Anja Stefanski

Proteome‐wide identification of mycobacterial pupylation targets

Mycobacteria use a unique system for covalently modifying proteins based on the conjugation of a small protein, referred to as prokaryotic ubiquitin‐like protein (PUP). In this study, we report a proteome‐wide analysis of endogenous pupylation targets in the model organism Mycobacterium smegmatis. On affinity capture, a total of 243 candidate pupylation targets were identified by two complementary proteomics approaches. For 41 of these protein targets, direct evidence for a total of 48 lysine‐mediated pupylation acceptor sites was obtained by collision‐induced dissociation spectra. For the majority of these pupylation targets (38 of 41), orthologous genes are found in the M. tuberculosis genome. Interestingly, approximately half of these proteins are involved in intermediary metabolism and respiration pathways. A considerable fraction of the remaining targets are involved in lipid metabolism, information pathways, and virulence, detoxification and adaptation. Approximately one‐third of the genes encoding these targets are located in seven gene clusters, indicating functional linkages of mycobacterial pupylation targets. A comparison of the pupylome under different cell culture conditions indicates that substrate targeting for pupylation is rather dynamic.

Tra2-Mediated Recognition of HIV-1 5′ Splice Site D3 as a Key Factor in the Processing of vpr mRNA

ABSTRACT Small noncoding HIV-1 leader exon 3 is defined by its splice sites A2 and D3. While 3′ splice site (3′ss) A2 needs to be activated for vpr mRNA formation, the location of the vpr start codon within downstream intron 3 requires silencing of splicing at 5′ss D3. Here we show that the inclusion of both HIV-1 exon 3 and vpr mRNA processing is promoted by an exonic splicing enhancer (ESE vpr ) localized between exonic splicing silencer ESSV and 5′ss D3. The ESE vpr sequence was found to be bound by members of the Transformer 2 (Tra2) protein family. Coexpression of these proteins in provirus-transfected cells led to an increase in the levels of exon 3 inclusion, confirming that they act through ESE vpr . Further analyses revealed that ESE vpr supports the binding of U1 snRNA at 5′ss D3, allowing bridging interactions across the upstream exon with 3′ss A2. In line with this, an increase or decrease in the complementarity of 5′ss D3 to the 5′ end of U1 snRNA was accompanied by a higher or lower vpr expression level. Activation of 3′ss A2 through the proposed bridging interactions, however, was not dependent on the splicing competence of 5′ss D3 because rendering it splicing defective but still competent for efficient U1 snRNA binding maintained the enhancing function of D3. Therefore, we propose that splicing at 3′ss A2 occurs temporally between the binding of U1 snRNA and splicing at D3.

MicroRNA-138 promotes acquired alkylator resistance in glioblastoma by targeting the Bcl-2-interacting mediator BIM

Glioblastoma is the most aggressive brain tumor in adults with a median survival below 12 months in population-based studies. The main reason for tumor recurrence and progression is constitutive or acquired resistance to the standard of care of surgical resection followed by radiotherapy with concomitant and adjuvant temozolomide (TMZ/RT→TMZ). Here, we investigated the role of microRNA (miRNA) alterations as mediators of alkylator resistance in glioblastoma cells. Using microarray-based miRNA expression profiling of parental and TMZ-resistant cultures of three human glioma cell lines, we identified a set of differentially expressed miRNA candidates. From these, we selected miR-138 for further functional analyses as this miRNA was not only upregulated in TMZ-resistant versus parental cells, but also showed increased expression in vivo in recurrent glioblastoma tissue samples after TMZ/RT→TMZ treatment. Transient transfection of miR-138 mimics in glioma cells with low basal miR-138 expression increased glioma cell proliferation. Moreover, miR-138 overexpression increased TMZ resistance in long-term glioblastoma cell lines and glioma initiating cell cultures. The apoptosis regulator BIM was identified as a direct target of miR-138, and its silencing mediated the induced TMZ resistance phenotype. Altered sensitivity to apoptosis played only a minor role in this resistance mechanism. Instead, we identified the induction of autophagy to be regulated downstream of the miR-138/BIM axis and to promote cell survival following TMZ exposure. Our data thus define miR-138 as a glioblastoma cell survival-promoting miRNA associated with resistance to TMZ therapy in vitro and with tumor progression in vivo.

How Stemlike Are Sphere Cultures From Long-term Cancer Cell Lines? Lessons From Mouse Glioma Models

Abstract Cancer stem cells may mediate therapy resistance and recurrence in various types of cancer, including glioblastoma. Cancer stemlike cells can be isolated from long-term cancer cell lines, including glioma lines. Using sphere formation as a model for cancer cell stemness in vitro, we derived sphere cultures from SMA-497, SMA-540, SMA-560, and GL-261 glioma cells. Gene expression and proteomics profiling demonstrated that sphere cultures uniformly showed an elevated expression of stemness-associated genes, notably including CD44. Differences in neural lineage marker expression between nonsphere and sphere cultures were heterogeneous except for a uniform reduction of &bgr;-III-tubulin in sphere cultures. All sphere cultures showed slower growth. Self-renewal capacity was influenced by medium conditions but not nonsphere versus sphere culture phenotype. Sphere cultures were more resistant to irradiation, whereas both nonsphere and sphere cultures were highly resistant to temozolomide. Nonsphere cells formed more aggressive tumors in syngeneic mice than sphere cells in all models except SMA-560. There were no major differences in vascularization or infiltration by T cells or microglia/macrophages between nonsphere and sphere cell–derived tumors implanted in syngeneic hosts. Together, these data indicate that mouse glioma cell lines may be induced in vitro to form spheres that acquire features of stemness, but they do not exhibit a uniform biologic phenotype, thereby challenging the view that they represent a superior model system.

Simultaneous extraction of nucleic acids and proteins from tissue specimens by ultracentrifugation: A protocol using the high-salt protein fraction for quantitative proteome analysis.

Comprehensive molecular profiling of human tumor tissue specimens at the DNA, mRNA and protein level is often obstructed by a limited amount of available material. Homogenization of frozen tissue samples in guanidine isothiocyanate followed by ultracentrifugation over cesium chloride allows the simultaneous extraction of high‐molecular weight DNA and RNA. Here, we present a protocol for quantitative proteome analysis using the high‐salt protein fraction obtained as supernatant after ultracentrifugation for nucleic acid extraction. We applied this method to extracts from primary human brain tumors and demonstrate its successful application for protein expression profiling in these tumors using 2‐D DIGE, MS and Western blotting.

The fate of b-ions in the two worlds of collision-induced dissociation

Fragment analysis of proteins and peptides by mass spectrometry using collision-induced dissociation (CID) revealed that the pairwise generated N-terminal b- and C-terminal y-ions have different stabilities resulting in underrepresentation of b-ions. Detailed analyses of large-scale spectra databases and synthetic peptides underlined these observations and additionally showed that the fragmentation pattern depends on utilized CID regime. To investigate this underrepresentation further we systematically compared resonant excitation energy and beam-type CID facilitated on different mass spectrometer platforms: (i) quadrupole time-of-flight, (ii) linear ion trap and (iii) three-dimensional ion trap. Detailed analysis of MS/MS data from a standard tryptic protein digest revealed that b-ions are significantly underrepresented on all investigated mass spectrometers. By N-terminal acetylation of tryptic peptides we show for the first time that b-ion cyclization reaction significantly contributes to b-ion underrepresentation even on ion trap instruments and accounts for at most 16% of b-ion loss.

SIRT4 interacts with OPA1 and regulates mitochondrial quality control and mitophagy

The stress-responsive mitochondrial sirtuin SIRT4 controls cellular energy metabolism in a NAD+-dependent manner and is implicated in cellular senescence and aging. Here we reveal a novel function of SIRT4 in mitochondrial morphology/quality control and regulation of mitophagy. We report that moderate overexpression of SIRT4, but not its enzymatically inactive mutant H161Y, sensitized cells to mitochondrial stress. CCCP-triggered dissipation of the mitochondrial membrane potential resulted in increased mitochondrial ROS levels and autophagic flux, but surprisingly led to increased mitochondrial mass and decreased Parkin-regulated mitophagy. The anti-respiratory effect of elevated SIRT4 was accompanied by increased levels of the inner-membrane bound long form of the GTPase OPA1 (L-OPA1) that promotes mitochondrial fusion and thereby counteracts fission and mitophagy. Consistent with this, upregulation of endogenous SIRT4 expression in fibroblast models of senescence either by transfection with miR-15b inhibitors or by ionizing radiation increased L-OPA1 levels and mitochondrial fusion in a SIRT4-dependent manner. We further demonstrate that SIRT4 interacts physically with OPA1 in co-immunoprecipitation experiments. Overall, we propose that the SIRT4-OPA1 axis is causally linked to mitochondrial dysfunction and altered mitochondrial dynamics that translates into aging-associated decreased mitophagy based on an unbalanced mitochondrial fusion/fission cycle.

Redox proteomics reveal stress responsive proteins linking peroxiredoxin-1 status in glioma to chemosensitivity and oxidative stress.

The combined deletion of chromosomal arms 1p and 19q has been described as a prognostic marker for oligodendroglial tumors. These tumors show a better response to chemotherapy and radiotherapy. Recently, we found a lower abundance of peroxiredoxin 1 (PRDX1) in oligodendroglial tumors with 1p/19q deletion, suggesting a potential role of this enzyme in the clearance of therapy induced reactive oxygen species (ROS). Here, we confirmed the importance of PRDX1 in tumor cell survival by PRDX1 knockdown and overexpression in A-172 cells treated with the alkylating agent bis-chloroethyl nitrosourea (BCNU). Overexpression of PRDX1 resulted in a higher resistance of cells to BCNU treatment. In addition, BCNU challenged cells showed higher levels of ROS in PRDX1 knockdown cells. We applied a modified version of the redox two dimensional difference gel electrophoresis approach to analyze ROS mediated effects on protein thiols after BCNU treatment by labeling protein thiols with fluorescent dyes. Altogether eleven proteins were identified showing PRDX1 dependent altered labeling, many of them have been previously linked to stress response processes. Furthermore, 30 additional potentially redox active proteins were identified. The majority of them is involved in therapy associated processes like cellular stress response, DNA damage and regulation of cell death and therewith suggests that tumor cells maintain a network of redox sensitive proteins to escape chemotherapy. This article is part of a Special Issue entitled: Medical Proteomics.

Surveying the Oligomeric State of Arabidopsis thaliana Chloroplasts

Blue native-PAGE (BN-PAGE) resolves protein complexes in their native state. When combined with immunoblotting, it can be used to identify the presence of high molecular weight complexes at high resolution for any protein, given a suitable antibody. To identify proteins in high molecular weight complexes on a large scale and to bypass the requirement for specific antibodies, we applied a tandem mass spectrometry (MS/MS) approach to BN-PAGE-resolved chloroplasts. Fractionation of the gel into six bands allowed identification and label-free quantification of 1000 chloroplast proteins with native molecular weight separation. Significantly, this approach achieves a depth of identification comparable with traditional shotgun proteomic analyses of chloroplasts, indicating much of the known chloroplast proteome is amenable to MS/MS identification under our fractionation scheme. By limiting the number of fractionation bands to six, we facilitate scaled-up comparative analyses, as we demonstrate with the reticulata chloroplast mutant displaying a reticulated leaf phenotype. Our comparative proteomics approach identified a candidate interacting protein of RETICULATA as well as effects on lipid remodeling proteins, amino acid metabolic enzymes, and plastid division machinery. We additionally highlight selected proteins from each sub-compartment of the chloroplast that provide novel insight on known or hypothesized protein complexes to further illustrate the utility of this approach. Our results demonstrate the high sensitivity and reproducibility of this technique, which is anticipated to be widely adaptable to other sub-cellular compartments.