discharge current I, pulse time ton, and the time interval toff, on the surface roughness (Sa), the thickness of the white layer (WL), and the MRR, during the machining of tool steel 55NiCrMoV7. The surface and subsurface integrity were evaluated using an optical microscope and a scanning profilometer. Analysis of variance (ANOVA) was used to establish the statistical significance parameters. The calculated contribution indicated that the discharge current had the most influence (over the 50%) on the Sa, WL, and MRR, followed by the discharge time. The multi-response optimization was carried out using the desirability function for the three cases of EDM: Finishing, semi-finishing, and roughing. The confirmation test showed that maximal errors between the predicted and the obtained values did not exceed 6%.

Loss of expression of the recycling receptor, FcRn, promotes tumor cell growth by increasing albumin consumption.

Tumor cells rely on high concentrations of amino acids to support their growth and proliferation. Although increased macropinocytic uptake and lysosomal degradation of the most abundant serum protein, albumin, in Ras-transformed cells can meet these demands, it is not understood how the majority of tumor cells that express wild type Ras achieve this. In the current study we reveal that the neonatal Fc receptor, FcRn, regulates tumor cell proliferation through the ability to recycle its ligand, albumin. By contrast with normal epithelial cells, we show that human FcRn is present at very low or undetectable levels in the majority of tumor cell lines analyzed. Remarkably, shRNA-mediated ablation of FcRn expression in an FcRn-positive tumor cell line results in a substantial growth increase of tumor xenografts, whereas enforced expression of this receptor by lentiviral transduction has the reverse effect. Moreover, intracellular albumin and glutamate levels are increased by the loss of FcRn-mediated recycling of albumin, combined with hypoalbuminemia in tumor-bearing mice. These studies identify a novel role for FcRn as a suppressor of tumor growth and have implications for the use of this receptor as a prognostic indicator and therapeutic target.

Use of Fc-Engineered Antibodies as Clearing Agents to Increase Contrast During PET.

UNLABELLED: Despite promise for the use of antibodies as molecular imaging agents in PET, their long in vivo half-lives result in poor contrast and radiation damage to normal tissue. This study describes an approach to overcome these limitations. METHODS: Mice bearing human epidermal growth factor receptor type 2 (HER2)-overexpressing tumors were injected with radiolabeled ((124)I, (125)I) HER2-specific antibody (pertuzumab). Pertuzumab injection was followed 8 h later by the delivery of an engineered, antibody-based inhibitor of the receptor, FcRn. Biodistribution analyses and PET were performed at 24 and 48 h after pertuzumab injection. RESULTS: The delivery of the engineered, antibody-based FcRn inhibitor (or Abdeg, for antibody that enhances IgG degradation) results in improved tumor-to-blood ratios, reduced systemic exposure to radiolabel, and increased contrast during PET. CONCLUSION: Abdegs have considerable potential as agents to stringently regulate antibody dynamics in vivo, resulting in increased contrast during molecular imaging with PET.

The encephalitogenic, human myelin oligodendrocyte glycoprotein-induced antibody repertoire is directed toward multiple epitopes in C57BL/6-immunized mice.

Although Abs specific for myelin oligodendrocyte glycoprotein (MOG) have been detected in patients with multiple sclerosis (MS), their contribution to pathogenesis remains poorly understood. Immunization of C57BL/6 mice with recombinant human MOG (hMOG) results in experimental autoimmune encephalomyelitis involving MOG-specific, demyelinating Abs. This model is therefore informative for understanding anti-MOG humoral responses in MS. In the current study, we have characterized the hMOG-specific Ab repertoire in immunized C57BL/6 mice using both in vitro and in vivo approaches. We demonstrate that hMOG-specific mAbs are not focused on one specific region of MOG, but instead target multiple epitopes. Encephalitogenicity of the mAbs, assessed by the ability of the mAbs to exacerbate experimental autoimmune encephalomyelitis in mice, correlates with the activity of the mAbs in binding to CNS tissue sections, but not with other in vitro assays. The targeting of different MOG epitopes by encephalitogenic Abs has implications for disease pathogenesis, because it could result in MOG cross linking on oligodendrocytes and/or immune complex formation. These studies reveal several novel features concerning pathogenic, humoral responses that may have relevance to human MS.

Progastrin overexpression imparts tumorigenic/metastatic potential to embryonic epithelial cells: phenotypic differences between transformed and nontransformed stem cells.

We recently reported that overexpression of progastrin (PG) in embryonic epithelial cells (HEKmGAS cells) increased proliferation of the cells compared to that of control HEKC cells. Here, we report the novel finding that tumorigenic and metastatic potential of HEKmGAS cells is also increased significantly compared to that of HEKC cells. Cell surface-associated annexinA2 (CS-ANXA2) binds PG and is overexpressed on cancer cells, allowing us to successfully use fluorescently labeled PG peptide for enumerating metastatic lesions of transformed/cancer cells in vivo. Next, we examined the hypothesis that increased tumorigenic/metastatic potential of isogenic HEKmGAS versus HEKC cells maybe due to transformed phenotype of stem cells. FACSorting/FACScanning of cells demonstrated significant increases in percent doublecortin-CAM-kinase-like1 (DCLK1)/Lgr5-positive stem cells, coexpressing cluster of differentiation44 (CD44)/CS-ANXA2, in HEKmGAS versus HEKC cells. Distinct differences were noted in the morphology of HEKC versus HEKmGAS spheroidal growths on nonadherent cultures (selective for stem cells). HEKC spheroids were rounded with distinct perimeters (e.g., basement membranes), whereas HEKmGAS spheroids were amorphous with no perimeters. Relative levels of DCLK1/Lgr5/CD44 and ANXA2/ß-catenin/pNFκBp65/metalloproteinases were significantly increased in HEKmGAS versus HEKC cells, growing as monolayer cultures, 3D spheroids (in vitro), or xenografts (in vivo). Interestingly, HEKC cells enriched for CS-ANXA2 developed amorphous spheroids, whereas downregulation of ANXA2 in HEKmGAS clones resulted in loss of matrixmetalloproteinases (MMPs) and re-formation of rounded spheroids, suggesting that high levels of CS-ANXA2/MMPs may impact spheroid morphology. Downregulation of DCLK1 significantly attenuated activation of ß-catenin, with loss of proliferation of HEKmGAS and HEKC cells, suggesting that DCLK1 is required for maintaining proliferation of cells. Our results suggest the novel possibility that transformed stem cells, unlike nontransformed stem cells, coexpress stem cell markers DCLK1 and CD44 with CS-ANXA2.

Annexin A2 mediates up-regulation of NF-κB, ß-catenin, and stem cell in response to progastrin in mice and HEK-293 cells.

BACKGROUND & AIMS: Prograstrin induces proliferation in colon crypts by activating p65nuclear factor-κB (NF-κB) (p65) and ß-catenin. We investigated whether Annexin A2 (AnxA2), a progastrin receptor, activates NF-κB and ß-catenin in vivo. METHODS: ANXA2-null (ANXA2(-/-)) and wild-type (ANXA2(+/+)) mice were studied, along with clones of progastrin-responsive HEK-293 cells that stably expressed full-length progastrin (HEK-mGAS) or an empty vector (HEK-C). Small interfering RNA was used to down-regulate AnxA2, p65NF-κB, and ß-catenin in cells. RESULTS: Proliferation and activation of p65 and ß-catenin increased significantly in HEK-mGAS compared with HEK-C clones. HEK-mGAS cells had a 2- to 4-fold increase in relative levels of c-Myc, cyclooxygenase (COX)-2, CyclinD1, double cortin CAM kinase-like 1 (DCAMKL+1), and CD44, compared with HEK-C clones. Down-regulation of AnxA2 in HEK-mGAS clones reduced activation of NF-κB and ß-catenin, as well as levels of DCAMKL+1. Surprisingly, down-regulation of ß-catenin had no effect on activation of p65NF-κB, whereas down-regulation of p65 significantly reduced activation of ß-catenin in HEK-mGAS clones. Loss of either p65 or ß-catenin significantly reduced proliferation of HEK-mGAS clones, indicating that both factors are required for the proliferative effects of progastrin. Lengths of colon crypts and levels of p65, ß-catenin, DCAMKL+1, and CD44 were significantly higher in ANXA2(+/+) mice compared with either ANXA2(-/-) mice given progastrin or ANXA2(+/+) and ANXA2(-/-) mice given saline. CONCLUSIONS: AnxA2 expression is required for the biologic effects of progastrin in vivo and in vitro and mediates the stimulatory effect of progastrin on p65NF-κ, ß-catenin, and the putative stem cell markers DCAMKL+1 and CD44. AnxA2 might therefore mediate the hyperproliferative and cocarcinogenic effects of progastrin.

Elevated metals compromise repair of oxidative DNA damage via the base excision repair pathway: implications of pathologic iron overload in the brain on integrity of neuronal DNA.

Tissue-specific iron content is tightly regulated to simultaneously satisfy specialized metabolic needs and avoid cytotoxicity. In the brain, disruption of iron homeostasis may occur in acute as well as progressive injuries associated with neuronal dysfunction and death. We hypothesized that adverse effects of disrupted metal homeostasis on brain function may involve impairment of DNA repair processes. Because in the brain, the base excision repair (BER) pathway is central for handling oxidatively damaged DNA, we investigated effects of elevated iron and zinc on key BER enzymes. In vitro DNA repair assays revealed inhibitory effects of metals on BER activities, including the incision of abasic sites, 5'-flap cleavage, gap filling DNA synthesis and ligation. Using the comet assay, we showed that while metals at concentrations which inhibit BER activities in in vitro assays, did not induce direct genomic damage in cultured primary neurons, they significantly delayed repair of genomic DNA damage induced by sublethal exposure to H(2)O(2). Thus, in the brain even a mild transient metal overload, may adversely affect the DNA repair capacity and thereby compromise genomic integrity and initiate long-term deleterious sequelae including neuronal dysfunction and death.

Ribosomal protein rpS2 is hypomethylated in PRMT3-deficient mice.

PRMT3 is a type I arginine methyltransferase that resides in the cytoplasm. A large proportion of this cystosolic PRMT3 is found associated with ribosomes. It is tethered to the ribosomes through its interaction with rpS2, which is also its substrate. Here we show that mouse embryos with a targeted disruption of PRMT3 are small in size but survive after birth and attain a normal size in adulthood, thus displaying Minute-like characteristics. The ribosome protein rpS2 is hypomethylated in the absence of PRMT3, demonstrating that it is a bona fide, in vivo PRMT3 substrate that cannot be modified by other PRMTs. Finally, the levels 40 S, 60 S, and 80 S monosomes and polyribosomes are unaffected by the loss of PRMT3, but there are additional as yet unidentified proteins that co-fractionate with ribosomes that are also dedicated PRMT3 substrates.

Ribosomal protein S2 is a substrate for mammalian PRMT3 (protein arginine methyltransferase 3).

PRMT3 (protein arginine methyltransferase 3) is one of four type I arginine methyltransferases that catalyse the formation of asymmetric dimethylarginine. PRMT3 is unique in that its N-terminus harbours a C2H2 zinc-finger domain that is proposed to confer substrate specificity. In addition, PRMT3 is the only type I enzyme that is restricted to the cytoplasm. Known in vitro substrates for PRMT3 include GST-GAR (a glutathione S-transferase fusion protein containing the glycine- and arginine-rich N-terminal region of fibrillarin), Sam68 (Src-associated substrate during mitosis 68 kDa) and PABP-N1 [poly(A)-binding protein-N1; PABP2]. Here we report the identification of an in vivo substrate for mammalian PRMT3. We found that FLAG-tagged PRMT3 can 'pull down' a protein with a molecular mass of 30 kDa from HeLa cell extracts. MS identified this PRMT3-interacting protein as rpS2 (ribosomal protein S2). In vitro studies showed that the zinc-finger domain of PRMT3 is necessary and sufficient for binding to rpS2. In addition, rpS2 is methylated by PRMT3 in vitro and is also methylated in cell lines. Deletion analysis of the rpS2 amino acid sequence identified a N-terminal Arg-Gly repeat as the methylation site. Furthermore, both PRMT3 and rpS2 co-sediment with free ribosomal subunits. These studies implicate PRMT3 in ribosomal function and in the regulation of protein synthesis.