A new method of purification of proteasome substrates reveals polyubiquitination of 20 S proteasome subunits

The 26 S proteasome is implicated in the control of many major biological functions but a reliable method for the identification of its major substrates, i.e. polyubiquitin (Ub) conjugates, is still lacking. Based on the steps present in cells, i.e. recognition and deubiquitination, we developed an affinity matrix-based purification of polyUb conjugates suitable for any biological sample. Ub-conjugates were first purified from proteasome inhibitor-treated C2C12 cells using the Ub binding domains of the S5a proteasome subunit bound to an affinity matrix and then deubiquitinated by the catalytic domain of the USP2 enzyme. This two step purification of proteasome substrates involving both protein-protein interactions and enzyme-mediated release allowed highly specific isolation of polyUb 26 S proteasome substrates, which were then resolved on two-dimensional gels post-deubiquitination. To establish our method, we focused on a gel area where spots were best resolved. Surprisingly, spot analysis by mass spectrometry identified alpha2, alpha6, alpha7, beta2, beta3, beta4, and beta5 20 S proteasome subunits as potential substrates. Western blots using an anti-beta3 proteasome subunit antibody confirmed that high molecular weight forms of beta3 were present, particularly in proteasome inhibitor-treated cells. Sucrose gradients of cell lysates suggested that the proteasome was first disassembled before subunits were polyubiquitinated. Altogether, we provide a technique that enables large scale identification of 26 S proteasome substrates that should contribute to a better understanding of this proteolytic machinery in any living cell and/or organ/tissue. Furthermore, the data suggest that proteasome homeostasis involves an autoregulatory mechanism.     

C11orf83, a Mitochondrial Cardiolipin-Binding Protein Involved in bc1 Complex Assembly and Supercomplex Stabilization

Mammalian mitochondria may contain up to 1,500 different proteins, and many of them have neither been confidently identified nor characterized. In this study, we demonstrated that C11orf83, which was lacking experimental characterization, is a mitochondrial inner membrane protein facing the intermembrane space. This protein is specifically associated with the bc₁ complex of the electron transport chain and involved in the early stages of its assembly by stabilizing the bc₁ core complex. C11orf83 displays some overlapping functions with Cbp4p, a yeast bc₁ complex assembly factor. Therefore, we suggest that C11orf83, now called UQCC3, is the functional human equivalent of Cbp4p. In addition, C11orf83 depletion in HeLa cells caused abnormal crista morphology, higher sensitivity to apoptosis, a decreased ATP level due to impaired respiration and subtle, but significant, changes in cardiolipin composition. We showed that C11orf83 binds to cardiolipin by its α-helices 2 and 3 and is involved in the stabilization of bc₁ complex-containing supercomplexes, especially the III₂/IV supercomplex. We also demonstrated that the OMA1 metalloprotease cleaves C11orf83 in response to mitochondrial depolarization, suggesting a role in the selection of cells with damaged mitochondria for their subsequent elimination by apoptosis, as previously described for OPA1.     

Impact of a resistance gene against a fungal pathogen on the plant host residue microbiome: The case of the Leptosphaeria maculans–Brassica napus pathosystem

Oilseed rape residues are a crucial determinant of stem canker epidemiology as they support the sexual reproduction of the fungal pathogen Leptosphaeria maculans. The aim of this study was to characterize the impact of a resistance gene against L. maculans infection on residue microbial communities and to identify microorganisms interacting with this pathogen during residue degradation. We used near-isogenic lines to obtain healthy and infected host plants. The microbiome associated with the two types of plant residues was characterized by metabarcoding. A combination of linear discriminant analysis and ecological network analysis was used to compare the microbial communities and to identify microorganisms interacting with L. maculans. Fungal community structure differed between the two lines at harvest, but not subsequently, suggesting that the presence/absence of the resistance gene influences the microbiome at the base of the stem whilst the plant is alive, but that this does not necessarily lead to differential colonization of the residues by fungi. Direct interactions with other members of the community involved many fungal and bacterial amplicon sequence variants (ASVs). L. maculans appeared to play a minor role in networks, whereas one ASV affiliated to Plenodomus biglobosus (synonym Leptosphaeria biglobosa) from the Leptosphaeria species complex may be considered a keystone taxon in the networks at harvest. This approach could be used to identify and promote microorganisms with beneficial effects against residue-borne pathogens and, more broadly, to decipher the complex interactions between multispecies pathosystems and other microbial components in crop residues.     

Dissociation of human serum low density lipoproteins into several immunologically distinct subunits. Isolation and characterization of a B-III subunit

Low density lipoproteins (d = 1.030–1.055) were partially delipidated with ethyl-ether (LDLe). These LDLe exibit a spontaneous dissociation several days after dehpidanon. Four different immunoprecipitation complexes (B-I to B-IV) are observed when using two dimensional immunoelectrophoresis against anti LDLe immunoserum. The various subunits of LDLe have different behaviour upon electrophoresis. On disc gel electrophoresis containing urea three bonds can be seen; all are phospholipoproteins. The apolipoprotein moiety of LDL and LDLe have the same apparent molecular weight around 550 000. With time several subunits aappear in LDLe, the majority of them have a molecular weight around 370 000, 260 000 and 125 000.One of the components from dissociated LDLe containing the immunodeterminant B-III, has been separated by chromatography on heparin-agarose. This LDLe-B-III has the same phospholipid/protein ratio as total LDL protein moiety with an apparent molecular weight of 110 000. This part of apolipoprotein no affinity for heparin.     

Prevalence of grey matter pathology in early multiple sclerosis assessed by magnetization transfer ratio imaging

The aim of the study was to assess the prevalence, the distribution and the impact on disability of grey matter (GM) pathology in early multiple sclerosis. Eighty-eight patients with a clinically isolated syndrome with a high risk developing multiple sclerosis were included in the study. Forty-four healthy controls constituted the normative population. An optimized statistical mapping analysis was performed to compare each subject''s GM Magnetization Transfer Ratio (MTR) imaging maps with those of the whole group of controls. The statistical threshold of significant GM MTR decrease was determined as the maximum p value (p<0.05 FDR) for which no significant cluster survived when comparing each control to the whole control population. Using this threshold, 51% of patients showed GM abnormalities compared to controls. Locally, 37% of patients presented abnormalities inside the limbic cortex, 34% in the temporal cortex, 32% in the deep grey matter, 30% in the cerebellum, 30% in the frontal cortex, 26% in the occipital cortex and 19% in the parietal cortex. Stepwise regression analysis evidenced significant association (p = 0.002) between EDSS and both GM pathology (p = 0.028) and T2 white matter lesions load (p = 0.019). In the present study, we evidenced that individual analysis of GM MTR map allowed demonstrating that GM pathology is highly heterogeneous across patients at the early stage of MS and partly underlies irreversible disability.     

Endogenous neurotrophins and Trk signaling in diffuse large B cell lymphoma cell lines are involved in sensitivity to rituximab-induced apoptosis

Background

Diffuse large B-cell lymphoma (DLBCL) is a common and often fatal malignancy. Immunochemotherapy, a combination of rituximab to standard chemotherapy, has resulted in improved survival. However a substantial proportion of patients still fail to reach sustained remission. We have previously demonstrated that autocrine brain-derived neurotrophic factor (BDNF) production plays a function in human B cell survival, at least partly via sortilin expression. As neurotrophin receptor (Trks) signaling involved activation of survival pathways that are inhibited by rituximab, we speculated that neurotrophins may provide additional support for tumour cell survival and therapeutic resistance in DLBCL.

Methodology/Principal Findings

In the present study, we used two DLBCL cell lines, SUDHL4 and SUDHL6, known to be respectively less and more sensitive to rituximab. We found by RT-PCR, western blotting, cytometry and confocal microscopy that both cell lines expressed, in normal culture conditions, BDNF and to a lesser extent NGF, as well as truncated TrkB and p75^NTR/sortilin death neurotrophin receptors. Furthermore, BDNF secretion was detected in cell supernatants. NGF and BDNF production and Trk receptor expression, including TrkA, are regulated by apoptotic conditions (serum deprivation or rituximab exposure). Indeed, we show for the first time that rituximab exposure of DLBCL cell lines induces NGF secretion and that differences in rituximab sensitivity are associated with differential expression patterns of neurotrophins and their receptors (TrkA). Finally, these cells are sensitive to the Trk-inhibitor, K252a, as shown by the induction of apoptosis. Furthermore, K252a exhibits additive cytotoxic effects with rituximab.

Conclusions/Significance

Collectively, these data strongly suggest that a neurotrophin axis, such NGF/TrkA pathway, may contribute to malignant cell survival and rituximab resistance in DLBCL.     

Comparison of the influence of small GTPases Arl1 and Ypt6 on yeast cells' tolerance to various stress factors

The GTPases Arl1 and Ypt6 are involved in the intracellular transport of vesicles and their fusion with the trans-Golgi network. This work is focused on comparing the roles of these GTPases in the tolerance of Saccharomyces cerevisiae cells to an increased concentration of alkali metal cations and other stress factors. We studied the phenotypes of arl1 or ypt6 deletions in combination with the deletions of genes encoding alkali-metal-cation transporters (ena1-4, nha1, nhx1, and kha1). Salt sensitivity of the arl1 and ypt6 mutants was shown to be independent of the tested cation transporters and electrochemical membrane potential. Phenotype manifestations of ypt6 deletion were usually more prominent than those of arl1 (cells were more sensitive to KCl, NaCl, LiCl, hygromycin B, increased temperature, and increased pH). At suboptimal temperature, the growth inhibition of arl1 and ypt6 mutants was approximately the same, and low pH was the only condition where arl1 mutants grew even worse than ypt6 mutants. Overexpression of the ARL1 gene suppressed the phenotypes of ypt6 deletion; however, this did not work vice versa (additional copies of YPT6 could not replace ARL1). Our results suggest partially overlapping functions of the GTPases in resistance to various stress factors, with Ypt6 being more efficient under physiological conditions and Arl1 more versatile when overexpressed.