Proteotoxicity from aberrant ribosome biogenesis compromises cell fitness.

To achieve maximal growth, cells must manage a massive economy of ribosomal proteins (r-proteins) and RNAs (rRNAs) to produce thousands of ribosomes every minute. Although ribosomes are essential in all cells, natural disruptions to ribosome biogenesis lead to heterogeneous phenotypes. Here, we model these perturbations in Saccharomyces cerevisiae and show that challenges to ribosome biogenesis result in acute loss of proteostasis. Imbalances in the synthesis of r-proteins and rRNAs lead to the rapid aggregation of newly synthesized orphan r-proteins and compromise essential cellular processes, which cells alleviate by activating proteostasis genes. Exogenously bolstering the proteostasis network increases cellular fitness in the face of challenges to ribosome assembly, demonstrating the direct contribution of orphan r-proteins to cellular phenotypes. We propose that ribosome assembly is a key vulnerability of proteostasis maintenance in proliferating cells that may be compromised by diverse genetic, environmental, and xenobiotic perturbations that generate orphan r-proteins.

68Ga-NOTA-Functionalized Ubiquicidin: Cytotoxicity, Biodistribution, Radiation Dosimetry, and First-in-Human PET/CT Imaging of Infections.

Ubiquicidin is an antimicrobial peptide with great potential for nuclear imaging of infectious diseases, as its cationic-rich fragment TGRAKRRMQYNRR (UBI) has been functionalized with NOTA to allow complexation to 68Ga (68Ga-NOTA-UBI). We herein assess the cytotoxicity and radiation dosimetry for 68Ga-NOTA-UBI and a first-in-human evaluation to diagnose infectious processes. Methods: Cytotoxicity was evaluated in green monkey kidney epithelial (Vero) cells and MT-4 leukocytes. Tracer susceptibility was studied in vitro using different bacterial and fungal strains. PET/CT-based biodistribution, pharmacokinetics, and radiation dosimetry were performed on nonhuman primates. Two healthy volunteers and 3 patients with suspected infection underwent 68Ga-NOTA-UBI PET/CT imaging. Results: Negligible cytotoxicity was determined for NOTA-UBI. 68Ga-NOTA-UBI showed moderate blood clearance (29-min half-life) and predominant renal clearance in nonhuman primates. Human radiation dose estimates indicated the bladder wall as the dose-critical tissue (185 µSv/MBq), followed by the kidneys (23 µSv/MBq). The total absorbed body dose was low (<7 µSv/MBq); the effective dose was estimated at 17 µSv/MBq. 68Ga-NOTA-UBI could diagnose bone- and soft-tissue infection in 3 of 3 patients. Conclusion:68Ga-NOTA-UBI is considered a nontoxic, safe-to-administer radiopharmaceutical unlikely to cause adverse effects in humans. The favorable tracer biodistribution and the first-in-human results will make 68Ga-NOTA-UBI PET/CT an encouraging future diagnostic technique with auxiliary clinical relevance.

Anti-ribosomal-P antibodies accelerate lupus glomerulonephritis and induce lupus nephritis in naïve mice.

BACKGROUND: Lupus nephritis is known to be associated with several antibodies including autoantibodies that target the DNA, C1q and histone, α-actinin, and the nucleosome. In addition, circulating anti-phosphoribosomal protein antibodies (anti-Ribos.P) were found to be associated with lupus nephritis. STUDY OBJECTIVE: We have assessed the direct role of anti-Ribos.P in the development of glomerulonephritis in-vitro and in animal models. STUDY DESIGN: NZBxW/F1 lupus prone mice were immunized with recombinant Ribos.P0 (rRibos.P). Evaluation of renal disease included mice evaluation for proteinuria and histologic analysis of the kidneys. Anti-Ribos.P monoclonal Ab was prepared from the rRibos.P immunized NZBxW/F1 mice by hybridoma technology. MAPKs expression was analyzed by MAPKs protein array and confirmed by real-time PCR and western blot. To elucidate whether anti-Ribos.P induce glomerulonephritis, naïve C3H mice were immunized with recombinant rRibos.P and the glomerulonephritis was followed up as described above. RESULTS: The immunized NZBxW/F1 lupus prone mice developed anti-Ribos.P which was cross reactive with Sm and not dsDNA. The mice developed accelerated glomerulonephritis manifested by early proteinuria and immunoglobulin deposites in the mesangium of the kidneys. Anti-Ribos.P deposited in the glomerular mesangium were eluted from the kidney. The Ribos.P immunized naïve C3H/Hen mice developed glomerulonephritis manifested by circulating autoantibodies directed to Ribos.P, dsDNA and Sm. The anti Ribos.P were cross reactive with Sm but not with dsDNA, and were deposited in the glomeruli. Interestingly these mice developed alopecia. In vitro. Primary mesangial cells exposed to mouse anti-Ribos.P mAb originated from the immunized lupus mice and to human anti-Ribos.P Abs, induced activation of mesangial cells via p38α, JNK, AKT and HSP27 MAPKs expression pathway. CONCLUSIONS: Our data show for the first time that anti-Ribos.P are nephritogenic autoantibodies, as illustrated by in-vitro and in-vivo experiments: a) They accelerate the development of glomerulonephritis in lupus prone mice; b) They induce nephritis in naïve mice. c) Anti-Ribos.P Abs trigger MAPKs expression in primary mesangial cells. These data contribute a direct mechanistic link between anti-Ribos.P and nephritis in lupus mice.

A helix-PXXP-helix peptide with antibacterial activity without cytotoxicity against MDRPA-infected mice.

In response to the growing problem of multidrug-resistant pathogenic microbes, much attention is being paid to naturally occurring and synthetic antimicrobial peptides (AMPs) and the effects of their structural modification. Among these modifications, amino acid substitution is a simple approach to enhancing biological activity and reducing cytotoxicity. An earlier study indicated that HPA3, an analog of HP (2-20) derived from the N-terminus of Helicobacter pylori ribosomal protein L1, forms large pores and shows considerable cytotoxicity. However, HPA3P, in which a proline (Pro) is substituted for glutamic acid (Glu) at position 9 of HPA3, shows markedly less cytotoxicity. This may be attributable to the presence of a Pro-kink into middle of the HPA3P structure within the membrane environment. Unfortunately, HPA3P is not an effective antibacterial agent in vivo. We therefore designed a helix-PXXP-helix structure (HPA3P2), in which Pro was substituted for the Glu and phenylalanine (Phe) at positions 9 and 12 of HPA3, yielding a molecule with a flexible central hinge. As compared to HPA3P, HPA3P3 exhibited dramatically increased antibacterial activity in vivo. ICR mice infected with clinically isolated multidrug-resistant Pseudomonas aeruginosa showed 100% survival when administered one 0.5-mg/kg dose of HPA3P2 or three 0.1-mg/kg doses of HPA3P2. Moreover, in a mouse model of septic shock induced by P. aeruginosa LPS, HPA3P2 reduced production of pro-inflammatory mediators and correspondingly reduced lung (alveolar) and liver tissue damage. The changes in HPA3 behavior with the introduction of Pro likely reflects alterations of the mechanism of action: i) HPA3 forms pores in the bacterial cell membranes, ii) HPA3P permeates the cell membranes and binds to intracellular RNA and DNA, and iii) HPA3P2 acts on the outer cellular membrane component LPS. Collectively, these results suggest HPA3P2 has the potential to be an effective antibiotic for use against multidrug-resistant bacterial strains.

Nigrina b: una proteína inactivadora de ribosomas no tóxica del saúco. Utilidad farmacéutica en la construcción de inmunotoxinas y conjugados para la terapia del cáncer* / Nigrin b: a ribosome-inactivating protein from elder. Pharmaceutical utility in the construction of inmmunotoxins and conjugates for cancer therapy

El saúco posee una colección de proteínas inactivadoras de ribosomas, en particular las nigrinas, que parecen ser responsables de su toxicidad. La nigrina b (corteza) posee isoformas en frutos (nigrina f) y hojas (nigrina l) que se encuentran a mayor concentración en las fases iniciales del desarrollo. Nigrina b es 103-104 veces menos tóxica que la ricina, una proteína inactivadora de ribosomas relacionada estructuralmente con la nigrina b y extremadamente tóxica presente en Ricinus communis L., que se utiliza para la construcción de inmunotoxinas para la terapia del cáncer. Nigrina b se internaliza en las células superiores por una vía intracelular distinta a la de la ricina independiente de temperatura y de brefeldina A. La administración de dosis letales de nigrina b produce lesiones intestinales irreversibles específicas por destrucción de las criptas y desaparición del epitelio intestinal lo que ocasiona hemorragias intestinales letales. Los datos sobre estructura primaria de las nigrinas indican que la diferencia de toxicidad con la ricina se basa en cambios de aminoácidos clave en los dominios de fijación de galactosa en las cadenas B de ambas proteínas. Esta característica de la nigrina b es de enorme utilidad en la construcción de los denominados "proyectiles mágicos" o fármacos inteligentes capaces de interaccionar y destruir blancos específicos. Como ejemplos de dichos proyectiles mágicos se han construido conjugados transferrina- nigrina b/ebulina l que han mostrado ser efectivos contra células cancerosas que sobreexpresan el receptor de transferrina y una inmunotoxina antitumoral contra el CD105 de ratón que identifica a la células CD105+ y las destruye de manera selectiva tanto in vitro como in vivo. Ello convierte a la nigrina b en una herramienta útil en la inmunotoxiterapia del cáncer (AU)

Identity of the N-terminal sequences of the three A chains of mistletoe (Viscum album L.) lectins: homology with ricin-like plant toxins and single-chain ribosome-inhibiting proteins.

Mistletoe lectin (ML) I increases the production of cytokines by mononuclear cells and has been proposed as a useful biological response modifier in the treatment of cancer. Two other lectins, ML II and ML III, have been identified in mistletoe. We report that the N-terminal sequences of the three A chains of ML I, ML II and ML III are identical, and have interesting homology with the N-terminal sequences of the A chain of ricin-like toxins and of single-chain ribosome-inhibiting proteins. In addition, the three mistletoe lectins inhibit the growth of the human tumor cell line Molt 4, ML III being the most potent. followed by ML II and ML I. This inhibition is suppressed by addition of rabbit anti-ML I antibodies to the cultured cells. The data obtained suggest that the three lectins have amino acid sequences which show extensive homology and exert very similar biological effects. They may be derived from the same precursor.