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Tissue sections, which are widely used in research and diagnostic laboratories and have already been examined by immunohistochemistry (IHC), may subsequently provide a resource for proteomic studies, even though only small amount of protein is available. Therefore, we established a workflow for tandem mass spectrometry-based protein profiling of IHC specimens and characterized defined brain area sections. We investigated the CA1 region of the hippocampus dissected from brain slices of adult C57BL/6J mice. The workflow contains detailed information on sample preparation from brain slices, including removal of antibodies and cover matrices, dissection of region(s) of interest, protein extraction and digestion, mass spectrometry measurement, and data analysis. The Gene Ontology (GO) knowledge base was used for further annotation. Literature searches and Gene Ontology annotation of the detected proteins verify the applicability of this method for global protein profiling using formalin-fixed and embedded material and previously used IHC slides.
Mutations in genes coding for proteasome subunits and/or proteasome assembly helpers typically cause recurring autoinflammation referred to as chronic atypical neutrophilic dermatosis with lipodystrophy and elevated temperatures (CANDLE) or proteasome-associated autoinflammatory syndrome (PRAAS). Patients with CANDLE/PRAAS present with mostly chronically elevated type I interferon scores that emerge as a consequence of increased proteotoxic stress by mechanisms that are not fully understood. Here, we report on five unrelated patients with CANDLE/PRAAS carrying novel inherited proteasome missense and/or nonsense variants. Four patients were compound heterozygous for novel pathogenic variants in the known CANDLE/PRAAS associated genes, PSMB8 and PSMB10, whereas one patient showed additive loss-of-function mutations in PSMB8. Variants in two previously not associated proteasome genes, PSMA5 and PSMC5, were found in a patient who also carried the PSMB8 founder mutation, p.T75M. All newly identified mutations substantially impact the steady-state expression of the affected proteasome subunits and/or their incorporation into mature 26S proteasomes. Our observations expand the spectrum of PRAAS-associated genetic variants and improve a molecular diagnosis and genetic counseling of patients with sterile autoinflammation.
Introduction
Proteasome inhibition is first line therapy in multiple myeloma (MM). The immunological potential of cell death triggered by defects of the ubiquitin-proteasome system (UPS) and subsequent perturbations of protein homeostasis is, however, less well defined.
Methods
In this paper, we applied the protein homeostasis disruptors bortezomib (BTZ), ONX0914, RA190 and PR619 to various MM cell lines and primary patient samples to investigate their ability to induce immunogenic cell death (ICD).
Results
Our data show that while BTZ treatment triggers sterile type I interferon (IFN) responses, exposure of the cells to ONX0914 or RA190 was mostly immunologically silent. Interestingly, inhibition of protein de-ubiquitination by PR619 was associated with the acquisition of a strong type I IFN gene signature which relied on key components of the unfolded protein and integrated stress responses including inositol-requiring enzyme 1 (IRE1), protein kinase R (PKR) and general control nonderepressible 2 (GCN2). The immunological relevance of blocking de-ubiquitination in MM was further reflected by the ability of PR619-induced apoptotic cells to facilitate dendritic cell (DC) maturation via type I IFN-dependent mechanisms.
Conclusion
Altogether, our findings identify de-ubiquitination inhibition as a promising strategy for inducing ICD of MM to expand current available treatments.
Cancer stem cells (CSCs) represent a small subset of slowly dividing cells with tumor-initiating ability. They can self-renew and differentiate into all the distinct cell populations within a tumor. CSCs are naturally resistant to chemotherapy or radiotherapy. CSCs, thus, can repopulate a tumor after therapy and are responsible for recurrence of disease. Stemness manifests itself through, among other things, the expression of stem cell markers, the ability to induce sphere formation and tumor growth in vivo, and resistance to chemotherapeutics and irradiation. Stemness is maintained by keeping levels of reactive oxygen species (ROS) low, which is achieved by enhanced activity of antioxidant pathways. Here, cellular sources of ROS, antioxidant pathways employed by CSCs, and underlying mechanisms to overcome resistance are discussed.
Proteostasis, a portmanteau of the words protein and homeostasis, refers to the ability of
eukaryotic cells to maintain a stable proteome by acting on protein synthesis, quality control and/or
degradation. Over the last two decades, an increasing number of disorders caused by proteostasis
perturbations have been identified. Depending on their molecular etiology, such diseases may be
classified into ribosomopathies, proteinopathies and proteasomopathies. Strikingly, most—if not
all—of these syndromes exhibit an autoinflammatory component, implying a direct cause-and-effect
relationship between proteostasis disruption and the initiation of innate immune responses. In this
review, we provide a comprehensive overview of the molecular pathogenesis of these disorders and
summarize current knowledge of the various mechanisms by which impaired proteostasis promotes
autoinflammation. We particularly focus our discussion on the notion of how cells sense and integrate
proteostasis perturbations as danger signals in the context of autoinflammatory diseases to provide
insights into the complex and multiple facets of sterile inflammation.
Reduction and oxidation reactions are essential for biochemical processes. They are part of metabolic pathways and signal transduction. Reactive oxygen species (ROS) as second messengers and oxidative modifications of cysteinyl (Cys) residues are key to transduce and translate intracellular and intercellular signals. Dysregulation of cellular redox signaling is known as oxidative distress, which has been linked to various pathologies, including neurodegeneration. Alzheimer's disease (AD) is a neurodegenerative pathology linked to both, abnormal amyloid precursor protein (APP) processing, generating Aβ peptide, and Tau hyperphosphorylation and aggregation. Signs of oxidative distress in AD include: increase of ROS (H2O2, O2•−), decrease of the levels or activities of antioxidant enzymes, abnormal oxidation of macromolecules related to elevated Aβ production, and changes in mitochondrial homeostasis linked to Tau phosphorylation. Interestingly, Cys residues present in APP form disulfide bonds that are important for intermolecular interactions and might be involved in the aggregation of Aβ. Moreover, two Cys residues in some Tau isoforms have been shown to be essential for Tau stabilization and its interaction with microtubules. Future research will show the complexities of Tau, its interactome, and the role that Cys residues play in the progression of AD. The specific modification of cysteinyl residues in redox signaling is also tightly connected to the regulation of various metabolic pathways. Many of these pathways have been found to be altered in AD, even at very early stages. In order to analyze the complex changes and underlying mechanisms, several AD models have been developed, including animal models, 2D and 3D cell culture, and ex-vivo studies of patient samples. The use of these models along with innovative, new redox analysis techniques are key to further understand the importance of the redox component in Alzheimer's disease and the identification of new therapeutic targets in the future.
Chagas’ disease (CD), caused by the hemoflagellate protozoan, Trypanosoma cruzi, is endemic in most countries of Latin America. Heart failure (HF) is often a late manifestation of chronic CD, and is associated with high morbidity and mortality. Inflammatory processes mediated by cytokines play a key role in the pathogenesis and progression of CD. Keeping in view the inflammatory nature of CD, this study investigated the possible role of 21 different inflammatory cytokines as biomarkers for prediction and prognosis of CD. The plasma concentration of these cytokines was measured in a group of patients with CD (n = 94), and then compared with those measured in patients with dilated cardiomyopathy (DCM) from idiopathic causes (n = 48), and with control subjects (n = 25). Monovariately, plasma levels of cytokines such as stem cell growth factor beta (SCGF beta), hepatocyte growth factor (HGF), monokine induced by interferon gamma (CXCL9), and macrophage inhibitory factor (MIF) were significantly increased in CD patients with advanced HF compared to control group. None of the cytokines could demonstrate any prognostic potency in CD patients, and only MIF and stromal derived factor-1 alpha (CXCL12) showed significance in predicting mortality and necessity for heart transplant in DCM patients. However, multivariate analysis prognosticated a large proportion of CD and DCM patients. In CD patients, HGF and Interleukin-12p40 (IL-12p40) together separated 81.9% of 3-year survivors from the deceased, while in DCM patients, CXCL12, stem cell factor (SCF), and CXCL9 together discriminated 77.1% of survivors from the deceased. The significant increase in plasma concentrations of cytokines such as HGF and CXCL9 in CD patients, and the ability of these cytokines to prognosticate a large proportion of CD and DCM patients multivariately, encourages further studies to clarify the diagnostic and prognostic potential of cytokines in such patients.
Despite their very close structural similarity, CxxC/S-type (class I) glutaredoxins (Grxs) actas oxidoreductases, while CGFS-type (class II) Grxs act as FeS cluster transferases. Here weshow that the key determinant of Grx function is a distinct loop structure adjacent to theactive site. Engineering of a CxxC/S-type Grx with a CGFS-type loop switched its functionfrom oxidoreductase to FeS transferase. Engineering of a CGFS-type Grx with a CxxC/S-typeloop abolished FeS transferase activity and activated the oxidative half reaction of the oxi-doreductase. The reductive half-reaction, requiring the interaction with a second GSHmolecule, was enabled by switching additional residues in the active site. We explain howsubtle structural differences, mostly depending on the structure of one particular loop, act inconcert to determine Grx function.
The failure of insulin-producingβ-cells is the underlying cause of hyperglycemia in diabetes mellitus.β-cell decay has been linked to hypoxia, chronic inflammation,and oxidative stress. Thioredoxin (Trx) proteins are major actors in redox signaling and essential for signal transduction and the cellular stress response. We haveanalyzed the cytosolic, mitochondrial, and extracellular Trx system proteins in hypoxic and cytokine-induced stress usingβ-cell culture, isolated pancreatic islets, andpancreatic islet transplantation modelling low oxygen supply.Protein levels of cytosolic Trx1 and Trx reductase (TrxR) 1 significantly decreased, while mitochondrial Trx2 and TrxR2 increased upon hypoxia and reox-ygenation. Interestingly, Trx1 was secreted byβ-cells during hypoxia. Moreover, murine and human pancreatic islet grafts released Trx1 upon glucose stimulation.Survival of transplanted islets was substantially impaired by the TrxR inhibitor auranofin.Since a release was prominent upon hypoxia, putative paracrine effects of Trx1 onβ-cells were examined. In fact, exogenously added recombinant hTrx1 mitigatedapoptosis and preserved glucose sensitivity in pancreatic islets subjected to hypoxia and inflammatory stimuli, dependent on its redox activity. Human subjects werestudied, demonstrating a transient increase in extracellular Trx1 in serum after glucose challenge. This increase correlated with better pancreatic islet function.Moreover, hTrx1 inhibited the migration of primary murine macrophages.In conclusion, our study offers evidence for paracrine functions of extracellular Trx1 that improve the survival and function of pancreaticβ-cells.