Sucrose Gradient Ultracentrifugation Research Articles

Serological characterisation of Lagovirus virus-like particles originating from native and mutated VP60 of rabbit haemorrhagic disease virus 2 and European brown hare syndrome virus

Abstract Introduction Since lagoviruses cannot be cultivated in vitro, using expression systems is an alternative and promising way of producing diagnostic viral antigens. It opens up their use as active immunogens for vaccine production. Material and Methods Virus-like particles (VLPs) were produced in a baculovirus expression system in Spodoptera frugiperda 9 (Sf9) insect cells based on wild-type and mutated variants of the virus capsid VP60 protein from a Polish strain of European brown hare syndrome virus (EBHSV) and wild-type and mutated versions of this protein from a Polish strain of rabbit haemorrhagic disease virus 2 (RHDV2). The mutations were the substitution of an arginylglycylaspartic acid (Arg-Gly-Asp/RGD) motif in the P2 subdomain and, in the S or P2 domain, the substitution of three lysines. The VLPs were purified with sucrose gradient ultracentrifugation. Results Protein production was confirmed by Western blot analysis using rabbit or hare sera and ELISA tests with different types of monoclonal antibody. The haemagglutination properties of some VLPs were also evaluated. Electron microscopy of wild-type EBHSV, wild-type RHDV2 and the four VP60 variants produced in this experiment revealed the formation of characteristic VLP structures. Conclusion For the first time, mutated VLPs of RHDV2 with an RGD motif in the VP60 sequence were obtained, which could potentially be used to deliver cargo to eukaryotic cells. Virus-like particles based on the VP60 proteins of EBHSV and RHDV with a three-lysine substitution in the S or P2 domains were also obtained. Potential exists for VLPs of EBHSV and RHDV2 as vaccine candidates.

Comprehensive characterization of human brain-derived extracellular vesicles using multiple isolation methods: Implications for diagnostic and therapeutic applications.

Extracellular vesicles (EVs) have emerged as critical mediators of intercellular communication and promising biomarkers and therapeutics in the central nervous system (CNS). Human brain-derived EVs (BDEVs) provide a comprehensive snapshot of physiological changes in the brain's environment, however, the isolation of BDEVs and the comparison of different methods for this purpose have not been fully investigated. In this study, we compared the yield, morphology, subtypes and protein cargo composition of EVs isolated from the temporal cortex of aged human brains using three established separation methods: size-exclusion chromatography (SEC), phosphatidylserine affinity capture (MagE) and sucrose gradient ultracentrifugation (SG-UC). Our results showed that SG-UC method provided the highest yield and collected larger EVs compared to SEC and MagE methods as assessed by transmission electron microscopy and nanoparticle tracking analysis (NTA). Quantitative tandem mass-tag (TMT) mass spectrometry analysis of EV samples from three different isolation methods identified a total of 1158 proteins, with SG-UC showing the best enrichment of common EV proteins with less contamination of non-EV proteins. In addition, SG-UC samples were enriched in proteins associated with ATP activity and CNS maintenance, and were abundant in neuronal and oligodendrocytic molecules. In contrast, MagE samples were more enriched in molecules related to lipoproteins, cell-substrate junction and microglia, whereas SEC samples were highly enriched in molecules related to extracellular matrix, Alzheimer's disease and astrocytes. Finally, we validated the proteomic results by performing single-particle analysis using the super-resolution microscopy and flow cytometry. Overall, our findings demonstrate the differences in yield, size, enrichment of EV cargo molecules and single EV assay by different isolation methods, suggesting that the choice of isolation method will have significant impact on the downstream analysis and protein discovery.

Oligomerization and Adjuvant Activity of Peptides Derived from the VirB4-like ATPase of Clostridioides difficile.

In a previous study, we demonstrated that the Clostridioides difficile VirB4-like ATPase forms oligomers in vitro. In the current investigation, to study the observed phenomenon in more detail, we prepared a library of VirB4-derived peptides (delVirB4s) fused to a carrier maltose-binding protein (MBP). Using gel chromatography and polyacrylamide gel electrophoresis, we found a set of overlapping fragments that contribute most significantly to protein aggregation, which were represented as water-soluble oligomers with molecular masses ranging from ~300 kD to several megadaltons. Membrane filtration experiments, sucrose gradient ultracentrifugation, and dynamic light scattering measurements indicated the size of the soluble complex to be 15-100 nm. It was sufficiently stable to withstand treatment with 1 M urea; however, it dissociated in a 6 M urea solution. As shown by the changes in GFP fluorescence and the circular dichroism spectra, the attachment of the delVirB4 peptide significantly altered the structure of the partner MBP. The immunization of mice with the hybrid consisting of the selected VirB4-derived peptide and MBP, GST, or GFP resulted in increased production of specific antibodies compared to the peptide-free carrier proteins, suggesting significant adjuvant activity of the VirB4 fragment. This feature could be useful for the development of new vaccines, especially in the case of "weak" antigens that are unable to elicit a strong immune response by themselves.

Abstract 6259: Novel inhibitors of StAR related lipid transfer protein 5 inhibitors target plasma membrane lipid raft growth signaling

Abstract Background: Treatment of colorectal cancer (CRC) presents a major challenge due to chemotherapeutic resistance and recurrence, which are attributed to the presence of cancer stem cells (CSCs). Cholesterol metabolism is a critical regulator of CSCs. Lipid Rafts are cholesterol-rich microdomains in the plasma membrane (PM) that anchor various growth receptors tyrosine kinases (RTKs). STARD5 is a novel protein that helps transport cholesterol from the endoplasmic reticulum to the PM. We have identified specific small molecule inhibitors of the STARD5 which disrupt the lipid rafts, resulting in the inhibition of growth signaling pathways. Methods and Results: In-silico modeling studies using STARD5 crystal structure revealed 8 putative inhibitors (SD5i (A-H)). In vitro binding studies to determine secondary structural changes using circular dichroism spectroscopy revealed SD5iA and SD5iB as excellent STARD5 ligands. Both SD5iA and SD5iB caused a potent inhibition (IC50 0.3 -18 µM) of CSC-enriched 3D spheroids in 9 CRC cell lines representative of 3 consensus molecular subtypes. However, SD5iA but not SD5iB showed toxicity towards normal murine intestinal organoids at effective doses against CRC CSCs. Also, SD5iB enhanced cytotoxicity by oxaliplatin and panitumumab against CRC CSCs in HT-29 spheroids. Hence, SD5iB was advanced into in vivo studies. SD5iB caused no discernible toxicity in doses as high as 100 mg/kg in NCr nude mice at gross, serum chemistry, organ viability, or intestinal stem cell level (organoid formation). Subsequently, we generated s.c. xenograft using dual CSC marker sorted HT-29 cells and enriched them further with chemotherapy (5-fluorouracil and oxaliplatin weekly x 3) treatment followed by randomization to vehicle or SD5iB (100mg/kg i.p. 3/wk x 3 wks.). We observed xenograft growth inhibition and LGR5 expression in SD5iB-treated animals compared to vehicle controls. Mechanistically, we observed a decrease in free accessible PM cholesterol by SD5i measured by fluorescently tagged cholesterol-binding bacterial protein fALOD4 in HT-29 cells. Moreover, lipid raft staining (cholera toxin B staining) was significantly reduced in SD5iB-treated HT-29 cells compared to vehicle control using immunofluorescence. Further, lipid raft isolation was done by sucrose gradient ultracentrifugation to study the changes in the phosphorylation of RTKs SD5iB treatment in HT-29 spheroids resulted in decreased phosphorylation of EGFR, IGF1Rβ, and FGFR1 in isolated lipid rafts. Consequently, we observed a robust inhibition of ERK1/2 activation following SD5iB treatment compared to vehicle control. Conclusion: SD5i shows anti-CSC activity by targeting lipid-raft-driven growth factor signaling. Hence, STARD5 inhibitors represent a novel class of targeted therapy that will enhance the efficacy of FDA-approved therapies in CRC. Citation Format: Shoja Hajira, Chetna Sharon, Daniel RodriguezAgudo, Rio Boothelllo, Glenn Kellogg, Gregorio Gil, William Pandak, Bhaumik B. Patel. Novel inhibitors of StAR related lipid transfer protein 5 inhibitors target plasma membrane lipid raft growth signaling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6259.

Methods for analyzing positive cardiac troponin assay interference

ObjectivesThe cardiac damage biomarkers cardiac troponin T (cTnT) and troponin I (cTnI) are used to identify patients with myocardial infarction (MI). To make the correct clinical decisions it is important to identify false positive results due to troponin assay interference. Often interferences are caused by high-molecular weight immunocomplexes called macrotroponin that may result in false troponin elevations because of delayed troponin clearance, or heterophilic antibodies that crosslink troponin assay antibodies and generate troponin-independent signals. Design & MethodsWe describe and compare four methods for cTnI assay interference analysis using a protein G spin column method, gel filtration chromatography and two versions of a sucrose gradient ultracentrifugation for cTnI assay interference analysis on five patients with confirmed cTnI interference and one MI patient without cTnI interference from our troponin interference referral center. ResultsThe protein G spin column method had a high between run variability but was still able to identify all five patients with cTnI interference. The sucrose gradient ultracentrifugation methods and the gel filtration method had simlar performancec and correctly identified the immunocomplexes that caused the cTnI interference. ConclusionsOur experience is that these methods are sufficient to safely confirm or exclude positive cTnI assay interference.

Probing regulatory mechanisms of the de novo DNA methyltransferase DNMT3A.

DNA methyltransferase 3A (DNMT3A) is one of the two human de novo DNA methyltransferases responsible for establishing proper DNA methylation during development. Beyond this role, DNMT3A is frequently mutated in hematopoietic disorders, including clonal hematopoiesis and acute myeloid leukemia (AML). One hotspot mutation, R882H, likely causes a dominant loss-of-function phenotype, but its mechanism of doing so remains controversial. DNMT3A is typically found as a tetramer and R882 is found at one of its two oligomerization interfaces. However, it is debated in the field whether this mutation perturbs the multimeric state of DNMT3A, and if so whether it breaks up tetramers or promotes oligomerization. We aim to determine how the R882H mutation causes a dominant negative phenotype and to describe new mechanisms of DNMT3A regulation that might allow for reactivation of the mutant protein. Towards this goal, we have performed in vitro biochemical assays that demonstrate that DNMT3AR882H is more likely to form macro-oligomers than WT DNMT3A. We have also shown that this phenomenon holds true in cell lysate through sucrose gradient ultracentrifugation. In addition, we have performed a base editing mutational scan of DNMT3A in cells with WT DNMT3A as well as in cells harboring a heterozygous DNMT3AR882H mutation and have identified mutations to DNMT3A that boost activity with and without the R882H mutation. By further characterizing these mutants and DNMT3AR882H through crosslinking mass spectrometry, biochemical assays, and reporter-based activity assays in cells, we hope to elucidate novel mechanisms of DNMT3A regulation that could be exploited for the treatment of hematopoietic disorders.

New role of gramicidin A in RIG-I-like receptors-mediated IFN signalling.

The pattern recognition receptors (PRRs) sense exogenous molecular patterns most commonly derived from invading pathogens, to active the interferon (IFN) signalling. In the cytoplasm, the viral double-stranded RNAs (dsRNAs) are sensed by retinoic acid-inducible gene I (RIG-I) or melanoma differentiation-associated protein 5 (MDA5), depending on the length and chemical properties. Through the binding and oligomerizing onto the RNAs, they form filament to initiate the signalling cascade. Regulation of these receptors' activities are essential for manipulating the strength of IFN signalling. Here, through the virtual screening of chemical reagents using the published MDA5-dsRNA complex structure (PDB: 4GL2), we identified an antibiotic, gramicidin A as a stimulator that enhanced MDA5-mediated IFN signalling. Cytotoxic assay and IFN signalling assay suggested that disruption of lipid membrane, which is a well-defined mechanism of gramicidin A to perform its action, was dispensable in this process. Sucrose gradient ultracentrifugation assay showed that the gramicidin A treatment enhanced MDA5 oligomerization status in the presence of dsRNA. Our work implicated a new role of gramicidin A in innate immunity and presented a new tool to manipulate MDA5 activity.

Ribosomal RNA-based epitranscriptomic regulation of chondrocyte translation and proteome in osteoarthritis

Osteoarthritis-related cartilage extracellular matrix remodelling is dependent on changes in chondrocyte protein expression. Yet, the role of ribosomes in chondrocyte translation regulation is unknown. In this exploratory study, we investigated ribosomal RNA (rRNA) epitranscriptomic-based ribosome heterogeneity in human articular chondrocytes and its relevance for osteoarthritis. Sequencing-based rRNA 2'-O-methylation profiling analysis (RiboMethSeq) was performed on non-OA primary human articular chondrocytes (n=5) exposed for 14 days to osteoarthritic synovial fluid (14 donors, pooled, 20% v/v). The SW1353 SNORD71 KO cell pool was generated using LentiCRISPRv2/Cas9. The mode of translation initiation and fidelity were determined by dual-luciferase reporters. The cellular proteome was analysed by LC-MS/MS and collagen type I protein expression was evaluated by immunoblotting. Loading of COL1A1 mRNA into polysomes was determined by sucrose gradient ultracentrifugation and fractionation. We discovered that osteoarthritic synovial fluid instigates site-specific changes in the rRNA 2'-O-me profile of primary human articular chondrocytes. We identified five sites with differential 2'-O-me levels. The 2'-O-me status of 5.8S-U14 (one of identified differential 2'-O-me sites; decreased by 7.7%, 95% CI [0.9% to 14.5%]) was targeted by depleting the level of its guide snoRNA SNORD71 (50% decrease, 95% CI [33% to 64%]). This resulted in an altered ribosome translation modus (e.g CrPV IRES, FC 3, 95% CI [2.2 to 4.1]) and promoted translation of COL1A1 mRNA which led to increased levels of COL1A1 protein (FC 1.7, 95% CI [1.3 to 2.0]). Our data identify a novel concept suggesting that articular chondrocytes employ rRNA epitranscriptomic mechanisms in osteoarthritis development.

Structural Analyses of the Glycolipids in Lipid Rafts.

Lipid rafts are usually isolated from cells or tissues using sucrose gradient ultracentrifugation in the presence of detergents such as Triton X-100 at 4 °C. Although detergents should be removed for further structural characterization following fractionation, these compounds are often difficult to completely remove, especially from the glycolipids. In this chapter, we describe a novel method for the fast and convenient removal of detergents from lipid raft glycolipids following fraction and describe the application of this method.

Separating Inner and Outer Membranes of Escherichia coli by EDTA-free Sucrose Gradient Centrifugation.

The envelope of Gram-negative bacteria consists of an outer membrane (OM), a peptidoglycan cell wall, and an inner membrane (IM). The OM and IM have different components of proteins and lipids. Separating the IM and OM is a basic biochemical procedure to further study lipids and membrane proteins in different locations. Sucrose gradient ultracentrifugation of lysozyme/EDTA-treated total membrane is the most widely used method to separate the IM and OM of Gram-negative bacteria. However, EDTA is often harmful to protein structure and function. Here, we describe a relatively simple sucrose gradient ultracentrifugation method to separate the IM and OM of Escherichia coli. In this method, the cells are broken by a high-pressure microfluidizer, and the total cell membrane is collected by ultracentrifugation. The IM and OM are then separated on a sucrose gradient. Because EDTA is not used, this method is beneficial for subsequent membrane protein purification and functional study.

Ginseng-derived exosome-like nanovesicles extracted by sucrose gradient ultracentrifugation to inhibit osteoclast differentiation.

Plant-derived extracellular nanovesicles contain RNA and proteins with unique and diverse pharmacological mechanisms. The extracellular nanovesicles encapsulating plant extracts resemble exosomes as they have a round, lipid bilayer morphology. Ginseng is anti-inflammatory, anti-cancer, immunostimulant, and osteogenic/anti-osteoporotic. Here, we confirmed that ginseng-derived extracellular nanovesicles (GDNs) inhibit osteoclast differentiation and elucidated the associated molecular mechanisms. We isolated GDNs by centrifugation with a sucrose gradient. We measured their dynamic light scattering and zeta potentials and examined their morphology by transmission electron microscopy. We used bone marrow-derived macrophages (BMMs) to determine the potential cytotoxicity of GDNs and establish their ability to inhibit osteoclast differentiation. The GDNs treatment maintained high BMM viability and proliferation whilst impeding osteoclastogenesis. Tartrate-resistant acid phosphatase and F-actin staining revealed that GDNs at concentrations >1 μg mL-1 strongly hindered osteoclast differentiation. Moreover, they substantially suppressed the RANKL-induced IκBα, c-JUN n-terminal kinase, and extracellular signal-regulated kinase signaling pathways and the genes regulating osteoclast maturation. The GDNs contained elevated proportions of Rb1 and Rg1 ginsenosides and were more effective than either of them alone or in combination at inhibiting osteoclast differentiation. In vivo bone analysis via microcomputerized tomography, bone volume/total volume ratios, and bone mineral density and bone cavity measurements demonstrated the inhibitory effect of GDNs against osteoclast differentiation in lipopolysaccharide-induced bone resorption mouse models. The results of this work suggest that GDNs are anti-osteoporotic by inhibiting osteoclast differentiation and are, therefore, promising for use in the clinical prevention and treatment of bone loss diseases.

Antigenicity and immunogenicity of chikungunya virus-like particles from mosquito cells.

The spread of chikungunya virus (CHIKV) is reaching pandemic levels, and vaccines and antivirals to control CHIKV infection have yet to be approved. Virus-like particles (VLPs), a self-assembled native multi-subunit protein structure, could potentially be used as an antigen for serological detection and vaccine development. In the current study, we describe the production of novel CHIKV VLPs from mosquitoes using a Baculovirus/Mosquito (BacMos) system in a simple Biosafety Level-2 laboratory. Substantial envelope and capsid protein secretions were detected in culture medium. Co-fractionation of CHIKV E2, E1, and capsid proteins via sucrose gradient ultracentrifugation provided evidence of VLP formation. Transmission electron microscopy and dynamic light scattering analysis revealed the formation of VLPs in the form of spherical particles with a diameter of roughly 40nm in transduced cells and culture medium. VLP-based IgM capture ELISA in CHIKV patient sera revealed native epitopes on the VLPs. These non-purified VLPs were shown to act as an antigen in CHIKV-specific IgM capture ELISA. The immunization of CHIKV-VLPs alone in mice induced a balance CHIKV-specific IgG2a/IgG1 antibodies and neutralized antibody responses. The study provides support for the hypothesis that mosquito cell-derived CHIKV VLPs could serve as a novel antigen for serological detection and the development of vaccines against CHIKV infection. KEY POINTS: • CHIKV VLPs secreted from BacMos-CHIKV 26S-transduced mosquito cell. • This CHIKV VLPs potentially serve as an alternative capture antigen for MAC-ELISA. • Unadjuvanted CHIK VLPs induce CHIKV-specific IgG and NT responses in mice.

Investigation of Photosystem II Functional Size in Higher Plants under Physiological and Stress Conditions Using Radiation Target Analysis and Sucrose Gradient Ultracentrifugation.

The photosystem II (PSII) reaction centre is the critical supramolecular pigment–protein complex in the chloroplast which catalyses the light-induced transfer of electrons from water to plastoquinone. Structural studies have demonstrated the existence of an oligomeric PSII. We carried out radiation inactivation target analysis (RTA), together with sucrose gradient ultracentrifugation (SGU) of PSII, to study the functional size of PSII in diverse plant species under physiological and stress conditions. Two PSII populations, made of dimeric and monomeric core particles, were revealed in Pisum sativum, Spinacea oleracea, Phaseulus vulgaris, Medicago sativa, Zea mais and Triticum durum. However, this core pattern was not ubiquitous in the higher plants since we found one monomeric core population in Vicia faba and a dimeric core in the Triticum durum yellow-green strain, respectively. The PSII functional sizes measured in the plant seedlings in vivo, as a decay of the maximum quantum yield of PSII for primary photochemistry, were in the range of 75–101 ± 18 kDa, 2 to 3 times lower than those determined in vitro. Two abiotic stresses, heat and drought, imposed individually on Pisum sativum, increased the content of the dimeric core in SGU and the minimum functional size determined by RTA in vivo. These data suggest that PSII can also function as a monomer in vivo, while under heat and drought stress conditions, the dimeric PSII structure is predominant.

Characterization of anti-proliferative and anti-oxidant effects of nano-sized vesicles from Brassica oleracea L. (Broccoli)

In this in vitro study, we test our hypothesis that Broccoli-derived vesicles (BDVs), combining the anti-oxidant properties of their components and the advantages of their structure, can influence the metabolic activity of different cancer cell lines. BDVs were isolated from homogenized fresh broccoli (Brassica oleracea L.) using a sucrose gradient ultracentrifugation method and were characterized in terms of physical properties, such as particle size, morphology, and surface charge by transmission electron microscopy (TEM) and laser doppler electrophoresis (LDE). Glucosinolates content was assessed by RPLC–ESI–MS analysis. Three different human cancer cell lines (colorectal adenocarcinoma Caco-2, lung adenocarcinoma NCI-H441 and neuroblastoma SHSY5Y) were evaluated for metabolic activity by the MTT assay, uptake by fluorescence and confocal microscopy, and anti-oxidant activity by a fluorimetric assay detecting intracellular reactive oxygen species (ROS). Three bands were obtained with average size measured by TEM based size distribution analysis of 52 nm (Band 1), 70 nm (Band 2), and 82 nm (Band 3). Glucobrassicin, glucoraphanin and neoglucobrassicin were found mostly concentrated in Band 1. BDVs affected the metabolic activity of different cancer cell lines in a dose dependent manner compared with untreated cells. Overall, Band 2 and 3 were more toxic than Band 1 irrespective of the cell lines. BDVs were taken up by cells in a dose- and time-dependent manner. Pre-incubation of cells with BDVs resulted in a significant decrease in ROS production in Caco-2 and NCI-H441 stimulated with hydrogen peroxide and SHSY5Y treated with 6-hydroxydopamine, with all three Bands. Our findings open to the possibility to find a novel “green” approach for cancer treatment, focused on using vesicles from broccoli, although a more in-depth characterization of bioactive molecules is warranted.

Separation and purification of highly infectious enterovirus A71 particles using a strong anion-exchange column

Virions produced from cell culture is the primary source for production of formalin-inactivated whole virus vaccines for enteroviruses. EV-A71 particles produced from culture system comprise two major types, the immature/empty (E)-particle and the mature/full (F)-particle, which both exhibit low isoelectric point (pI) values but have distinct differences in infectivity and immunogenicity. Although EV-A71 particles can conventionally be separated into E-particle and F-particle using sucrose gradient ultracentrifugation, this procedure is cumbersome and difficult to put into practice for vaccine production. Methods based on ion-exchange chromatography have been exploited to improve the purification efficacy; however, none of them are capable of separating the E- and F-particles efficiently. In this study, we aimed to develop an approach to isolate and purify the highly immunogenic mature EV-A71 particles. By applying a step gradient elution procedure, we successfully isolated the viral structure protein VP0-cleaved particles of EV-A71 from a mixture of cultured viral solution using the Q-membrane anion-exchange chromatography. The elution started with 0.1x phosphate buffered saline (PBS) solution while increasing the percentage of 1x PBS containing 1M NaCl in sequential steps. By this procedure, the VP0-cleaved mature particles and VP0-uncleaved immature particles of EV-A71 could be separated into different fractions in Q-membrane with gradually increased NaCl concentration in elution buffer. The purified VP0-cleaved particles were shown to have characteristics equivalent to those of the highly infectious F-particles of EV-A71. The overall recovery rate for the mature EV-A71 particles by Q-membrane is 56% and its purity was shown to be equivalent to those isolated by the sucrose gradient ultracentrifugation. Our approach provides a simple and efficient purification method for recovering mature, highly infectious virus particles from the EV-A71 culture bulk.

Isolation of fucoxanthin chlorophyll protein complexes of the centric diatom Thalassiosira pseudonana associated with the xanthophyll cycle enzyme diadinoxanthin de-epoxidase.

In the present study, low concentrations of the very mild detergent n-dodecyl-α-d-maltoside in conjunction with sucrose gradient ultracentrifugation were used to prepare fucoxanthin chlorophyll protein (FCP) complexes of the centric diatom Thalassiosira pseudonana. Two main FCP fractions were observed in the sucrose gradients, one in the upper part and one at high sucrose concentrations in the lower part of the gradient. The first fraction was dominated by the 18 kDa FCP protein band in SDS-gels. Since this fraction also contained other protein bands, it was designated as fraction enriched in FCP-A complex. The second fraction contained mainly the 21 kDa FCP band, which is typical for the FCP-B complex. Determination of the lipid composition showed that both FCP fractions contained monogalactosyl diacylglycerol as the main lipid followed by the second galactolipid of the thylakoid membrane, namely digalactosyl diacylglycerol. The negatively charged lipids sulfoquinovosyl diacylglycerol and phosphatidyl glycerol were also present in both fractions in pronounced concentrations. With respect to the pigment composition, the fraction enriched in FCP-A contained a higher amount of the xanthophyll cycle pigments diadinoxanthin (DD) and diatoxanthin (Dt), whereas the FCP-B fraction was characterized by a lower ratio of xanthophyll cycle pigments to the light-harvesting pigment fucoxanthin. Protein analysis by mass spectrometry revealed that in both FCP fractions the xanthophyll cycle enzyme diadinoxanthin de-epoxidase (DDE) was present. In addition, the analysis showed an enrichment of DDE in the fraction enriched in FCP-A but only a very low amount of DDE in the FCP-B fraction. In-vitro de-epoxidation assays, employing the isolated FCP complexes, were characterized by an inefficient conversion of DD to Dt. However, in line with the heterogeneous DDE distribution, the fraction enriched in FCP-A showed a more pronounced DD de-epoxidation compared with the FCP-B.

Targeting CRABP-II overcomes pancreatic cancer drug resistance by reversing lipid raft cholesterol accumulation and AKT survival signaling

BackgroundResistance to standard therapy is a major reason for the poor prognosis of pancreatic ductal adenocarcinoma (PDAC). Developing novel therapy to overcome PDAC drug-resistance is urgently needed. CRABP-II was highly expressed in all PDAC but not expressed in normal pancreatic tissues and chronic pancreatitis. CRABP-II was shown to promote PDAC migration and metastasis while its potential role in promoting PDAC drug-resistance was not known.MethodsA paired cohort of human primary and relapsing PDAC tissues was assessed for CRABP-II expression by immunohistochemistry. CRISPR/cas9 gene editing was used to establish CRABP-II knockout cell lines and MTT assays were performed to assess gemcitabine sensitivity in vitro. Cleaved caspase-3/PARP blots and Annexin V staining were conducted to detect cell apoptosis. Gene expression microarray, Q-PCR, western blots, Co-IP and RNA-IP were used to study the molecular function of CRABP-II. Sucrose gradient ultracentrifugation was applied to isolate lipid rafts and LC–MS-MS was used to assess cholesterol content. Both subcutaneous CDX models and orthotopic PDX models were established to examine the efficacy of SNIPER-11 and the synergistic effect between SNIPER-11 and gemcitabine in vivo.ResultsA higher expression of CRABP-II was found in relapsing PDAC tissue and was associated with poor prognosis. Gemcitabine-resistant cell lines exhibited increased level of CRABP-II, while CRABP-II knockout resensitized PDAC cells to gemcitabine. Mechanistically, aberrant expression of CRABP-II increased the stability of SREBP-1c mRNA through cooperation with HuR and upregulated the downstream genes of SREBP-1c to favor cholesterol uptake and accumulation in lipid rafts. Increased lipid raft cholesterol accumulation facilitated ATK survival signaling and PDAC drug resistance. The small compound SNIPER-11 treatment effectively induced CRABP-II protein degradation, induced apoptosis, and suppressed tumor growth. Combination of SNIPER-11 and gemcitabine significantly reduced the lipid raft cholesterol content in CDX/PDX and profoundly inhibited tumor progression.ConclusionsThese findings identified CRABP-II as a novel regulator of cholesterol metabolism and suggested that CRABP-II is a selective target for overcoming PDAC drug resistance.

Invariable Ribosome Stoichiometry During Murine Erythroid Differentiation: Implications for Understanding Ribosomopathies.

Heterogeneity of the main ribosomal composition represents an emerging, yet debatable, mechanism of gene expression regulation with a purported role in ribosomopathies, a group of disorders caused by mutations in ribosomal protein genes (RPs). Ribosomopathies, mysteriously relate with tissue-specific symptoms (mainly anemia and cancer predisposition), despite the ubiquitous expression and necessity of the associated RPs. An outstanding question that may shed light into disease pathogenicity and provide potential pharmacological interventions, is whether and how the ribosomal composition is modified during, the highly affected by RP mutations, process of erythroid differentiation. To address this issue, we analyzed ribosome stoichiometry using an established model of erythroid differentiation, through sucrose gradient ultracentrifugation and quantitative proteomics. We found that differentiation associates with an extensive reprogramming of the overall ribosomal levels, characterized by an increase in monosomes and a decrease in polysomes. However, by calculating a stoichiometry score for each independent ribosomal protein, we found that the main ribosomal architecture remained invariable between immature and differentiated cells. In total, none of the 78 Ribosomal Proteins (RPs- 74 core RPs, Rack1, Fau and 2 paralogs) detected was statistically different between the samples. This data was further verified through antibody-mediated quantification of 6 representative RPs. Moreover, bioinformatic analysis of whole cell proteomic data derived out of 4 additional models of erythropoiesis revealed that RPs were co-regulated across these cell types, too. In conclusion, ribosomes maintain an invariant protein stoichiometry during differentiation, thus excluding ribosome heterogeneity from a potential mechanism of toxicity in ribosomopathies and other erythroid disorders.

Centrosome as Center for Proteolytic Activity and Dysfunctions Associated with Pathogenesis of Human Disease.

Among the multiple and intriguing roles of centrosomes in cellular functions is the ubiquitin-proteasome-mediated protein degradation. It has been shown that proteasomes are concentrated at the mammalian centrosome which led to further studies to view the centrosome as a proteolytic center (Wojcik et al. 1996; Wigley et al. 1999; reviewed in Badano et al. 2005). Proteasomal components that are concentrated around the centrosome include ubiquitin, the 20S and 19S subunits of the proteasome, as well as the E3 enzyme parkin. These proteasomal components colocalize with the centrosomal marker γ-tubulin and co-purify with γ-tubulin in the centrosomal fractions after sucrose-gradient ultracentrifugation (Wigley et al. 1999). The localization, accumulation, and concentration of proteasomal components around centrosomes appear to be microtubule independent which has been shown experimentally by inhibiting microtubule functions. When intracellular levels of misfolded proteins were experimentally increased by either proteasome inhibition with drugs such as lactacystin, or by overexpression of misfolded mutant proteins, the centrosome-associated proteasome network became expanded and proteolytic components were recruited from the cytosol without involvement of microtubules. These studies revealed a critical role of centrosomes in the organization and subcellular localization of proteasomes (Wigley et al. 1999; Fabunmi et al. 2000).

Isolation and Characterization of Intact Phycobilisome in Cyanobacteria.

In cyanobacteria, phycobilisome is a vital antenna protein complex that harvests light and transfers energy to photosystem I and II for photochemistry. Studying the structure and composition of phycobilisome is of great interest to scientists because it reveals the evolution and divergence of photosynthesis in cyanobacteria. This protocol provides a detailed and optimized method to break cyanobacterial cells at low cost by a bead-beater efficiently. The intact phycobilisome can then be isolated from the cell extract by sucrose gradient ultracentrifugation. This method has demonstrated being suitable for both model and non-model cyanobacteria with different cell types. A step-by-step procedure is also provided to confirm the integrity and property of phycobiliproteins by 77K fluorescence spectroscopy and SDS-PAGE stained by zinc sulfate and Coomassie Blue. The isolated phycobilisome can also be subjected to further structural and compositional analyses. Overall, this protocol provides a helpful starting guide that allows researchers unfamiliar with cyanobacteria to quickly isolate and characterize intact phycobilisome.