Ribonuclease zymogen induces cytotoxicity upon HIV-1 infection

Background Targeting RNA is a promising yet underdeveloped modality for the selective killing of cells infected with HIV-1. The secretory ribonucleases (RNases) found in vertebrates have cytotoxic ribonucleolytic activity that is kept in check by a cytosolic ribonuclease inhibitor protein, RI. Methods We engineered amino acid substitutions that enable human RNase 1 to evade RI upon its cyclization into a zymogen that is activated by the HIV-1 protease. In effect, the zymogen has an HIV-1 protease cleavage site between the termini of the wild-type enzyme, thereby positioning a cleavable linker over the active site that blocks access to a substrate. Results The amino acid substitutions in RNase 1 diminish its affinity for RI by 106-fold and confer high toxicity for T-cell leukemia cells. Pretreating these cells with the zymogen leads to a substantial drop in their viability upon HIV-1 infection, indicating specific toxicity toward infected cells. Conclusions These data demonstrate the utility of ribonuclease zymogens as biologic prodrugs.

LRR protein RNH1 inhibits inflammasome activation through proteasome-mediated degradation of Caspase-1 and is associated with adverse clinical outcomes in COVID-19 patients.

Inflammasomes are cytosolic innate immune sensors that, upon activation, induce caspase-1 mediated inflammation. Although inflammation is protective, uncontrolled excessive inflammation can cause inflammatory diseases and is also detrimental in COVID-19 infection. However, the underlying mechanisms that control inflammasome activation are incompletely understood. Here we report that the leucine rich repeat (LRR) protein Ribonuclease inhibitor (RNH1), which shares homology with LRRs of NOD-like receptor family pyrin domain (PYD)-containing (NLRP) proteins, attenuates inflammasome activation. Mechanistically, RNH1 decreased pro-IL1b expression and induced proteasome-mediated caspase-1 degradation. Corroborating this, mouse models of monosodium urate (MSU)-induced peritonitis and LPS-induced endotoxemia, which are dependent on caspase-1, respectively showed increased neutrophil infiltration and lethality in Rnh1-/- mice compared to WT mice. Further, RNH1 protein levels were negatively correlated with inflammation and disease severity in hospitalized COVID-19 patients. We propose that RNH1 is a new inflammasome regulator with relevance to COVID-19 severity.

Angiogenin (ANG)—Ribonuclease Inhibitor (RNH1) System in Protein Synthesis and Disease

Protein synthesis is a highly complex process executed by well-organized translation machinery. Ribosomes, tRNAs and mRNAs are the principal components of this machinery whereas RNA binding proteins and ribosome interacting partners act as accessory factors. Angiogenin (ANG)—Ribonuclease inhibitor (RNH1) system is one such accessory part of the translation machinery that came into focus afresh due to its unconventional role in the translation. ANG is conventionally known for its ability to induce blood vessel formation and RNH1 as a “sentry” to protect RNAs from extracellular RNases. However, recent studies suggest them to be important in translation regulation. During cell homeostasis, ANG in the nucleus promotes rRNA transcription. While under stress, ANG translocates to the cytosol and cleaves tRNA into fragments which inhibit ribosome biogenesis and protein synthesis. RNH1, which intimately interacts with ANG to inhibit its ribonucleolytic activity, can also bind to the 40S ribosomes and control translation by yet to be known mechanisms. Here, we review recent advancement in the knowledge of translation regulation by the ANG-RNH1 system. We also gather information about this system in cell homeostasis as well as in pathological conditions such as cancer and ribosomopathies. Additionally, we discuss the future research directions and therapeutic potential of this system.

The Role of Ribonuclease 1 and Ribonuclease Inhibitor 1 in Acute Kidney Injury after Open and Endovascular Thoracoabdominal Aortic Aneurysm Repair

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.

Development of a method for determining the levels of normalized expression of structural and functional genes in patients with knee joint arthropathy

Objective : to develop a method of molecular genetic analysis for determining the normalized expression levels of COL2A1, COL6A1, MMP-2, and MMP-9 genes in the biological material of patients with gonarthrosis. Material and methods . The biopsy samples of knee joint cartilage of 10 patients with gonarthrosis were used as a biological material for the study. Nucleic acids were isolated from the samples of the biological material using TRIZol Reagent. Reverse transcription was performed using the SuperScript III reverse transcriptase kit, dNTP and Ribonuclease inhibitor (Invitrogen, USA). Results . The usage of VectorNTI software has made it possible to select pairs of primers (forward and reverse) and TaqMan probes for COL2A1, COL6A1, MMP-2 , and MMP-9 genes, and to study the possibility of the use of the selected pairs of primers and probes for the purpose of identification of each of the studied genes. In-house test systems in the multiplex format have been designed for reference and target genes. Conclusion . High specificity (100 %) of the selected oligonucleotide primer sets was confirmed using the online application NCBI/Blast. The developed molecular genetic method can be applied to determine the normalized expression levels of COL2A1, COL6A1, MMP-2 , and MMP-9 genes in knee joint arthropathy

Higher Vertebrate Specific Gene Ribonuclease Inhibitor (RNH1) Is Essential for Adult Hematopoietic Stem Cell Function and Cell Cycle Regulation

Hematopoietic stem cells (HSC) in higher vertebrate species, especially in mammals, maintain hematopoiesis throughout adult life and require critical cell cycle regulation for their self-renewal and cell fate decisions. Although cell cycle pathways are quite conserved across animal species, it is unknown whether a higher vertebrate specific cell cycle regulation exists in adult mammalian HSCs. Recently, we have published that Ribonuclease inhibitor (RNH1) regulates erythropoiesis by controlling GATA1 mRNA translation. Here, we report that RNH1, which is present only in higher vertebrates regulates HSC cell cycle and HSC function. To study the role of RNH1 in hematopoiesis, we generated hematopoietic-specific knockout mice by backcrossing Rnh1FL/FL mice with Vav1-iCre and Mx1-Cre mice, respectively. Rnh1-deficiency (Rnh1FL/FLVav1-iCre mice) resulted in hematopoietic alterations resembling emergency myelopoiesis. At 15 weeks of age Rnh1-deficient mice had reduced hemoglobin levels (144.4 ± 2.6 vs 165.0 ± 4.2 g/L, p = 0.005), decreased lymphocytes (4.1 ± 0.8 vs 9.6 ± 1.6 K/µL, p = 0.023), increased neutrophils (3.2 ± 0.6 vs 1.5 ± 0.2 K/µL, p = 0.046) and monocytes (0.65 ± 0.05 vs 0.09 ± 0.02 K/µL, p = 0.0001) in the peripheral blood. Total bone-marrow (BM) cellularity was similar in wild type andRnh1-deficient mice, however the number of erythroid cells and lymphoid cells (T and B cells) was significantly decreased, whereas myeloid cells were significantly increased. Rnh1-deficient spleens were significantly larger than wild type controls and showed extramedullary hematopoiesis. Surprisingly, although Rnh1-deficient mice showed myeloproliferation they survived normally and did not show progression to leukemia. However, they did not tolerate even little stress, such as 35 µg LPS administration, which lead to early mortality. We analysed the progenitor populations in the BM. In line with the myelopoiesis dominant phenotype granulocyte-monocyte progenitor (GMP) cell numbers were increased but common lymphoid progenitor (CLP) and megakaryocyte-erythrocyte progenitor (MEP) cell numbers were decreased. Cell extrinsic factors such as growth factors and the bone marrow niche play a critical role in shaping lineage choice. To exclude this, we performed bone marrow transplantation experiments (BMT) by transplanting wild type (Rnh1FL/FL) and Rnh1-deficient (Rnh1FL/FLMx1-Cre+) bone marrow into lethally irradiated CD45.1 congenic mice. After reconstitution Rnh1 was deleted by administration of polyinosinic:polycytidylic acid (polyI:C). We observed a similar myelopoiesis dominant phenotype in Rnh1-deleted mice. Interestingly, we found increased numbers of long term HSCs (LT-HSCs) and short term HSCs (ST-HSCs) in Rnh1-deficient mouse BM, suggesting that RNH1 could affect HSC function. Supporting this Rnh1-deficient HSCs failed to engraft lethally irradiated mice in competitive BMT experiments. Furthermore, Rnh1-deficient HSCs produced significantly less and smaller colonies in in-vitro colony forming cell (CFC) assays. Transcriptome analysis showed increased expression of genes related to cell cycle, kinetochore, DNA damage and decreased expression of genes related to stem cell function in Rnh1-deficient LT-HSCs and ST-HSCs. Corroborating this, Rnh1-deficient LT-HSCs and ST-HSCs showed increased S/G2/M phase in cell cycle analysis. In line with this, at the molecular level, we found that RNH1 directly binds to cell-cycle related proteins such as cyclin-dependent kinase 1 (CDK1), cell-division cycle protein 20 (CDC20) and mitotic checkpoint protein BUB3, suggesting direct involvement of RNH1 in cell cycle regulation. Confirming this, pharmacological inhibition of CDK1 (RO-3306, 10 µM) in Rnh1-deficinet ST-HSCs restored colony size in CFC assays, suggesting that RNH1 and CDK1 inhibition have a synergistic effect in ST-HSCs. In summary, our results demonstrate that RNH1, which is present only in higher vertebrates, is essential for HSC cell cycle regulation and steady state hematopoiesis. Disclosures No relevant conflicts of interest to declare.