Association of serum cortisol and cortisone levels and risk of recurrence after endocrine treatment in breast cancer.

Metabolic reprogramming in breast cancer involves changes in steroid hormone synthesis and metabolism. Alterations in estrogen levels in both breast tissue and blood may influence carcinogenesis, breast cancer growth, and response to therapy. Our aim was to examine whether serum steroid hormone concentrations could predict the risk of recurrence and treatment-related fatigue in patients with breast cancer. This study included 66 postmenopausal patients with estrogen receptor-positive breast cancer who underwent surgery, radiotherapy, and adjuvant endocrine treatment. Serum samples were collected at six different time points [before the start of radiotherapy (as baseline), immediately after radiotherapy, and then 3, 6, 12 months, and 7-12 years after radiotherapy]. Serum concentrations of eight steroid hormones (cortisol, cortisone, 17α-hydroxyprogesterone, 17ß-estradiol, estrone, androstenedione, testosterone, and progesterone) were measured using a liquid chromatography-tandem mass spectrometry-based method. Breast cancer recurrence was defined as clinically proven relapse/metastatic breast cancer or breast cancer-related death. Fatigue was assessed with the QLQ-C30 questionnaire. Serum steroid hormone concentrations measured before and immediately after radiotherapy differed between relapse and relapse-free patients [(accuracy 68.1%, p = 0.02, and 63.2%, p = 0.03, respectively, partial least squares discriminant analysis (PLS-DA)]. Baseline cortisol levels were lower in patients who relapsed than in those who did not (p < 0.05). The Kaplan-Meier analysis showed that patients with high baseline concentrations of cortisol (≥ median) had a significantly lower risk of breast cancer recurrence than patients with low cortisol levels (

Proteomic analysis reveals mechanisms underlying increased efficacy of bleomycin by photochemical internalization in bladder cancer cells.

Photochemical internalization (PCI) is a promising new technology for site-specific drug delivery, developed from photodynamic therapy (PDT). In PCI, light-induced activation of a photosensitizer trapped inside endosomes together with e.g. chemotherapeutics, nucleic acids or immunotoxins, allows cytosolic delivery and enhanced local therapeutic effect. Here we have evaluated the photosensitizer meso-tetraphenyl chlorine disulphonate (TPCS2a/fimaporfin) in a proteome analysis of AY-27 rat bladder cancer cells in combination with the chemotherapeutic drug bleomycin (BML). We find that BLMPCI attenuates oxidative stress responses induced by BLM alone, while concomitantly increasing transcriptional repression and DNA damage responses. BLMPCI also mediates downregulation of bleomycin hydrolase (Blmh), which is responsible for cellular degradation of BLM, as well as several factors known to be involved in fibrotic responses. PCI-mediated delivery might thus allow reduced dosage of BLM and alleviate unwanted side effects from treatment, including pulmonary fibrosis.

An optimized MALDI MSI protocol for spatial detection of tryptic peptides in fresh frozen prostate tissue.

MALDI MS imaging (MSI) is a powerful analytical tool for spatial peptide detection in heterogeneous tissues. Proper sample preparation is crucial to achieve high quality, reproducible measurements. Here we developed an optimized protocol for spatially resolved proteolytic peptide detection with MALDI time-of-flight MSI of fresh frozen prostate tissue sections. The parameters tested included four different tissue washes, four methods of protein denaturation, four methods of trypsin digestion (different trypsin densities, sprayers, and incubation times), and five matrix deposition methods (different sprayers, settings, and matrix concentrations). Evaluation criteria were the number of detected and excluded peaks, percentage of high mass peaks, signal-to-noise ratio, spatial localization, and average intensities of identified peptides, all of which were integrated into a weighted quality evaluation scoring system. Based on these scores, the optimized protocol included an ice-cold EtOH+H2 O wash, a 5 min heating step at 95°C, tryptic digestion incubated for 17h at 37°C and CHCA matrix deposited at a final amount of 1.8 µg/mm2 . Including a heat-induced protein denaturation step after tissue wash is a new methodological approach that could be useful also for other tissue types. This optimized protocol for spatial peptide detection using MALDI MSI facilitates future biomarker discovery in prostate cancer and may be useful in studies of other tissue types.

UDP-glucose dehydrogenase expression is upregulated following EMT and differentially affects intracellular glycerophosphocholine and acetylaspartate levels in breast mesenchymal cell lines.

Metabolic rewiring is one of the indispensable drivers of epithelial-mesenchymal transition (EMT) involved in breast cancer metastasis. In this study, we explored the metabolic changes during spontaneous EMT in three separately established breast EMT cell models using a proteomic approach supported by metabolomic analysis. We identified common proteomic changes, including the expression of CDH1, CDH2, VIM, LGALS1, SERPINE1, PKP3, ATP2A2, JUP, MTCH2, RPL26L1 and PLOD2. Consistently altered metabolic enzymes included the following: FDFT1, SORD, TSTA3 and UDP-glucose dehydrogenase (UGDH). Of these, UGDH was most prominently altered and has previously been associated with breast cancer patient survival. siRNA-mediated knock-down of UGDH resulted in delayed cell proliferation and dampened invasive potential of mesenchymal cells and downregulated expression of the EMT transcription factor SNAI1. Metabolomic analysis revealed that siRNA-mediated knock-down of UGDH decreased intracellular glycerophosphocholine (GPC), whereas levels of acetylaspartate (NAA) increased. Finally, our data suggested that platelet-derived growth factor receptor beta (PDGFRB) signalling was activated in mesenchymal cells. siRNA-mediated knock-down of PDGFRB downregulated UGDH expression, potentially via NFkB-p65. Our results support an unexplored relationship between UGDH and GPC, both of which have previously been independently associated with breast cancer progression.

ALKBH3 partner ASCC3 mediates P-body formation and selective clearance of MMS-induced 1-methyladenosine and 3-methylcytosine from mRNA.

BACKGROUND: Reversible enzymatic methylation of mammalian mRNA is widespread and serves crucial regulatory functions, but little is known to what degree chemical alkylators mediate overlapping modifications and whether cells distinguish aberrant from canonical methylations. METHODS: Here we use quantitative mass spectrometry to determine the fate of chemically induced methylbases in the mRNA of human cells. Concomitant alteration in the mRNA binding proteome was analyzed by SILAC mass spectrometry. RESULTS: MMS induced prominent direct mRNA methylations that were chemically identical to endogenous methylbases. Transient loss of 40S ribosomal proteins from isolated mRNA suggests that aberrant methylbases mediate arrested translational initiation and potentially also no-go decay of the affected mRNA. Four proteins (ASCC3, YTHDC2, TRIM25 and GEMIN5) displayed increased mRNA binding after MMS treatment. ASCC3 is a binding partner of the DNA/RNA demethylase ALKBH3 and was recently shown to promote disassembly of collided ribosomes as part of the ribosome quality control (RQC) trigger complex. We find that ASCC3-deficient cells display delayed removal of MMS-induced 1-methyladenosine (m1A) and 3-methylcytosine (m3C) from mRNA and impaired formation of MMS-induced P-bodies. CONCLUSIONS: Our findings conform to a model in which ASCC3-mediated disassembly of collided ribosomes allows demethylation of aberrant m1A and m3C by ALKBH3. Our findings constitute first evidence of selective sanitation of aberrant mRNA methylbases over their endogenous counterparts and warrant further studies on RNA-mediated effects of chemical alkylators commonly used in the clinic.

Cancer-induced muscle atrophy is determined by intrinsic muscle oxidative capacity.

We tested the hypothesis that cancer cachexia progression would induce oxidative post-translational modifications (Ox-PTMs) associated with skeletal muscle wasting, with different responses in muscles with the prevalence of glycolytic and oxidative fibers. We used cysteine-specific isotopic coded affinity tags (OxICAT) and gel-free mass spectrometry analysis to investigate the cysteine Ox-PTMs profile in the proteome of both plantaris (glycolytic) and soleus (oxidative) muscles in tumor-bearing and control rats. Histological analysis revealed muscle atrophy in type II fibers in plantaris muscle, with no changes in plantaris type I fibers and no differences in both soleus type I and II fibers in tumor-bearing rats when compared to healthy controls. Tumor progression altered the Ox-PTMs profile in both plantaris and soleus. However, pathway analysis including the differentially oxidized proteins revealed tricarboxylic acid cycle and oxidative phosphorylation as main affected pathways in plantaris muscle from tumor-bearing rats, while the same analysis did not show main metabolic pathways affected in the soleus muscle. In addition, cancer progression affected several metabolic parameters such as ATP levels and markers of oxidative stress associated with muscle atrophy in plantaris muscle, but not in soleus. However, isolated soleus from tumor-bearing rats had a reduced force production capacity when compared to controls. These novel findings demonstrate that tumor-bearing rats have severe muscle atrophy exclusively in glycolytic fibers. Cancer progression is associated with cysteine Ox-PTMs in the skeletal muscle, but these modifications affect different pathways in a glycolytic muscle compared to an oxidative muscle, indicating that intrinsic muscle oxidative capacity determines the response to cancer cachectic effects.

Potential Antiviral Options against SARS-CoV-2 Infection.

As of June 2020, the number of people infected with severe acute respiratory coronavirus 2 (SARS-CoV-2) continues to skyrocket, with more than 6.7 million cases worldwide. Both the World Health Organization (WHO) and United Nations (UN) has highlighted the need for better control of SARS-CoV-2 infections. However, developing novel virus-specific vaccines, monoclonal antibodies and antiviral drugs against SARS-CoV-2 can be time-consuming and costly. Convalescent sera and safe-in-man broad-spectrum antivirals (BSAAs) are readily available treatment options. Here, we developed a neutralization assay using SARS-CoV-2 strain and Vero-E6 cells. We identified the most potent sera from recovered patients for the treatment of SARS-CoV-2-infected patients. We also screened 136 safe-in-man broad-spectrum antivirals against the SARS-CoV-2 infection in Vero-E6 cells and identified nelfinavir, salinomycin, amodiaquine, obatoclax, emetine and homoharringtonine. We found that a combination of orally available virus-directed nelfinavir and host-directed amodiaquine exhibited the highest synergy. Finally, we developed a website to disseminate the knowledge on available and emerging treatments of COVID-19.

Exercise training reverses cancer-induced oxidative stress and decrease in muscle COPS2/TRIP15/ALIEN.

OBJECTIVE: We tested the hypothesis that exercise training would attenuate metabolic impairment in a model of severe cancer cachexia. METHODS: We used multiple in vivo and in vitro methods to explore the mechanisms underlying the beneficial effects induced by exercise training in tumor-bearing rats. RESULTS: Exercise training improved running capacity, prolonged lifespan, reduced oxidative stress, and normalized muscle mass and contractile function in tumor-bearing rats. An unbiased proteomic screening revealed COP9 signalosome complex subunit 2 (COPS2) as one of the most downregulated proteins in skeletal muscle at the early stage of cancer cachexia. Exercise training normalized muscle COPS2 protein expression in tumor-bearing rats and mice. Lung cancer patients with low endurance capacity had low muscle COPS2 protein expression as compared to age-matched control subjects. To test whether decrease in COPS2 protein levels could aggravate or be an intrinsic compensatory mechanism to protect myotubes from cancer effects, we performed experiments in vitro using primary myotubes. COPS2 knockdown in human myotubes affected multiple cellular pathways, including regulation of actin cytoskeleton. Incubation of cancer-conditioned media in mouse myotubes decreased F-actin expression, which was partially restored by COPS2 knockdown. Direct repeat 4 (DR4) response elements have been shown to positively regulate gene expression. COPS2 overexpression decreased the DR4 activity in mouse myoblasts, and COPS2 knockdown inhibited the effects of cancer-conditioned media on DR4 activity. CONCLUSIONS: These studies demonstrated that exercise training may be an important adjuvant therapy to counteract cancer cachexia and uncovered novel mechanisms involving COPS2 to regulate myotube homeostasis in cancer cachexia.

HDACi mediate UNG2 depletion, dysregulated genomic uracil and altered expression of oncoproteins and tumor suppressors in B- and T-cell lines.

BACKGROUND: HDAC inhibitors (HDACi) belong to a new group of chemotherapeutics that are increasingly used in the treatment of lymphocyte-derived malignancies, but their mechanisms of action remain poorly understood. Here we aimed to identify novel protein targets of HDACi in B- and T-lymphoma cell lines and to verify selected candidates across several mammalian cell lines. METHODS: Jurkat T- and SUDHL5 B-lymphocytes were treated with the HDACi SAHA (vorinostat) prior to SILAC-based quantitative proteome analysis. Selected differentially expressed proteins were verified by targeted mass spectrometry, RT-PCR and western analysis in multiple mammalian cell lines. Genomic uracil was quantified by LC-MS/MS, cell cycle distribution analyzed by flow cytometry and class switch recombination monitored by FACS in murine CH12F3 cells. RESULTS: SAHA treatment resulted in differential expression of 125 and 89 proteins in Jurkat and SUDHL5, respectively, of which 19 were commonly affected. Among these were several oncoproteins and tumor suppressors previously not reported to be affected by HDACi. Several key enzymes determining the cellular dUTP/dTTP ratio were downregulated and in both cell lines we found robust depletion of UNG2, the major glycosylase in genomic uracil sanitation. UNG2 depletion was accompanied by hyperacetylation and mediated by increased proteasomal degradation independent of cell cycle stage. UNG2 degradation appeared to be ubiquitous and was observed across several mammalian cell lines of different origin and with several HDACis. Loss of UNG2 was accompanied by 30-40% increase in genomic uracil in freely cycling HEK cells and reduced immunoglobulin class-switch recombination in murine CH12F3 cells. CONCLUSION: We describe several oncoproteins and tumor suppressors previously not reported to be affected by HDACi in previous transcriptome analyses, underscoring the importance of proteome analysis to identify cellular effectors of HDACi treatment. The apparently ubiquitous depletion of UNG2 and PCLAF establishes DNA base excision repair and translesion synthesis as novel pathways affected by HDACi treatment. Dysregulated genomic uracil homeostasis may aid interpretation of HDACi effects in cancer cells and further advance studies on this class of inhibitors in the treatment of APOBEC-expressing tumors, autoimmune disease and HIV-1.

Complex alternative splicing of human Endonuclease V mRNA, but evidence for only a single protein isoform.

Endonuclease V (ENDOV) is a ribonuclease with affinity for inosine which is the deamination product of adenosine. The genomes of most organisms, including human, encode ENDOV homologs, yet knowledge about in vivo functions and gene regulation is sparse. To contribute in this field, we analyzed mRNA and protein expression of human ENDOV (hENDOV). Analyses of public sequence databases revealed numerous hENDOV transcript variants suggesting extensive alternative splicing. Many of the transcripts lacked one or more exons corresponding to conserved regions of the ENDOV core domain, suggesting that these transcripts do not encode for active proteins. Three complete transcripts were found with open reading frames encoding 282, 308 and 309 amino acids, respectively. Recombinant hENDOV 308 and hENDOV 309 share the same cleavage activity as hENDOV 282 which is the variant that has been used in previous studies of hENDOV. However, hENDOV 309 binds inosine-containing RNA with stronger affinity than the other isoforms. Overexpressed GFP-fused isoforms were found in cytoplasm, nucleoli and arsenite induced stress granules in human cells as previously reported for hENDOV 282. RT-qPCR analysis of the 3'-termini showed that hENDOV 308 and hENDOV 309 transcripts are more abundant than hENDOV 282 transcripts in immortalized cell lines, but not in primary cells, suggesting that cells regulate hENDOV mRNA expression. In spite of the presence of all three full-length transcripts, mass spectrometry analyses identified peptides corresponding to the hENDOV 309 isoform only. This result suggests that further studies of human ENDOV should rather encompass the hENDOV 309 isoform.

A targeted mass spectrometry immunoassay to quantify osteopontin in fresh-frozen breast tumors and adjacent normal breast tissues.

Osteopontin (OPN) is a multifunctional protein that can activate cell-signaling pathways and lead to cancer development and metastasis. Elevated OPN expression was reported in different cancer types, including breast tumors. Here, we present a new immuno-mass spectrometry method for OPN quantification in fresh-frozen malignant and adjacent normal human breast tissues. For quantification we used two proteotypic peptides: OPN-peptide-1 and OPN-peptide-2. Peptide concentrations were determined by liquid chromatography-tandem mass spectrometry (LC-MS/MS) in multiple reaction monitoring (MRM) mode with stable isotope standards (SIS) and immuno-affinity enrichment for isolation of OPN peptides. Based on the OPN-peptide-1, the average OPN concentration in normal breast tissue was 19.42 µg/g, while the corresponding level in breast tumors was 603.9 µg/g. Based on OPN-peptide-2, the average concentration in normal breast tissue was 19.30 µg/g and in breast tumors 535.0 µg/g. In ER/PR/HER2(-) patients the OPN levels in breast tumors were significantly higher than in corresponding normal breast tissue samples, whereas in the single ER/PR/HER2(+) patient the OPN concentration in tumor samples was lower than in normal breast tissue sample. In conclusion, the current method is considered promising for the quantification of OPN in research and in clinical settings and should be further studied in breast cancer patients. SIGNIFICANCE: A new immuno-mass spectrometry method was successfully developed and applied to determine OPN concentrations in malignant tumor and normal breast tissues from six patients, and the method is promising for OPN quantification in both research and clinical settings.

A Novel Truncated Form of Nephronectin Is Present in Small Extracellular Vesicles Isolated from 66cl4 Cells.

Extracellular vesicles are emerging as biomarkers in breast cancer. Our recent report suggested that an intracellular granular staining pattern of the extracellular matrix protein nephronectin (NPNT) in breast tumor sections correlated with a poor prognosis. Furthermore, the results showed that NPNT is localized in extracellular vesicles derived from mouse breast cancer cells. In this study, we performed proteomic analysis that revealed that several proteins, including tumor-promoting molecules, are differentially expressed in the cargo of small extracellular vesicles (sEVs) derived from NPNT-expressing mouse breast cancer cells. We also identified three different forms of NPNT at 80, 60, and 20 kDa. We report that the native form of NPNT at 60 kDa becomes further glycosylated and is detected as the 80 kDa NPNT, which may be processed by matrix metalloproteinases to a shorter form of around 20 kDa, which has not previously been described. Although both 80 and 20 kDa NPNT are detected in sEVs derived from breast cancer cells, the 20 kDa form of NPNT is concentrated in sEVs. In summary, we show that a novel truncated form of NPNT is found in sEVs derived from breast cancer cells.

Differential regulation of cysteine oxidative post-translational modifications in high and low aerobic capacity.

Given the association between high aerobic capacity and the prevention of metabolic diseases, elucidating the mechanisms by which high aerobic capacity regulates whole-body metabolic homeostasis is a major research challenge. Oxidative post-translational modifications (Ox-PTMs) of proteins can regulate cellular homeostasis in skeletal and cardiac muscles, but the relationship between Ox-PTMs and intrinsic components of oxidative energy metabolism is still unclear. Here, we evaluated the Ox-PTM profile in cardiac and skeletal muscles of rats bred for low (LCR) and high (HCR) intrinsic aerobic capacity. Redox proteomics screening revealed different cysteine (Cys) Ox-PTM profile between HCR and LCR rats. HCR showed a higher number of oxidized Cys residues in skeletal muscle compared to LCR, while the opposite was observed in the heart. Most proteins with differentially oxidized Cys residues in the skeletal muscle are important regulators of oxidative metabolism. The most oxidized protein in the skeletal muscle of HCR rats was malate dehydrogenase (MDH1). HCR showed higher MDH1 activity compared to LCR in skeletal, but not cardiac muscle. These novel findings indicate a clear association between Cys Ox-PTMs and aerobic capacity, leading to novel insights into the role of Ox-PTMs as an essential signal to maintain metabolic homeostasis.

Photodynamic treatment with hexyl-aminolevulinate mediates reversible thiol oxidation in core oxidative stress signaling proteins.

Photodynamic therapy (PDT) is a highly selective two-step cancer treatment involving a photosensitizer and illumination with visible light in the presence of molecular oxygen. PDT is clinically approved worldwide for treating several premalignant conditions and cancer forms, especially endoscopically accessible tumors and dermatological malignancies. PDT-mediated cytotoxicity takes place via autophagy, apoptosis and necrosis, but the exact trigger mechanisms for various death-pathways are still unknown. PDT induces reactive oxygen species (ROS) through photochemical reactions. ROS can react with different macromolecules resulting in cellular damage, including oxidation of proteins. One of the known protein modifications is reversible oxidation of cysteine thiols (-SH), which in many cases constitute a redox switch to modulate protein activity and cellular signaling. Here we have examined the role of reversible oxidation of protein thiols as a potential mediator of cytotoxicity after hexylaminolevulinate-mediated photodynamic treatment (HAL-PDT) in the human epidermoid carcinoma cell line A431. Nearly 2300 proteins were found to be reversibly oxidized after HAL-PDT, of which 374 high-confidence proteins were further allocated to cellular compartments and functional networks. 115 of the high confidence proteins were associated with apoptosis and 257 have previously not been reported to be reversibly oxidized on cysteines. We find an enrichment of DNA damage checkpoint and oxidative stress response proteins. Many of these constitute potential signaling hubs in apoptosis, including ATM, p63, RSK1 p38, APE1/Ref-1 and three 14-3-3 family members. Our study represents the first comprehensive mapping of reversibly oxidized proteins subsequent to HAL-PDT. Several of the proteins constitute potentially novel redox-regulated apoptotic triggers as well as potential targets for adjuvants that may improve the efficacy of HAL-PDT and PDT using other photosensitizers.

Studies of the photosensitizer disulfonated meso-tetraphenyl chlorin in an orthotopic rat bladder tumor model.

BACKGROUND: Photochemical internalization (PCI) is a novel technology for the release of a therapeutic molecule from endocytic vesicles into the cytosol of a cell. The release of molecules occurs after activation of an endocytic membrane-embedded photosensitizer by light. In this study uptake and localization of the photosensitizer disulfonated tetraphenyl chlorin (TPCS2a) were explored to optimize a PCI protocol in an orthotopic rat bladder tumor model. METHODS: Female Fischer F344 rats were intravesically instilled with 0.4×10(6) AY-27 transitional carcinoma cells before allowing tumor growth for 14 days. The photosensitizer TPCS2a was intravesically instilled at different concentrations, and bladders were excised after different time intervals. The retention, penetration, and localization of intratumoral TPCS2a were explored ex vivo using fluorescence spectroscopy and fluorescence microscopy to determine an optimal PCI protocol. These results were compared to histological analysis of necrotic areas after activation of intratumoral TPCS2a by red light (652nm, 0.5J/cm(2)). RESULTS: A superficial distribution pattern of the photosensitizer TPCS2a was seen in bladder tumor tissue, and TPCS2a was almost cleared from the tumors after 72h. The highest retention of TPCS2a was found at 24h after instillation when using a concentration of 3mg/ml. CONCLUSION: An optimal PCI protocol was defined for the tumor model, including a 24-h TPCS2a-to-light interval and a dose of 3mg/ml TPCS2a. This protocol will be utilized for the study of PCI-enhanced therapeutic effects on non-muscle invasive bladder cancer, using a potent chemotherapeutic under an optimal light dose.

Enhanced efficacy of bleomycin in bladder cancer cells by photochemical internalization.

Bleomycin is a cytotoxic chemotherapeutic agent widely used in cancer treatment. However, its efficacy in different cancers is low, possibly due to limited cellular internalization. In this study, a novel approach known as photochemical internalization (PCI) was explored to enhance bleomycin delivery in bladder cancer cells (human T24 and rat AY-27), as bladder cancer is a potential indication for use of PCI with bleomycin. The PCI technique was mediated by the amphiphilic photosensitizer disulfonated tetraphenyl chlorin (TPCS(2a)) and blue light (435 nm). Two additional strategies were explored to further enhance the cytotoxicity of bleomycin; a novel peptide drug ATX-101 which is known to impair DNA damage responses, and the protease inhibitor E-64 which may reduce bleomycin degradation by inhibition of bleomycin hydrolase. Our results demonstrate that the PCI technique enhances the bleomycin effect under appropriate conditions, and importantly we show that PCI-bleomycin treatment leads to increased levels of DNA damage supporting that the observed effect is due to increased bleomycin uptake. Impairing the DNA damage responses by ATX-101 further enhances the efficacy of the PCI-bleomycin treatment, while inhibiting the bleomycin hydrolase does not.

Psoriasis pathogenesis - Pso p27 is generated from SCCA1 with chymase.

Psoriasis is a chronic inflammatory skin disease with unknown aetiology. Infiltration of inflammatory cells as the initial event in the development of new psoriatic plaques together with the defined inflamed areas of such lesions argues for an immunological disease with a local production of a causal antigen. The auto-antigen Pso p27 is a protein expressed in the skin lesions. We recently demonstrated that Pso p27 is homologous to the core amino acid sequences of squamous cell carcinoma antigens 1 and 2 (SCCA1/2) and it is apparently generated from SCCA molecules by digestion with highly specific endoproteases. In this communication we demonstrate the generation of Pso p27 from SCCA1 with extracts from psoriatic scale and even more remarkably, the generation of Pso p27 from SCCA1 in the presence of mast cell associated chymase. These findings open up for new therapeutic strategies in psoriasis and probably also in other autoimmune diseases as Pso p27 epitopes have been detected in diseased tissues from patients with various chronic inflammatory diseases.

The UNG2 Arg88Cys variant abrogates RPA-mediated recruitment of UNG2 to single-stranded DNA.

In human cell nuclei, UNG2 is the major uracil-DNA glycosylase initiating DNA base excision repair of uracil. In activated B cells it has an additional role in facilitating mutagenic processing of AID-induced uracil at Ig loci and UNG-deficient patients develop hyper-IgM syndrome characterized by impaired class-switch recombination and disturbed somatic hypermutation. How UNG2 is recruited to either error-free or mutagenic uracil processing remains obscure, but likely involves regulated interactions with other proteins. The UNG2 N-terminal domain contains binding motifs for both proliferating cell nuclear antigen (PCNA) and replication protein A (RPA), but the relative contribution of these interactions to genomic uracil processing is not understood. Interestingly, a heterozygous germline single-nucleotide variant leading to Arg88Cys (R88C) substitution in the RPA-interaction motif of UNG2 has been observed in humans, but with unknown functional relevance. Here we demonstrate that UNG2-R88C protein is expressed from the variant allele in a lymphoblastoid cell line derived from a heterozygous germ line carrier. Enzyme activity as well as localization in replication foci of UNG2-R88C was similar to that of WT. However, binding to RPA was essentially abolished by the R88C substitution, whereas binding to PCNA was unaffected. Moreover, we show that disruption of the PCNA-binding motif impaired recruitment of UNG2 to S-phase replication foci, demonstrating that PCNA is a major factor for recruitment of UNG2 to unperturbed replication forks. Conversely, in cells treated with hydroxyurea, RPA mediated recruitment of UNG2 to stalled replication forks independently of functional PCNA binding. Modulation of PCNA- versus RPA-binding may thus constitute a functional switch for UNG2 in cells subsequent to genotoxic stress and potentially also during the processing of uracil at the immunoglobulin locus in antigen-stimulated B cells.

Photodynamic therapy with hexyl aminolevulinate induces carbonylation, posttranslational modifications and changed expression of proteins in cell survival and cell death pathways.

Photodynamic therapy (PDT) using blue light and the potent precursor for protoporphyrin IX, hexyl aminolevulinate (HAL), has been shown to induce apoptosis and necrosis in cancer cells, but the mechanism remains obscure. In the present study, we examined protein carbonylation, expression levels and post-translational modifications in rat bladder cells (AY-27) after PDT with HAL. Altered levels of expression and/or post-translational modifications induced by PDT were observed for numerous proteins, including proteins required for cell mobility, energy supply, cell survival and cell death pathways, by using two-dimensional difference gel electrophoresis (2D-DIGE) and mass spectrometry (MS). Moreover, 10 carbonylated proteins associated with cytoskeleton, transport, oxidative stress response, protein biosynthesis and stability, and DNA repair were identified using immunoprecipitation, two-dimensional gel electrophoresis and MS. Overall, the results indicate that HAL-mediated PDT triggers a complex cellular response involving several biological pathways. Our findings may account for the elucidation of mechanisms modulated by PDT, paving the way to improve clinic PDT-efficacy.