Combined treatment of human multiple myeloma cells with bortezomib and doxorubicin alters the interactome of 20S proteasomes

The proteasome is the key player in targeted degradation of cellular proteins and serves as a therapeutic target for treating several blood malignancies. Although in general, degradation of proteins via the proteasome requires their ubiquitination, a subset of proteins can be degraded independently of their ubiquitination by direct interaction with subunits of the 20S proteasome core. Thus, investigation of the proteasome-associated proteins may help identify novel targets of proteasome degradation and provide important insights into the mechanisms of malignant cell proteostasis. Here, using biochemical purification of proteasomes from multiple myeloma (MM) cells followed by mass-spectrometry we have uncovered 77 proteins in total that specifically interacted with the 20S proteasome via its PSMA3 subunit. Our GST pull-down assays followed by western blots validated the interactions identified by mass-spectrometry. Eleven proteins were confirmed to bind PSMA3 only upon apoptotic conditions induced by a combined treatment with the proteasome inhibitor, bortezomib, and genotoxic drug, doxorubicin. Nine of these eleven proteins contained bioinformatically predicted intrinsically disordered regions thus making them susceptible to ubiquitin-independent degradation. Importantly, among those proteins five interacted with the ubiquitin binding affinity matrix suggesting that these proteins may also be ubiquitinylated and hence degraded via the ubiquitin-dependent pathway. Collectively, these PSMA3-interacting proteins represent novel potential substrates for 20S proteasomes upon apoptosis. Furthermore, these data may shed light on the molecular mechanisms of cellular response to chemotherapy.

Abbreviations: BD: bortezomib/doxorubicin treatment; CDK: cyclin-dependent kinases; CHCA: α-cyanohydroxycinnamic acid; IDP: intrinsically disordered proteins; IDR: intrinsically disordered regions; IPG: immobilized pI gradient; MALDI TOF/TOF: matrix-assisted laser desorption/ionization time-of-flight tandem mass-spectrometry; MM: multiple myeloma; ODC: ornithine decarboxylase; PI: proteasomal inhibitors; PSMA: alpha-type 20S proteasome subunits; PTMs: post-translational modifications; SDS-PAGE: sodium dodecylsulphate polyacrylamide gel electrophoresis; UIP: ubiquitin-independent proteasomal proteolysis.     

Biosynthesis of polar steroids from the Far Eastern starfish Patiria (=Asterina) pectinifera. Cholesterol and cholesterol sulfate are converted into polyhydroxylated sterols and monoglycoside asterosaponin P1 in feeding experiments

For the first time, it is experimentally established that the dietary cholesterol and cholesterol sulfate are biosynthetic precursors of polyhydroxysteroids and related low molecular weight glycosides in starfishes. These deuterium labeled precursors were converted into partly deuterated 5α-cholestane-3β,6α,7α,8,15α,16β,26-heptaol, 5α-cholestane-3β,4β,6α,7α,8,15β,16β,26-octaol, and steroid monoside asterosaponin P₁ in result of feeding experiments on the Far Eastern starfish Patiria (=Asterina) pectinifera. The incorporations of deuterium were established by MS and NMR spectroscopy. Scheme of the first stages of biosynthesis of polar steroids in these animals was suggested on the basis of inclusion of three from six deuterium atoms and determination of their positions in biosynthetic products, when [2,2,3,4,4,6-²H₆]cholesterol 3-sulfate was used as precursor. It was also shown that labeled cholesterol is transformed into Δ⁷-cholesterol (lathosterol) in digestive organs and gonads of the starfish.     

Embryo structure,seed traits,and productivity of relict vine Aristolochia contorta (Aristolochiaceae)

The herbaceous vine Aristolochia contorta (Aristolochiaceae) is a rare plant with a fragmented area in East Asia. Confocal laser-scanning microscopy and stereomicroscopy were employed to examine the seed and embryo structure. This is the first research on embryo anatomy in Aristolochia. Shape and structure of this embryo may be phylogenetically significant. The seed structure of A. contorta is similar to that of other Aristolochia species. Considerable variation is found for productivity traits; the average seed set is rather low (26.7%). The studied seed and embryo traits of A. contorta are found to be variable, but may be potentially informative at the genus and species level. The restricted productivity and reduced population size threaten the continued survival of A. contorta and, perhaps, the butterfly that depends on it.     

Genome-wide study identifies two loci associated with lung function decline in mild to moderate COPD

Accelerated lung function decline is a key COPD phenotype; however, its genetic control remains largely unknown. We performed a genome-wide association study using the Illumina Human660W-Quad v.1_A BeadChip. Generalized estimation equations were used to assess genetic contributions to lung function decline over a 5-year period in 4,048 European American Lung Health Study participants with largely mild COPD. Genotype imputation was performed using reference HapMap II data. To validate regions meeting genome-wide significance, replication of top SNPs was attempted in independent cohorts. Three genes (TMEM26, ANK3 and FOXA1) within the regions of interest were selected for tissue expression studies using immunohistochemistry. Two intergenic SNPs (rs10761570, rs7911302) on chromosome 10 and one SNP on chromosome 14 (rs177852) met genome-wide significance after Bonferroni. Further support for the chromosome 10 region was obtained by imputation, the most significantly associated imputed SNPs (rs10761571, rs7896712) being flanked by observed markers rs10761570 and rs7911302. Results were not replicated in four general population cohorts or a smaller cohort of subjects with moderate to severe COPD; however, we show novel expression of genes near regions of significantly associated SNPS, including TMEM26 and FOXA1 in airway epithelium and lung parenchyma, and ANK3 in alveolar macrophages. Levels of expression were associated with lung function and COPD status. We identified two novel regions associated with lung function decline in mild COPD. Genes within these regions were expressed in relevant lung cells and their expression related to airflow limitation suggesting they may represent novel candidate genes for COPD susceptibility.     

Genome sequence of Frateuria aurantia type strain (Kond? 67T), a xanthomonade isolated from Lilium auratium Lindl.

Frateuria aurantia (ex Kondô and Ameyama 1958) Swings et al. 1980 is a member of the bispecific genus Frateuria in the family Xanthomonadaceae, which is already heavily targeted for non-type strain genome sequencing. Strain Kondô 67^T was initially (1958) identified as a member of ‘Acetobacter aurantius’, a name that was not considered for the approved list. Kondô 67^T was therefore later designated as the type strain of the newly proposed acetogenic species Frateuria aurantia. The strain is of interest because of its triterpenoids (hopane family). F. aurantia Kondô 67^T is the first member of the genus Frateura whose genome sequence has been deciphered, and here we describe the features of this organism, together with the complete genome sequence and annotation. The 3,603,458-bp long chromosome with its 3,200 protein-coding and 88 RNA genes is a part of the Genomic Encyclopedia of Bacteria and Archaea project.     

Crystal Structures of a Plant Trypsin Inhibitor from Enterolobium contortisiliquum (EcTI) and of Its Complex with Bovine Trypsin

A serine protease inhibitor from Enterolobium contortisiliquum (EcTI) belongs to the Kunitz family of plant inhibitors, common in plant seeds. It was shown that EcTI inhibits the invasion of gastric cancer cells through alterations in integrin-dependent cell signaling pathway. We determined high-resolution crystal structures of free EcTI (at 1.75 Å) and complexed with bovine trypsin (at 2 Å). High quality of the resulting electron density maps and the redundancy of structural information indicated that the sequence of the crystallized isoform contained 176 residues and differed from the one published previously. The structure of the complex confirmed the standard inhibitory mechanism in which the reactive loop of the inhibitor is docked into trypsin active site with the side chains of Arg64 and Ile65 occupying the S1 and S1′ pockets, respectively. The overall conformation of the reactive loop undergoes only minor adjustments upon binding to trypsin. Larger deviations are seen in the vicinity of Arg64, driven by the needs to satisfy specificity requirements. A comparison of the EcTI-trypsin complex with the complexes of related Kunitz inhibitors has shown that rigid body rotation of the inhibitors by as much as 15° is required for accurate juxtaposition of the reactive loop with the active site while preserving its conformation. Modeling of the putative complexes of EcTI with several serine proteases and a comparison with equivalent models for other Kunitz inhibitors elucidated the structural basis for the fine differences in their specificity, providing tools that might allow modification of their potency towards the individual enzymes.     

Multipronged approach to managing beta‐glucan contaminants in the downstream process: Control of raw materials and filtration with charge‐modified nylon 6,6 membrane filters

(1→3)‐β‐d ‐Glucans (beta‐glucans) have been found in raw materials used in the manufacture of recombinant therapeutics. Because of their biological activity, beta‐glucans are considered process contaminants and consequently their level in the product needs to be controlled. Although beta‐glucans introduced into the cell culture process can readily be removed by bind‐and‐elute chromatography process steps, beta‐glucans can also be introduced into the purification process through raw materials containing beta‐glucans as well as leachables from filters made from cellulose. This article reports a multipronged approach to managing the beta‐glucan contamination in the downstream process. Raw material screening and selection can be used to effectively limit the level of beta‐glucan introduced into the downstream process. Placement of a cellulosic filter upstream of the last bind‐and‐elute column step or effective preuse flushing can also limit the level of contaminant introduced. More importantly, this article reports the active removal of beta‐glucan from the downstream process when necessary. It was discovered that the Posidyne^® filter, a charge‐modified nylon 6,6 membrane filter, was able to effectively remove beta‐glucans from buffers at relatively low pH and salt concentrations. An approach of using low beta‐glucan buffer components combined with filtration of the buffer with a Posidyne membrane has been successfully demonstrated at preparative scale. Additionally, the feasibility of active removal of beta‐glucan from in‐process product pools by Posidyne membrane filtration has also been demonstrated. Based on the data presented, a mechanism for binding is proposed, as well as a systematic approach for sizing of the Posidyne filter. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:672–680, 2013     

T cell exosomes induce cholesterol accumulation in human monocytes via phosphatidylserine receptor

Activated T lymphocytes release vesicles, termed exosomes, enriched in cholesterol and exposing phosphatidylserine (PS) at their outer membrane leaflet. Although CD4(+) activated T lymphocytes infiltrate an atherosclerotic plaque, the effects of T cell exosomes on the atheroma-associated cells are not known. We report here that exosomes isolated from the supernatants of activated human CD4(+) T cells enhance cholesterol accumulation in cultured human monocytes and THP-1 cells. Lipid droplets found in the cytosol of exosome-treated monocytes contained both cholesterol ester and free cholesterol. Anti-phosphatidylserine receptor antibodies recognized surface protein on the monocyte plasma membrane and prevented exosome-induced cholesterol accumulation, indicating that exosome internalization is mediated via endogenous phosphatidylserine receptor. The production of proinflammatory cytokine TNF-alpha enhanced in parallel with monocyte cholesterol accumulation. Our data strongly indicate that exosomes released by activated T cells may represent a powerful, previously unknown, atherogenic factor.