Redox Proteomic Profile of Tirapazamine-Resistant Murine Hepatoma Cells.

3-Amino-1,2,4-benzotriazine-1,4-dioxide (tirapazamine, TPZ) and other heteroaromatic N-oxides (ArN→O) exhibit tumoricidal, antibacterial, and antiprotozoal activities. Their action is attributed to the enzymatic single-electron reduction to free radicals that initiate the prooxidant processes. In order to clarify the mechanisms of aerobic mammalian cytotoxicity of ArN→O, we derived a TPZ-resistant subline of murine hepatoma MH22a cells (resistance index, 5.64). The quantitative proteomic of wild-type and TPZ-resistant cells revealed 5818 proteins, of which 237 were up- and 184 down-regulated. The expression of the antioxidant enzymes aldehyde- and alcohol dehydrogenases, carbonyl reductases, catalase, and glutathione reductase was increased 1.6-5.2 times, whereas the changes in the expression of glutathione peroxidase, superoxide dismutase, thioredoxin reductase, and peroxiredoxins were less pronounced. The expression of xenobiotics conjugating glutathione-S-transferases was increased by 1.6-2.6 times. On the other hand, the expression of NADPH:cytochrome P450 reductase was responsible for the single-electron reduction in TPZ and for the 2.1-fold decrease. These data support the fact that the main mechanism of action of TPZ under aerobic conditions is oxidative stress. The unchanged expression of intranuclear antioxidant proteins peroxiredoxin, glutaredoxin, and glutathione peroxidase, and a modest increase in the expression of DNA damage repair proteins, tend to support non-site-specific but not intranuclear oxidative stress as a main factor of TPZ aerobic cytotoxicity.

Independent human mesenchymal stromal cell-derived extracellular vesicle preparations differentially attenuate symptoms in an advanced murine graft-versus-host disease model.

BACKGROUND AIMS: Extracellular vesicles (EVs) harvested from conditioned media of human mesenchymal stromal cells (MSCs) suppress acute inflammation in various disease models and promote regeneration of damaged tissues. After successful treatment of a patient with acute steroid-refractory graft-versus-host disease (GVHD) using EVs prepared from conditioned media of human bone marrow-derived MSCs, this study focused on improving the MSC-EV production for clinical application. METHODS: Independent MSC-EV preparations all produced according to a standardized procedure revealed broad immunomodulatory differences. Only a proportion of the MSC-EV products applied effectively modulated immune responses in a multi-donor mixed lymphocyte reaction (mdMLR) assay. To explore the relevance of such differences in vivo, at first a mouse GVHD model was optimized. RESULTS: The functional testing of selected MSC-EV preparations demonstrated that MSC-EV preparations revealing immunomodulatory capabilities in the mdMLR assay also effectively suppress GVHD symptoms in this model. In contrast, MSC-EV preparations, lacking such in vitro activities, also failed to modulate GVHD symptoms in vivo. Searching for differences of the active and inactive MSC-EV preparations, no concrete proteins or miRNAs were identified that could serve as surrogate markers. CONCLUSIONS: Standardized MSC-EV production strategies may not be sufficient to warrant manufacturing of MSC-EV products with reproducible qualities. Consequently, given this functional heterogeneity, every individual MSC-EV preparation considered for the clinical application should be evaluated for its therapeutic potency before administration to patients. Here, upon comparing immunomodulating capabilities of independent MSC-EV preparations in vivo and in vitro, we found that the mdMLR assay was qualified for such analyses.

Mitochondrial dysfunction rapidly modulates the abundance and thermal stability of cellular proteins.

Cellular functionality relies on a well-balanced, but highly dynamic proteome. Dysfunction of mitochondrial protein import leads to the cytosolic accumulation of mitochondrial precursor proteins which compromise cellular proteostasis and trigger a mitoprotein-induced stress response. To dissect the effects of mitochondrial dysfunction on the cellular proteome as a whole, we developed pre-post thermal proteome profiling. This multiplexed time-resolved proteome-wide thermal stability profiling approach with isobaric peptide tags in combination with a pulsed SILAC labelling elucidated dynamic proteostasis changes in several dimensions: In addition to adaptations in protein abundance, we observed rapid modulations of the thermal stability of individual cellular proteins. Different functional groups of proteins showed characteristic response patterns and reacted with group-specific kinetics, allowing the identification of functional modules that are relevant for mitoprotein-induced stress. Thus, our new pre-post thermal proteome profiling approach uncovered a complex response network that orchestrates proteome homeostasis in eukaryotic cells by time-controlled adaptations of the abundance and the conformation of proteins.

Extended N-Terminal Acetyltransferase Naa50 in Filamentous Fungi Adds to Naa50 Diversity.

Most eukaryotic proteins are N-terminally acetylated by a set of Nα acetyltransferases (NATs). This ancient and ubiquitous modification plays a fundamental role in protein homeostasis, while mutations are linked to human diseases and phenotypic defects. In particular, Naa50 features species-specific differences, as it is inactive in yeast but active in higher eukaryotes. Together with NatA, it engages in NatE complex formation for cotranslational acetylation. Here, we report Naa50 homologs from the filamentous fungi Chaetomium thermophilum and Neurospora crassa with significant N- and C-terminal extensions to the conserved GNAT domain. Structural and biochemical analyses show that CtNaa50 shares the GNAT structure and substrate specificity with other homologs. However, in contrast to previously analyzed Naa50 proteins, it does not form NatE. The elongated N-terminus increases Naa50 thermostability and binds to dynein light chain protein 1, while our data suggest that conserved positive patches in the C-terminus allow for ribosome binding independent of NatA. Our study provides new insights into the many facets of Naa50 and highlights the diversification of NATs during evolution.

Increased levels of mitochondrial import factor Mia40 prevent the aggregation of polyQ proteins in the cytosol.

The formation of protein aggregates is a hallmark of neurodegenerative diseases. Observations on patient samples and model systems demonstrated links between aggregate formation and declining mitochondrial functionality, but causalities remain unclear. We used Saccharomyces cerevisiae to analyze how mitochondrial processes regulate the behavior of aggregation-prone polyQ protein derived from human huntingtin. Expression of Q97-GFP rapidly led to insoluble cytosolic aggregates and cell death. Although aggregation impaired mitochondrial respiration only slightly, it considerably interfered with the import of mitochondrial precursor proteins. Mutants in the import component Mia40 were hypersensitive to Q97-GFP, whereas Mia40 overexpression strongly suppressed the formation of toxic Q97-GFP aggregates both in yeast and in human cells. Based on these observations, we propose that the post-translational import of mitochondrial precursor proteins into mitochondria competes with aggregation-prone cytosolic proteins for chaperones and proteasome capacity. Mia40 regulates this competition as it has a rate-limiting role in mitochondrial protein import. Therefore, Mia40 is a dynamic regulator in mitochondrial biogenesis that can be exploited to stabilize cytosolic proteostasis.

Stroma Transcriptomic and Proteomic Profile of Prostate Cancer Metastasis Xenograft Models Reveals Prognostic Value of Stroma Signatures.

Resistance acquisition to androgen deprivation treatment and metastasis progression are a major clinical issue associated with prostate cancer (PCa). The role of stroma during disease progression is insufficiently defined. Using transcriptomic and proteomic analyses on differentially aggressive patient-derived xenografts (PDXs), we investigated whether PCa tumors predispose their microenvironment (stroma) to a metastatic gene expression pattern. RNA sequencing was performed on the PCa PDXs BM18 (castration-sensitive) and LAPC9 (castration-resistant), representing different disease stages. Using organism-specific reference databases, the human-specific transcriptome (tumor) was identified and separated from the mouse-specific transcriptome (stroma). To identify proteomic changes in the tumor (human) versus the stroma (mouse), we performed human/mouse cell separation and subjected protein lysates to quantitative Tandem Mass Tag labeling and mass spectrometry. Tenascin C (TNC) was among the most abundant stromal genes, modulated by androgen levels in vivo and highly expressed in castration-resistant LAPC9 PDX. The tissue microarray of primary PCa samples (n = 210) showed that TNC is a negative prognostic marker of the clinical progression to recurrence or metastasis. Stroma markers of osteoblastic PCa bone metastases seven-up signature were induced in the stroma by the host organism in metastatic xenografts, indicating conserved mechanisms of tumor cells to induce a stromal premetastatic signature. A 50-gene list stroma signature was identified based on androgen-dependent responses, which shows a linear association with the Gleason score, metastasis progression and progression-free survival. Our data show that metastatic PCa PDXs, which differ in androgen sensitivity, trigger differential stroma responses, which show the metastasis risk stratification and prognostic biomarker potential.

The Hsp90 isoforms from S. cerevisiae differ in structure, function and client range.

The molecular chaperone Hsp90 is an important regulator of proteostasis. It has remained unclear why S. cerevisiae possesses two Hsp90 isoforms, the constitutively expressed Hsc82 and the stress-inducible Hsp82. Here, we report distinct differences despite a sequence identity of 97%. Consistent with its function under stress conditions, Hsp82 is more stable and refolds more efficiently than Hsc82. The two isoforms also differ in their ATPases and conformational cycles. Hsc82 is more processive and populates closed states to a greater extent. Variations in the N-terminal ATP-binding domain modulate its dynamics and conformational cycle. Despite these differences, the client interactomes are largely identical, but isoform-specific interactors exist both under physiological and heat shock conditions. Taken together, changes mainly in the N-domain create a stress-specific, more resilient protein with a shifted activity profile. Thus, the precise tuning of the Hsp90 isoforms preserves the basic mechanism but adapts it to specific needs.

Asparagine endopeptidase cleaves tau at N167 after uptake into microglia.

Tau cleavage by different proteolytic enzymes generates short, aggregation-prone fragments that have been implicated in the pathogenesis of Alzheimer's disease (AD). Asparagine endopeptidase (AEP) activity in particular has been associated with tau dysfunction and aggregation, and the activity of the protease is increased in both aging and AD. Using a mass spectrometry approach, we identified a novel tau cleavage site at N167 and confirmed its processing by AEP. In combination with the previously known site at N368, we show that AEP cleavage yields a tau fragment that is present in both control and AD brains at similar levels. AEP is a lysosomal enzyme, and our data suggest that it is expressed in microglia rather than in neurons. Accordingly, we observe tau cleavage at N167 and N368 after endocytotic uptake into microglia, but not neurons. However, tau168-368 does not accumulate in microglia and we thus conclude that the fragment is part of a proteolytic cascade leading to tau degradation. While we confirm previous studies showing increased overall AEP activity in AD brains, our data suggests that AEP-mediated cleavage of tau is a physiological event occurring during microglial degradation of the secreted neuronal protein. As a consequence, we caution against preventing AEP-mediated tau cleavage as a therapeutic approach in AD.