Functional Sites of Ribosome Modulation Factor (RMF) Involved in the Formation of 100S Ribosome

One of the important cellular events in all organisms is protein synthesis, which is catalyzed by ribosomes. The ribosomal activity is dependent on the environmental situation of the cell. Bacteria form 100S ribosomes, lacking translational activity, to survive under stress conditions such as nutrient starvation. The 100S ribosome is a dimer of two 70S ribosomes bridged through the 30S subunits. In some pathogens of gammaproteobacteria, such as Escherichia coli, Yersinia pestis, and Vibrio cholerae, the key factor for ribosomal dimerization is the small protein, ribosome modulation factor (RMF). When ribosomal dimerization by RMF is impaired, long-term bacterial survival is abolished. This shows that the interconversion system between active 70S ribosomes and inactive 100S ribosomes is an important survival strategy for bacteria. According to the results of several structural analyses, RMF does not directly connect two ribosomes, but binds to them and changes the conformation of their 30S subunits, thus promoting ribosomal dimerization. In this study, conserved RMF amino acids among 50 bacteria were selectively altered by mutagenesis to identify the residues involved in ribosome binding and dimerization. The activities of mutant RMF for ribosome binding and ribosome dimerization were measured using the sucrose density gradient centrifugation (SDGC) and western blotting methods. As a result, some essential amino acids of RMF for the ribosomal binding and dimerization were elucidated. Since the induction of RMF expression inhibits bacterial growth, the data on this protein could serve as information for the development of antibiotic or bacteriostatic agents.

Intestine epithelial cell-derived extracellular vesicles alleviate inflammation induced by Clostridioides difficile TcdB through the activity of TGF-β1

Objective: Clostridioides difficile infection (CDI) has been primarily associated with the toxin B (TcdB), which can activate the intestinal immune system and lead to pathological damage. Even though the biological functions of intestine epithelial cell-derived extracellular vesicles (I-Evs) have been well documented, the role of I-Evs in the process of CDI is still unknown. Methods: I-Evs were isolated from mouse intestine tissues by ultracentrifugation protocol, identified by electron microscopy, nanoparticle tracking, sucrose density gradient centrifugation, and western blotting. Intestinal pathological damage was measured after intraperitoneal injection of TcdB into mice. Results: We isolated I-Evs ranging from 100–200 nm in mean diameter, with a density of 1.09-1.17 g/mL. These I-Evs expressed the extracellular vesicle-associated specific surface markers, CD63 and TSG101. In vitro, 50 µg I-Evs decreased the expression of IL-6, TNF-a, IL-1β, and IL-22 induced by 0.8 ng/mL C. difficile TcdB, and increased expression of TGF-b1. In vivo, I-Evs also promoted regulatory T cell induction, which improved the survival rate of mice up to 80% relative to C. difficile TcdB mice, dependent on the TGF-b1 signalling pathway. Conclusion: As an emerging immunotherapy, I-Evs can reduce the intraperitoneal infection induced by C. difficile TcdB and improve survival in mice.

Prion protein oligomers cause neuronal cytoskeletal damage in rapidly progressive Alzheimer’s disease

BackgroundHigh-density oligomers of the prion protein (HDPs) have previously been identified in brain tissues of patients with rapidly progressive Alzheimer’s disease (rpAD). The current investigation aims at identifying interacting partners of HDPs in the rpAD brains to unravel the pathological involvement of HDPs in the rapid progression.MethodsHDPs from the frontal cortex tissues of rpAD brains were isolated using sucrose density gradient centrifugation. Proteins interacting with HDPs were identified by co-immunoprecipitation coupled with mass spectrometry. Further verifications were carried out using proteomic tools, immunoblotting, and confocal laser scanning microscopy.ResultsWe identified rpAD-specific HDP-interactors, including the growth arrest specific 2-like 2 protein (G2L2). Intriguingly, rpAD-specific disturbances were found in the localization of G2L2 and its associated proteins i.e., the end binding protein 1, α-tubulin, and β-actin.DiscussionThe results show the involvement of HDPs in the destabilization of the neuronal actin/tubulin infrastructure. We consider this disturbance to be a contributing factor for the rapid progression in rpAD.

Retinol binding protein 4 (Rbp4) mRNA translation in hepatocytes is enhanced by activation of mTORC1

Increased expression of the peptide hormone retinol-binding protein 4 (RBP4) has been implicated in the development of insulin resistance, type 2 diabetes, and visual dysfunction. Prior investigations of the mechanisms that influence RBP4 synthesis have focused solely on changes in mRNA abundance. Yet, the production of many secreted proteins is controlled at the level of mRNA translation, as it allows for a rapid and reversible change in expression. Herein, we evaluated Rbp4 mRNA translation using sucrose density gradient centrifugation. In the liver of fasted rodents, Rbp4 mRNA translation was low. In response to re-feeding, Rbp4 mRNA translation was enhanced and RBP4 levels in serum were increased. In H4IIE cells, refreshing culture medium promoted Rbp4 mRNA translation and expression of the protein. Rbp4 mRNA abundance was not increased by either experimental manipulation. Enhanced Rbp4 mRNA translation was associated with activation of the kinase mTORC1 and enhanced phosphorylation of the translational repressor 4E-BP1. In H4IIE cells, expression of a 4E-BP1 variant that is unable to be phosphorylated by mTORC1 or suppression of mTORC1 with rapamycin attenuated activity of a luciferase reporter encoding the Rbp4 mRNA 5′-untranslated region (UTR). Purine substitutions to disrupt a terminal oligopyrimidine (TOP)-like sequence in the Rbp4 5′-UTRprevented the suppressive effect of rapamycin on reporter activity. Rapamycin also prevented upregulation of Rbp4 mRNA translation in the liver, and reduced serum levels of RBP4 in response to feeding. Overall, the findings support a model in which nutrient-induced activation of mTORC1 up regulates Rbp4 mRNA translation to promote RBP4 synthesis.

The non-coding RNA BC200 associates with polysomes to positively regulate mRNA translation in tumour cells

BC200 is a non-coding RNA elevated in a broad spectrum of tumour cells that is critical for cell viability, invasion, and migration. Over-expression studies have implicated BC200 and the rodent analog BC1 as negative regulators of translation in both cell-based and in vitro translation assays. While consistent, these studies have not been confirmed in knock-down studies and direct evidence for this function is lacking. Herein, we have demonstrated that BC200 knock-down is correlated with a decrease in global translation rates. As this conflicts with the hypothesis that BC200 is a translational suppressor, we overexpressed BC200 by transfection of in vitro transcribed RNA and transient expression from transfected plasmids. In this context BC200 suppressed translation; however, an innate immune response confounded the data. To overcome this, breast cancer cells stably overexpressing BC200 and various control RNAs were developed by selection for genomic incorporation of a plasmid co-expressing BC200 and the neomycin resistance gene. Stable overexpression of BC200 was associated with elevated translation levels in pooled stable cell lines and isolated single-cell clones. Crosslinking sucrose density gradient centrifugation demonstrated an association of BC200 and its reported binding partners SRP9/14, CSDE1, DHX36 and PABPC1 with both ribosomal subunits and polysomal RNA, an association not previously observed due to the labile nature of the interactions. In summary, these data present a novel understanding of BC200 function as well as optimized methodology that has far reaching implications in the study of non-coding RNAs, particularly within the context of translational regulatory mechanisms.

Preparation and optimization of rapid and sensitive coagglutination test for detection of infectious pancreatic necrosis virus (IPNV)

The aim of this study is the development of a coagglutination test for rapid diagnosis of infectious pancreatic necrosis (IPN) virus which causes an acute infectious viral disease with high mortality in young salmonid fish. Serotypes Sp, Ab, WB of the IPNV were cultivated in BF-2 cells and purified by linear sucrose density gradient centrifugation. Hyperimmune sera against purified IPNV serotypes were collected from immunized guinea pigs. Coagglutination test conjugates were prepared by using sensitized S. aureus and hyperimmune sera. Cell culture derived salmonid pathogens such as infectious hematopoietic necrosis virus (IHNV), viral haemorrhagic septicaemia virus (VHSV) and epizootic hematopoietic necrosis virus (EHNV) were used to determine the specificity of the test as control. Upward comparison of the preferability for the rapid diagnosis was also carried out with ELISA and RT-PCR. The validation studies of newly developed coagglutination test were carried out in accordance with the Manual of Diagnostic Tests for Aquatic Animals. Consequently, determined detection limit of the test was approximately 105.25 TCID50/mL for the Sp and Ab serotypes and 107.75 TCID50/mL for the WB in laboratory conditions. The results indicated that newly developed coagglutination test was compatible with ELISA and RT-PCR in positive cell culture supernatants. It was concluded that this test would be an economic, rapid and reliable method for the diagnosis of IPN virus in cell culture.

Potent Protective Immune Responses to Senecavirus Induced by Virus-Like Particle Vaccine in Pigs

Senecavirus A (SVA) is the pathogen that has recently caused porcine idiopathic vesicular disease (PIVD). The clinical symptoms of PIVD are similar to those of acute foot-and-mouth disease and also can result in the death of newborn piglets, thus entailing economic losses. Vaccine immunization is the most effective way to prevent and control SVA. Among all SVA vaccines reported, only the SVA inactivated vaccine has been successfully developed. However, to ensure the elimination of this pathogen, safer and more effective vaccines are urgently required. A virus-like particles (VLPs)-based vaccine is probably the best alternative to inactivated vaccine. To develop an SVA VLPs vaccine and evaluate its immune effect, a prokaryotic expression system was used to produce SVA capsid protein and assemble VLPs. The VLPs were characterized by affinity chromatography, sucrose density gradient centrifugation, ZetaSizer and transmission electron microscopy. Meanwhile, the SVA CH-HB-2017 strain was used to infect pigs and to determine infection routes and dose. Experimental pigs were then immunized with the SVA VLPs vaccine emulsified in an ISA 201 adjuvant. The results showed that the VLPs vaccine induced neutralizing and specific antibodies at similar levels as an inactivated SVA vaccine after immunization. The level of INF-γ induced by the VLPs vaccine gradually decreased—similar to that of inactivated vaccine. These results indicated that VLPs vaccine may simultaneously cause both cellular and humoral immune responses. Importantly, after the challenge, the VLPs vaccine provided similar levels of protection as the inactivated SVA vaccine. In this study, we successfully obtained novel SVA VLPs and confirmed their highly immunogenicity, thus providing a superior candidate vaccine for defense and elimination of SVA, compared to the inactivated vaccine.

Derlin-3 Is Required for Changes in ERAD Complex Formation under ER Stress

Endoplasmic reticulum (ER)-associated protein degradation (ERAD) is a quality control system that induces the degradation of ER terminally misfolded proteins. The ERAD system consists of complexes of multiple ER membrane-associated and luminal proteins that function cooperatively. We aimed to reveal the role of Derlin-3 in the ERAD system using the liver, pancreas, and kidney obtained from different mouse genotypes. We performed coimmunoprecipitation and sucrose density gradient centrifugation to unravel the dynamic nature of ERAD complexes. We observed that Derlin-3 is exclusively expressed in the pancreas, and its deficiency leads to the destabilization of Herp and accumulation of ERAD substrates. Under normal conditions, Complex-1a predominantly contains Herp, Derlin-2, HRD1, and SEL1L, and under ER stress, Complex-1b contains Herp, Derlin-3 (instead of Derlin-2), HRD1, and SEL1L. Complex-2 is upregulated under ER stress and contains Derlin-1, Derlin-2, p97, and VIMP. Derlin-3 deficiency suppresses the transition of Derlin-2 from Complex-1a to Complex-2 under ER stress. In the pancreas, Derlin-3 deficiency blocks Derlin-2 transition. In conclusion, the composition of ERAD complexes is tissue-specific and changes in response to ER stress in a Derlin-3-dependent manner. Derlin-3 may play a key role in changing ERAD complex compositions to overcome ER stress.

Separation and Paired Proteome Profiling of Plant Chloroplast and Cytoplasmic Ribosomes

Conventional preparation methods of plant ribosomes fail to resolve non-translating chloroplast or cytoplasmic ribosome subunits from translating fractions. We established preparation of these ribosome complexes from Arabidopsis thaliana leaf, root, and seed tissues by optimized sucrose density gradient centrifugation of protease protected plant extracts. The method co-purified non-translating 30S and 40S ribosome subunits separated non-translating 50S from 60S subunits, and resolved assembled monosomes from low oligomeric polysomes. Combining ribosome fractionation with microfluidic rRNA analysis and proteomics, we characterized the rRNA and ribosomal protein (RP) composition. The identity of cytoplasmic and chloroplast ribosome complexes and the presence of ribosome biogenesis factors in the 60S-80S sedimentation interval were verified. In vivo cross-linking of leaf tissue stabilized ribosome biogenesis complexes, but induced polysome run-off. Omitting cross-linking, the established paired fractionation and proteome analysis monitored relative abundances of plant chloroplast and cytoplasmic ribosome fractions and enabled analysis of RP composition and ribosome associated proteins including transiently associated biogenesis factors.

A2E Distribution in RPE Granules in Human Eyes

A2E (N-retinylidene-N-retinylethanolamine) is a major fluorophore in the RPE (retinal pigment epithelium). To identify and characterize A2E-rich RPE lipofuscin, we fractionated RPE granules from human donor eyes into five fractions (F1–F5 in ascending order of density) by discontinuous sucrose density gradient centrifugation. The dry weight of each fraction was measured and A2E was quantified by liquid chromatography/mass spectrometry (LC/MS) using a synthetic A2E homolog as a standard. Autofluorescence emission was characterized by a customer-built spectro-fluorometer system. A significant A2E level was detected in every fraction, and the highest level was found in F1, a low-density fraction that makes up half of the total weight of all RPE granules, contains 67% of all A2E, and emits 75% of projected autofluorescence by all RPE granules. This group of RPE granules, not described previously, is therefore the most abundant RPE lipofuscin granule population. A progressive decrease in autofluorescence was observed from F2 to F4, whereas no autofluorescence emission was detected from the heavily pigmented F5. The identification of a novel and major RPE lipofuscin population could have significant implications in our understanding of A2E and lipofuscin in human RPE.