Engineered human angiogenin mutations in the placental ribonuclease inhibitor complex for anticancer therapy: Insights from enhanced sampling simulations.
ABSTRACT: Targeted human cytolytic fusion proteins (hCFPs) represent a new generation of immunotoxins (ITs) for the specific targeting and elimination of malignant cell populations. Unlike conventional ITs, hCFPs comprise a human/humanized target cell-specific binding moiety (e.g., an antibody or a fragment thereof) fused to a human proapoptotic protein as the cytotoxic domain (effector domain). Therefore, hCFPs are humanized ITs expected to have low immunogenicity. This reduces side effects and allows long-term application. The human ribonuclease angiogenin (Ang) has been shown to be a promising effector domain candidate. However, the application of Ang-based hCFPs is largely hampered by the intracellular placental ribonuclease inhibitor (RNH1). It rapidly binds and inactivates Ang. Mutations altering Ang's affinity for RNH1 modulate the cytotoxicity of Ang-based hCFPs. Here we perform in total 2.7 µs replica-exchange molecular dynamics simulations to investigate some of these mutations-G85R/G86R (GGRRmut ), Q117G (QGmut ), and G85R/G86R/Q117G (GGRR/QGmut ). GGRRmut turns out to perturb greatly the overall Ang-RNH1 interactions, whereas QGmut optimizes them. Combining QGmut with GGRRmut compensates the effects of the latter. Our results explain the in vitro finding that, while Ang GGRRmut -based hCFPs resist RNH1 inhibition remarkably, Ang WT- and Ang QGmut -based ones are similarly sensitive to RNH1 inhibition, whereas Ang GGRR/QGmut -based ones are only slightly resistant. This work may help design novel Ang mutants with reduced affinity for RNH1 and improved cytotoxicity.
Project description:Human angiogenin (ANG) is a homologue of bovine pancreatic ribonuclease (RNase A) that induces neovascularization. ANG is the only human angiogenic factor that possesses ribonucleolytic activity. To stimulate blood vessel growth, ANG must be transported to the nucleus and must retain its catalytic activity. Like other mammalian homologues of RNase A, ANG forms a femtomolar complex with the cytosolic ribonuclease inhibitor protein (RI). To determine whether RI affects ANG-induced angiogenesis, we created G85R/G86R ANG, which possesses 10(6)-fold lower affinity for RI but retains wild-type ribonucleolytic activity. The neovascularization of rabbit corneas by G85R/G86R ANG was more pronounced and more rapid than by wild-type ANG. These findings provide the first direct evidence that RI serves to regulate the biological activity of ANG in vivo.
Project description:Angiogenin (ANG) is a secretory ribonuclease that promotes the proliferation of endothelial cells, leading to angiogenesis. This function relies on its ribonucleolytic activity, which is low for simple RNA substrates. Upon entry into the cytosol, ANG is sequestered by the ribonuclease inhibitor protein (RNH1). We find that ANG is a potent cytotoxin for RNH1-knockout HeLa cells, belying its inefficiency as a nonspecific catalyst. The toxicity does, however, rely on the ribonucleolytic activity of ANG and a cytosolic localization, which lead to the accumulation of particular tRNA fragments (tRFs), such as tRF-5 Gly-GCC. These up-regulated tRFs are highly cytotoxic at physiological concentrations. Although ANG is well-known for its promotion of cell growth, our results reveal that ANG can also cause cell death.
Project description:Angiogenin (ANG) promotes cell growth and survival. Under growth conditions, ANG undergoes nuclear translocation and accumulates in the nucleolus where it stimulates rRNA transcription. When cells are stressed, ANG mediates the production of tRNA-derived stress-induced small RNA (tiRNA), which reprograms protein translation into a survival mechanism. The ribonucleolytic activity of ANG is essential for both processes but how this activity is regulated is unknown. We report here that ribonuclease/angiogenin inhibitor 1 (RNH1) controls both the localization and activity of ANG. Under growth conditions, ANG is located in the nucleus and is not associated with RNH1 so that the ribonucleolytic activity is retained to ensure rRNA transcription. Cytoplasmic ANG is associated with and inhibited by RNH1 so that random cleavage of cellular RNA is prevented. Under stress conditions, ANG is localized to the cytoplasm and is concentrated in stress granules where it is not associated with RNH1 and thus remains enzymatically active for tiRNA production. By contrast, nuclear ANG is associated with RNH1 in stressed cells to ensure that the enzymatic activity is inhibited and no unnecessary rRNA is produced to save anabolic energy. Knockdown of RNH1 abolished stress-induced relocalization of ANG and decreased cell growth and survival.
Project description:Adaptation to endoplasmic reticulum (ER) stress depends on the activation of the sensor inositol-requiring enzyme 1? (IRE1), an endoribonuclease that splices the mRNA of the transcription factor XBP1 (X-box-binding protein 1). To better understand the protein network that regulates the activity of the IRE1 pathway, we systematically screened the proteins that interact with IRE1 and identified a ribonuclease inhibitor called ribonuclease/angiogenin inhibitor 1 (RNH1). RNH1 is a leucine-rich repeat domains-containing protein that binds to and inhibits ribonucleases. Immunoprecipitation experiments confirmed this interaction. Docking experiments indicated that RNH1 physically interacts with IRE1 through its cytosolic RNase domain. Upon ER stress, the interaction of RNH1 with IRE1 in the ER increased at the expense of the nuclear pool of RNH1. Inhibition of RNH1 expression using siRNA mediated RNA interference upon ER stress led to an increased splicing activity of XBP1. Modulation of IRE1 RNase activity by RNH1 was recapitulated in a cell-free system, suggesting direct regulation of IRE1 by RNH. We conclude that RNH1 attenuates the activity of IRE1 by interacting with its ribonuclease domain. These findings have implications for understanding the molecular mechanism by which IRE1 signaling is attenuated upon ER stress.
Project description:Angiogenin (ANG) is a secreted ribonuclease that cleaves tRNA to initiate a stress-response program in mammalian cells. Here we show that ANG inhibits protein synthesis and promotes arsenite- and pateamine A-induced assembly of stress granules (SGs). These effects are abrogated in cells transfected with the ANG inhibitor RNH1. Transfection of natural or synthetic 5'- but not 3'-tRNA fragments (tRNA-derived stress-induced RNAs; tiRNAs) induces the phospho-eukaryotic translation initiation factor 2alpha-independent assembly of SGs. Natural 5'-tiRNAs but not 3'-tiRNAs are capped with a 5'-monophosphate that is required for optimal SG assembly. These findings reveal that SG assembly is a component of the ANG- and tiRNA-induced stress response program.
Project description:Ribonuclease inhibitor (RNH1) is a cytosolic protein that binds with femtomolar affinity to human ribonuclease 1 (RNase 1) and homologous secretory ribonucleases. RNH1 contains 32 cysteine residues and has been implicated as an antioxidant. Here, we use CRISPR-Cas9 to knock out RNH1 in HeLa cells. We find that cellular RNH1 affords marked protection from the lethal ribonucleolytic activity of RNase 1 but not from oxidants. We conclude that RNH1 protects cytosolic RNA from invading ribonucleases.
Project description:Ribonuclease Inhibitor (RI also known as Rnh1) is a 50 kDa, ubiquitously expressed leucine-rich repeat (LRR) protein. It is localized in cytosol and binds to pancreatic-type ribonucleases and inhibit their function. However the entire biological role for Rnh1 is unknown. We generated Rnh1 knock out mice by homologous recombination. Here we studied differential gene expression from wild type (Rnh1 +/+), Heterozygous (Rnh1+/-) and Knock out (Rnh1-/-) yolk sacs isolated from embryonic day 9.5 (E9.5). We used microarrays to study global gene expression regulated by Rnh1 in yolk sacs. Total RNA was isolated from E9.5 yolk sacs of Rnh1 Wild type, heterozygous and knock out.
Project description:Ribosomal proteins (RP) regulate specific gene expression by selectively translating subsets of mRNAs. Indeed, in Diamond-Blackfan anemia and 5q- syndrome, mutations in RP genes lead to a specific defect in erythroid gene translation and cause anemia. Little is known about the molecular mechanisms of selective mRNA translation and involvement of ribosomal-associated factors in this process. Ribonuclease inhibitor 1 (RNH1) is a ubiquitously expressed protein that binds to and inhibits pancreatic-type ribonucleases. Here, we report that RNH1 binds to ribosomes and regulates erythropoiesis by controlling translation of the erythroid transcription factor GATA1. Rnh1-deficient mice die between embryonic days E8.5 and E10 due to impaired production of mature erythroid cells from progenitor cells. In Rnh1-deficient embryos, mRNA levels of Gata1 are normal, but GATA1 protein levels are decreased. At the molecular level, we found that RNH1 binds to the 40S subunit of ribosomes and facilitates polysome formation on Gata1 mRNA to confer transcript-specific translation. Further, RNH1 knockdown in human CD34+ progenitor cells decreased erythroid differentiation without affecting myelopoiesis. Our results reveal an unsuspected role for RNH1 in the control of GATA1 mRNA translation and erythropoiesis.
Project description:Summary: Ribonuclease Inhibitor (RI also known as Rnh1) is a 50 kDa, ubiquitously expressed leucine-rich repeat (LRR) protein. It is localized in cytosol and binds to pancreatic-type ribonucleases and inhibit their function. However, the entire biological role for Rnh1 is unknown. We generated RNH1 knock out K562 cells by CRISPR/Cas9 method. Here we studied differential gene expression from wild type and RNH1 knock out K562 cells by RNA-Seq analysis. Overall design: Total RNA was isolated from wild type and RNH1 deficient K562 cells.
Project description:Acute kidney injury (AKI) is one of the most common post-operative complications and is closely associated with increased mortality after open and endovascular thoracoabdominal aortic aneurysm (TAAA) repair. Ribonuclease (RNase) 1 belongs to the group of antimicrobial peptides elevated in septic patients and indicates the prediction of two or more organ failures. The role of RNase 1 and its antagonist RNase inhibitor 1 (RNH1) after TAAA repair is unknown. In this study, we analyzed RNase 1 and RNH1 serum levels in patients undergoing open (n = 14) or endovascular (n = 19) TAAA repair to determine their association with post-operative AKI and in-hospital mortality. Increased RNH1 serum levels after open TAAA repair as compared with endovascular TAAA repair immediately after surgery and 12, 48, and 72 h after surgery (all p < 0.05) were observed. Additionally, elevated RNase 1 and RNH1 serum levels 12, 24, and 48 h after surgery were shown to be significantly associated with AKI (all p < 0.05). RNH1 serum levels before and RNase 1 serum levels 12 h after TAAA repair were significantly correlated with in-hospital mortality (both p < 0.05). On the basis of these findings, RNase 1 and RNH1 may be therapeutically relevant and may represent biomarkers for post-operative AKI and in-hospital mortality.