Beyond cells: The extracellular circulating 20S proteasomes.

Accumulating evidence arising from numerous clinical studies indicate that assembled and functional 20S proteasome complexes circulate freely in plasma. Elevated levels of this core proteolytic complex have been found in the plasma of patients suffering from blood, skin and solid cancers, autoimmune disorders, trauma and sepsis. Moreover, in various diseases, there is a positive correlation between circulating 20S proteasome (c20S) levels and treatment efficacy and survival rates, suggesting the involvement of this under-studied c20S complex in pathophysiology. However, many aspects of this system remain enigmatic, as we still do not know the origin, biological role or mechanisms of extracellular transport and regulation of c20S proteasomes. In this review, we provide an overview of the current understanding of the c20S proteasome system and discuss the remaining gaps in knowledge.

Elevated plasma 20S proteasome chymotrypsin-like activity is correlated with IL-8 levels and associated with an increased risk of death in glial brain tumor patients.

INTRODUCTION: In cancer treatment an attempt has been made to pharmacologically regulate the proteasome functions, thus the aim was to test whether 20S proteasome chymotrypsin-like (ChT-L) activity has a role in glial brain tumors. Furthermore, we analyzed the correlation between proteasome activity and IL-8, CCL2, NF-κB1 and NF-κB2 concentrations, which impact on brain tumors has already been indicated. METHODS: Plasma 20S proteasome ChT-L activity was assayed using the fluorogenic peptide substrate Suc-Leu-Leu-Val-Tyr-AMC in the presence of SDS. IL-8, CCL2, NF-κB1 and NF-κB2 concentration was analyzed with the use of ELISA method. Immunohistochemistry for IDH1-R132H was done on 5-microns-thick formalin-fixed, paraffin-embedded tumor sections with the use of antibody specific for the mutant IDH1-R132H protein. Labelled streptavidin biotin kit was used as a detection system. RESULTS: Brain tumor patients had statistically higher 20S proteasome ChT-L activity (0.649 U/mg) compared to non-tumoral individuals (0.430 U/mg). IDH1 wild-type patients had statistically higher 20S proteasome ChT-L activity (1.025 U/mg) compared to IDH1 mutants (0.549 U/mg). 20S proteasome ChT-L activity in brain tumor patients who died as the consequence of a tumor (0.649) in the following 2 years was statistically higher compared to brain tumor patients who lived (0.430 U/mg). In brain tumor patients the 20S proteasome ChT-L activity positively correlated with IL-8 concentration. CONCLUSIONS: Elevated 20S proteasome ChT-L activity was related to the increased risk of death in glial brain tumor patients. A positive correlation between 20S proteasome ChT-L activity and IL-8 concentration may indicate the molecular mechanisms regulating glial tumor biology. Thus research on proteasomes may be important and should be carried out to verify if this protein complexes may represent a potential therapeutic target to limit brain tumor invasion.

PSMD11, PTPRM and PTPRB as novel biomarkers of pancreatic cancer progression.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) has the lowest survival rate of all major cancers. Surgery is the only curative intent therapy, but the majority of patients experience disease relapse. Thus, patients who do not benefit from highly morbid surgical resection needs to be identified and offered palliative chemotherapy instead. In this pilot study, we aimed to identify differentially regulated proteins in plasma and plasma derived microparticles from PDAC patients with poor and good prognosis. METHODS: Plasma and plasma derived microparticle samples were obtained before surgical resection from PDAC patients. Sequential Windowed Acquisition of all Theoretical fragment ion spectra - Mass Spectrometry (SWATH-MS) proteomic analysis was performed to identify and quantify proteins in these samples. Statistical analysis was performed to identify biomarkers for poor prognosis. RESULTS: A total of 482 and 1024 proteins were identified from plasma and microparticle samples, respectively, by SWATH-MS analysis. Statistical analysis of the data further identified nine and six differentially (log2ratio > 1, p < .05) expressed proteins in plasma and microparticles, respectively. Protein tyrosine phosphatases, PTPRM and PTPRB, were decreased in plasma of patients with poor PDAC prognosis, while proteasomal subunit PSMD11 was increased in microparticles of patients with poor prognosis. CONCLUSION AND GENERAL SIGNIFICANCE: A novel blood-based biomarker signature for PDAC prognosis was identified.

Biomarker panel for early detection of endometrial cancer in the Prostate, Lung, Colorectal, and Ovarian cancer screening trial.

BACKGROUND: Endometrial cancer is the most common gynecological cancer in the United States. However, no early detection test exists for asymptomatic women at average risk for endometrial cancer. OBJECTIVE: We sought to identify early detection biomarkers for endometrial cancer using prediagnostic serum. STUDY DESIGN: We performed a nested case-control study of postmenopausal women in the Prostate, Lung, Colorectal, and Ovarian cancer screening trial (n = 78,216), including 112 incident endometrial cancer cases and 112 controls. Prediagnostic serum was immunodepleted of high-abundance proteins and digested with sequencing grade porcine trypsin via pressure cycling technology. Quantitative proteomics and phosphoproteomics was performed using high-resolution liquid chromatography-tandem mass spectrometry and highly multiplexed isobaric mass tag combined with basic reversed-phase liquid chromatography. A set of proteins able to predict cancer status was identified with an integrated score assessed by receiver-operator curve analysis. RESULTS: Mean time from blood draw to endometrial cancer diagnosis was 3.5 years (SD, 1.9 years). There were 47 differentially abundant proteins between cases and controls (P < .05). Protein alterations with high predictive potential were selected by regression analysis and compiled into an aggregate score to determine the ability to predict endometrial cancer. An integrated risk score of 6 proteins was directly related to disease incidence in cases with blood draw ≤2 years, >2 years to ≤5 years or >5 years prior to cancer diagnosis. The integrated score distinguished cases from controls with an area under the curve of 0.80 (95% confidence interval, 0.72-0.88). CONCLUSION: An integrated score of 6 proteins using prediagnostic serum from the Prostate, Lung, Colorectal, and Ovarian cancer screening trial distinguishes postmenopausal endometrial cancer cases from controls. Validation is needed to evaluate whether this test can improve prediction or detection of endometrial cancer among postmenopausal women.

A novel mechanism for immune regulation after human lung transplantation.

OBJECTIVE: Lung transplantation is therapeutic for end-stage lung disease, but survival is limited due to bronchiolitis obliterans syndrome and restrictive chronic lung allograft dysfunction. We sought a common denominator in lung transplant recipients, analyzing risk factors that trigger immune responses that lead to bronchiolitis obliterans syndrome. METHODS: We collected blood from patients who underwent lung transplant at our institution. Exosomes were isolated from the sera of recipients with risk factors for chronic rejection and from stable recipients. Exosomes were analyzed with western blot, using antibodies to lung self-antigens K alpha 1 tubulin and collagen-V, costimulatory molecules (costimulatory molecule 80, costimulatory molecule 86), transcription factors (nuclear factor kappa-light-chain-enhancer of activated B cells, hypoxia-inducible factor 1α, Class II Major Histocompatibility Complex Transactivator), and 20S proteasome. RESULTS: Of the 90 patients included, we identified 5 with grade 3 primary graft dysfunction, 5 without, 15 with respiratory viral infection, 10 with acute rejection, 10 with donor-specific antibodies (DSA), 5 without DSA, and 10 who were stable for exosome isolation. Recipients with grade 3 primary graft dysfunction, respiratory viral infection, acute rejection, and DSA had exosomes containing self-antigens; exosomes from stable recipients did not. Exosomes from recipients with grade 3 primary graft dysfunction, acute rejection, and DSA also demonstrated costimulatory molecule 80, costimulatory molecule 86, major histocompatibility complex class II, transcription factor, and 20S proteasome. CONCLUSIONS: Transplanted lungs with grade 3 primary graft dysfunction, symptomatic respiratory viral infection, acute rejection, and immune responses induce exosomes that contain self-antigens, costimulatory molecules, major histocompatibility complex class II, transcription factors, and 20S proteasome. Release of circulating exosomes post-transplant from the aforementioned stress-inducing insults augment immunity and may play an important role in the pathogenesis of bronchiolitis obliterans syndrome.

Proteasome Activity and C-Reactive Protein Concentration in the Course of Inflammatory Reaction in Relation to the Type of Abdominal Operation and the Surgical Technique Used.

Surgical tissue damage and the accompanying inflammatory response lead to proteasome activation, initiation of damaged protein degradation, and induction of acute-phase inflammatory response. The aim of this study was to investigate the rate of change in proteasome chymotrypsin-like (ChT-L) activity and C-reactive protein concentration depending on the degree of tissue damage and their correlation with prealbumin concentrations in children before and after abdominal surgery. This experimental study included children who underwent abdominal surgery between 2015 and 2017. Plasma prealbumin concentrations and C-reactive protein levels (CRP) were determined by standard biochemical laboratory procedures. Proteasome activity was assessed using a Suc-Leu-Leu-Val-Tyr-AMC peptide substrate. Elevation of plasma proteasome activity was noted in children after laparoscopic and open abdominal surgeries. However, 20S proteasome activity in children undergoing conventional open surgery was significantly higher (P < 0.05) than in patients subjected to laparoscopy. At the same time, an increase in the CRP level was observed. However, there was no correlation between C-reactive protein concentrations and the type of abdominal surgery while there was a correlation observed in the case of proteasomes. Proteasome activity correlates with the degree of surgical tissue damage and prealbumin concentrations. More invasive surgery leads to a stronger activation of the proteasome involved in removing proteins that were damaged due to the surgical procedure. Proteasomes are more specific markers because there is a correlation between proteasome activity and the type of abdominal surgery in contrast to C-reactive protein concentrations which are not different in response to surgery performed in regard to ovarian cysts or cholelithiasis.

Determination of the concentration of cathepsin B by SPRI biosensor in children with appendicitis, and its correlation with proteasomes.

BACKGROUND: Cathepsin B (CatB) belongs to a family of lysosomal cysteine proteases and plays an important role in intracellular proteolysis. OBJECTIVES: The concentration of CatB and 20S proteasome was evaluated in the serum of children with appendicitis, before and after surgery, on a basis of an innovative method for determining biomolecules concentration - surface plasmon resonance imaging (SPRI) biosensor. MATERIAL AND METHODS: Forty-two children with acute appendicitis, who were treated at the Department of Pediatric Surgery (Medical University of Bialystok, Poland), were randomly included into the study (age: 5-17 years, mean age: 11.5 ±1 year). There were 15 girls and 27 boys in the study group. Eighteen healthy, age-matched subjects, admitted for planned surgeries, served as controls. Exclusion criteria were the following: severe preexisting infections, immunological or cardiovascular diseases that required longterm medication, and complicated cases of appendicitis with perforation of the appendix and/or peritonitis. RESULTS: The CatB concentrations in the blood plasma of patients with acute appendicitis were elevated before surgery, they were the highest 24 h after surgery, and were above the range of concentrations measured in controls; the difference was statistically significant. The CatB concentration measured 72 h after the operation was decreased, but still did not reach the normal range when compared with the concentration measured in controls (p < 0.05). CONCLUSIONS: Cathepsin B concentration may reflect the metabolic response to acute state of inflammation, surgical intervention in the abdominal cavity and the process of gradual ebbing of the inflammation. The method of operation - classic open appendectomy or laparoscopic appendectomy - does not influence the general trend in the CatB concentration in children with appendicitis. There is a strong positive correlation between the CatB and 20S proteasome concentrations 24 h after surgery. The SPRI method can be successfully used for determining the concentration of active forms of enzymes presented in lysosomes in the diagnosis of inflammatory conditions in the abdominal cavity.

26S proteasome and insulin-like growth factor-1 in serum of dogs suffering from malignant tumors.

Studies in humans have shown that the ubiquitin-proteasome pathway and the insulin-like growth factor axis are involved in carcinogenesis, thus, components of these systems might be useful as prognostic markers and constitute potential therapeutic targets. In veterinary medicine, only a few studies exist on this topic. Here, serum concentrations of 26S proteasome (26SP) and insulin-like growth factor-1 (IGF-1) were measured by canine enzyme-linked immunosorbent assay (ELISA) in 43 dogs suffering from malignant tumors and 21 clinically normal dogs (control group). Relationships with tumor size, survival time, body condition score (BCS), and tumor entity were assessed. The median 26SP concentration in the tumor group was non-significantly higher than in the control group. However, dogs with mammary carcinomas displayed significantly increased 26SP levels compared to the control group and dogs with tumor size less than 5 cm showed significantly increased 26SP concentrations compared to dogs with larger tumors and control dogs. 26SP concentrations were not correlated to survival time or BCS. No significant difference in IGF-1 levels was found between the tumor group and the control group; however, IGF-1 concentrations displayed a larger range of values in the tumor group. Dogs with tumors greater than 5 cm showed significantly higher IGF-1 levels than dogs with smaller tumors. The IGF-1 concentrations were positively correlated to survival time, but no correlation with BCS was found. Consequently, serum 26SP concentrations seem to be increased in some dogs suffering from malignant tumors, especially in dogs with mammary carcinoma and smaller tumors. Increased serum IGF-1 concentrations could be an indication of large tumors and a poor prognosis.

Des études chez l'humain ont démontré que la voie ubiquitine-protéasome et l'axe du facteur de croissance apparenté à l'insuline sont impliqués dans la carcinogénèse, et ainsi, des composantes de ces systèmes pourraient être utiles en tant que marqueurs du pronostic et constituer des cibles thérapeutiques potentielles. En médecine vétérinaire, seules quelques études existent sur ce sujet. Dans cette étude, les concentrations sériques de protéasome 26S (26SP) et du facteur de croissance 1 apparenté à l'insuline (IGF-1) ont été mesurés par réaction immunoenzymatique (ELISA) chez 43 chiens souffrant de tumeurs malignes et 21 chiens cliniquement normaux (groupe témoin). Les associations entre la taille des tumeurs, le temps de survie, le pointage de la condition corporelle (PCC) et le type de tumeurs ont été évaluées. La concentration médiane de 26SP dans le groupe avec tumeur était plus élevée que celle du groupe témoin mais de manière non-significative. Toutefois, les chiens avec des carcinomes mammaires montraient des quantités significativement augmentées de 26SP comparativement au groupe témoin et les chiens avec des tumeurs dont la taille était de moins de 5 cm avaient des concentrations de 26SP significativement augmentées comparativement aux chiens avec des tumeurs plus grosses et aux chiens du groupe témoin. Les concentrations de 26SP n'étaient pas corrélées au temps de survie ou au PCC. Aucune différence significative dans les niveaux d'IGF-1 ne fut trouvée entre le groupe avec tumeur et le groupe témoin; toutefois, les concentrations d'IGF-1 s'étendaient sur un plus large spectre dans le groupe avec tumeur. Les chiens avec des tumeurs plus large que 5 cm avaient des concentrations d'IGF-1 significativement plus élevées que les chiens avec des tumeurs plus petites. Les concentrations d'IGF-1 étaient corrélées positivement avec le temps de survie, mais aucune corrélation avec le PCC ne fut trouvée. Conséquemment, les concentrations de 26SP semblent être augmentées chez quelques chiens souffrant de tumeurs malignes, et plus spécialement les chiens avec des carcinomes mammaires et des plus petites tumeurs. Des concentrations augmentées d'IGF-1 pourraient être une indication d'une grosse tumeur et d'un pronostic sombre.(Traduit par Docteur Serge Messier).

Selective Substrates and Activity-Based Probes for Imaging of the Human Constitutive 20S Proteasome in Cells and Blood Samples.

The proteasome is an enzyme complex critical for maintaining protein homeostasis. Perturbed proteasome function leads to pathologies including cancer and autoimmune and neurodegenerative disease. Therefore, the proteasome constitutes an excellent molecular target for pharmaceutical development. Here, using the HyCoSuL approach, we designed and synthesized novel and selective fluorogenic substrates for each of these three constitutive 20S proteasome activities and applied them to assess inhibition of proteasome subunits by MG-132 and a clinically used inhibitor bortezomib. Our results confirm the utility of designed substrates in biochemical assays. Furthermore, selective peptide sequences obtained in this manner were used to construct fluorophore-labeled activity-based probes and then utilized to detect each constitutive 20S proteasome subunit simultaneously in lysates of HEK-293F cells and red blood cells. Overall, we describe a simple and rapid method useful to measure constitutive 20S proteasome activity in whole human blood samples that could enable early diagnosis of pathological states associated with aberrantly upregulated proteasome activity.

Proteasome 20S in multiple myeloma: comparison of concentration and chymotrypsin-like activity in plasma and serum.

The ubiquitin-proteasome system is relevant in the pathobiology of many haematological malignancies, including multiple myeloma. The assessment of proteasome concentration and chymotrypsin-like (ChT-L) activity might constitute a new approach to diagnosis, prognosis and monitoring of anticancer treatment of patients with haematological malignancies and other diseases. The aim of our study was to determine which material, plasma or serum, is better for measuring chymotrypsin-like (ChT-L) activity and proteasome concentration. We analysed proteasome concentration and chymotrypsin-like (ChT-L) activity in 70 plasma and serum samples drawn from 28 patients at different treatment stages for multiple myeloma (MM) and 31 healthy volunteers. Proteasome ChT-L activity and concentration in multiple myeloma patients were significantly higher in plasma compared to serum. In this group we observed significant and positive correlations both between the plasma and serum proteasome ChT-L activity and plasma and serum proteasome concentration. The higher values of proteasome concentration and ChT-L activity in plasma than in serum and their better correlations with parameters of tumour load and prognosis suggest that plasma constitutes a better biological material for measuring ChT-L activity and proteasome concentration than serum in multiple myeloma patients.

Concentration of Proteasome in the Blood Plasma of Children with Acute Appendicitis, Before and After Surgery, and Its Correlation with CRP.

OBJECTIVE: The determination of 20S proteasome concentration in the blood plasma of children with appendicitis and its correlation with CRP. DESIGN AND SETTING: Thirty-one children with acute appendicitis, were randomly included into the study (age 5 years up to 17 years, mean age 11.5 + 1 years). PARTICIPANTS: There were 17 girls and 14 boys. Eighteen healthy, age-matched subjects, admitted for planned surgeries served as controls. Exclusion criteria were: severe preexisting infections, immunological or cardiovascular diseases that required long-term medication, and complicated cases of appendicitis with perforation of appendix and/or peritonitis. MAIN OUTCOME MEASURES: The 20S proteasome concentrations in the blood plasma of patients with acute appendicitis were highest before the surgery and were above the range of concentrations measured in controls, and the difference was statistically significant. RESULTS: The 20S proteasome concentration measured 24 and 72 h after the operation, slowly decreased over time, and still did not reach the normal range, when compared with the concentration measured in controls (p < 0.05). CONCLUSIONS: 20S proteasome concentration may reflect the metabolic response to acute state inflammation, and the process of gradual ebbing of the inflammation. The method of operation-classic open appendectomy, or laparoscopic appendectomy, does not influence the general trend in 20S proteasome concentration.

Immunoproteasome in the blood plasma of children with acute appendicitis, and its correlation with proteasome and UCHL1 measured by SPR imaging biosensors.

The aim of this study was to determinate the immunoproteasome concentration in the blood plasma of children with appendicitis, and its correlation with circulating proteasome and ubiquitin carboxyl-terminal hydrolase L1 (UCHL1). Twenty-seven children with acute appendicitis, managed at the Paediatric Surgery Department, were included randomly into the study (age 2 years 9 months up to 14 years, mean age 9·5 ± 1 years). There were 10 girls and 17 boys; 18 healthy, age-matched subjects, admitted for planned surgeries served as controls. Mean concentrations of immunoproteasome, 20S proteasome and UCHL1 in the blood plasma of children with appendicitis before surgery 24 h and 72 h after the appendectomy were higher than in the control group. The immunoproteasome, 20S proteasome and UCHL1 concentrations in the blood plasma of patients with acute appendicitis were highest before surgery. The immunoproteasome, 20S proteasome and UCHL1 concentration measured 24 and 72 h after the operation decreased slowly over time and still did not reach the normal range (P < 0·05). There was no statistical difference between immunoproteasome, 20S proteasome and UCHL1 concentrations in children operated on laparoscopically and children after classic appendectomy. The immunoproteasome concentration may reflect the metabolic response to acute state inflammation, and the process of gradual ebbing of the inflammation may thus be helpful in the assessment of the efficacy of treatment. The method of operation - classic open appendectomy or laparoscopic appendectomy - does not influence the general trend in immunoproteasome concentration in children with appendicitis.

Down-regulation of TYK2, CBLB and LMP7 genes expression in relapsing-remitting multiple sclerosis patients treated with interferon-beta.

This study aimed to examine the expression of TYK2, CBLB and LMP7 genes at both mRNA and protein levels in relapsing-remitting MS (RRMS) patients in compare with healthy controls. Seventy-eight RRMS patients treated with IFNß-1a and 79 age- and ethnic-matched healthy subjects were studied. The mRNA expression levels of TYK2, CBLB and LMP7 in PBMCs were quantified by real-time PCR and plasma concentrations of three molecules were measured by ELISA. Results were compared between patients and controls, IFNß-responders and non-responders. Forty-nine of 78 patients were classified as IFNß-responders and 29 cases were non-responders. Significantly down-regulated expression of TYK2, CBLB and LMP7 genes was found in the patients group versus controls (P<0.001). Decreased plasma levels of three molecules were observed in patients compared to controls (P<0.001). IFNß-responders had significantly higher expressions for CBLB (P=0.001) and LMP7 (P=0.02) than non-responders. Also, we observed increased expressions of LMP7 (P=0.39) and CBLB (P=0.02) genes in patients under 30y and increased expression of TYK2 in patients >40years (P=0.002). Our results suggest that expression analysis of TYK2, CBLB and LMP7 genes could be useful for evaluation of T cells immunity and clinical response to IFNß-therapy in RRMS patients.

Proteasome and C-reactive protein inflammatory response in children undergoing shorter and longer lasting laparoscopic cholecystectomy.

Operations of varying duration cause the release of a number of inflammatory mediators, in particular cytokines which lead to proteasome and acute-phase reactions. The purpose of this novel human study, was to characterize inflammatory response in children undergoing laparoscopic cholecystectomy, by analyzing changes in selected inflammatory mediators: C-reactive protein concentration and circulating 20S proteasome activity following surgical injury and to correlate them with the duration of the surgical procedure. Plasma C-reactive protein concentration (CRP) was determined by standard biochemical laboratory procedures. Proteasome activity in the plasma of children was assessed using Suc-Leu-Leu-Val-Tyr-AMC peptide substrate. Statistically significant increase in the plasma proteasome activity and C-reactive protein concentration, was noted (p < .05) in children after laparoscopic cholecystectomy. We found the correlation between the 20S proteasome activity and the length of the procedure. In children undergoing longer lasting laparoscopic cholecystectomy the proteasome activity was much higher than in patients having shorter surgical procedure. The CRP concentration and 20S proteasome activity significantly increase after surgery, but only 20S proteasome activity correlate with the length of the surgery. This may confirm that CRP is only an indicator of pathological state, while the function of the proteasomes is more complex because of their participation in the processes of repair and wound healing, and in the removal of damaged proteins.

Circulating Proteasomes in the Pathogenesis of Head and Neck Squamous Cell Carcinoma.

Increased proteasome activity was revealed in blood serum of patients with stage T1N0M0 head and neck squamous cell carcinoma in comparison with patients with chronic diseases of the larynx and laryngopharynx. This opens prospects of using chymotrypsin-like activity measurement for differential diagnosis of squamous cell carcinoma, screening for high-risk groups, and evaluation of the degree of tumor differentiation.

Proteasome activity related with the daily physical activity of COPD patients.

BACKGROUND: COPD is a debilitating disease that affects patients' daily lives. One's daily physical activity (DPA) decreases due to multifactorial causes, and this decrease is correlated with a poor prognosis in COPD patients. Muscle wasting may at least be partly due to increased activity of the ubiquitin proteasome pathway and apoptosis. METHODS: This study investigated the relationships among DPA, circulating proteasome activity, and protein carbonyl in COPD patients and healthy subjects (HSs). This study included 57 participants (42 patients and 15 healthy subjects). Ambulatory DPA was measured using actigraphy, and oxygen saturation was measured with a pulse oximeter. RESULTS: COPD patients had lower DPA, lower 6 min walking distance (6MWD), lower delta saturation pulse oxygenation (SpO2) during the 6MWT, and lower delta SpO2 during DPA than HSs. COPD patients had higher proteasome activity and protein carbonyl than HSs. Circulating proteasome activity was significantly negatively correlated with DPA (r=-0.568, P<0.05) in COPD patients, whereas delta SpO2 during the 6MWT was significantly positively correlated with proteasome activity (r=0.685, P<0.05) in HSs. Protein carbonyl was significantly negatively correlated with the body mass index (r=-0.318, P<0.05), mid-arm circumference (r=0.350, P<0.05), calf circumference (r=0.322, P<0.05), forced expiratory volume in the first second (r=-0.441, P<0.01), and 6MWD (r=-0.313, P<0.05) in COPD patients. Our results showed no significant difference in inflammatory markers (interleukin-6 and tumor necrosis factor-α) or ubiquitin between the two groups. CONCLUSION: COPD patients had lower DPA levels and higher circulating proteasome activity than HSs, and a negative correlation of DPA with circulating proteasome activity.

Plasma Immunoproteasome Predicts Early Hemorrhagic Transformation in Acute Ischemic Stroke Patients.

BACKGROUND AND PURPOSE: Currently, blood biomarkers associated with an increased hemorrhagic transformation (HT) risk remain uncertain. We aimed to determine the significance of immunoproteasome as predictors of early HT in acute ischemic stroke patients. METHODS: This study enrolled 316 patients with ischemic stroke. HT was assessed by computed tomography examination performed on day 5 ± 2 after stroke onset or immediately in case of clinical deterioration (CD). Plasma immunoproteasome subunits low molecular mass peptide 2 (LMP2), multicatalytic endopeptidase complex-like 1 (MECL-1), LMP7, interleukin-1ß (IL1ß), and high-sensitivity C-reactive protein (Hs-CRP) were measured with quantitative sandwich enzyme-linked immunosorbent assay kits. Factors associated with HT were analyzed using a multivariate logistic regression analysis. RESULTS: There were 42 (13.3%, 42 of 316) patients who experienced HT. Compared with those patients without HT, plasma LMP2, MECL-1, LMP7, IL1ß, and Hs-CRP concentrations on admission were significantly increased in patients with subsequent HT (P < .001). These protein concentrations increased with hemorrhage severity. Patients with CD caused by HT had the highest levels of LMP2 (1679.5 [1394.6-136.6] pg/mL), MECL-1 (992.5 [849.7-1075.8] pg/mL), LMP7 (822.6 [748.6-1009.5] pg/mL), IL1ß (113.2 [90.6-194.5] pg/mL), and Hs-CRP (30.0 [12.8-75.6] mg/L) (P < .05). Logistic regression analysis showed cardioembolism, LMP2, MECL-1, and LMP7 as independent predictors of HT (P < .05). Receiver operating characteristic curve analysis demonstrated LMP2 ≥ 988.3 pg/mL, MECL-1 ≥ 584.7 pg/mL, and LMP7 ≥ 509.0 pg/mL as independent factors associated with HT (P < .001). CONCLUSION: Evaluation of plasma levels of immunoproteasome could be helpful in the early prediction of HT in acute ischemic stroke patients.

Enzymatic activities of circulating plasma proteasomes in newly diagnosed multiple myeloma patients treated with carfilzomib, lenalidomide and dexamethasone.

The proteasome inhibitor carfilzomib is highly effective in the treatment of multiple myeloma. It irreversibly binds the chymotrypsin-like active site in the ß5 subunit of the 20S proteasome. Despite impressive response rates when carfilzomib is used in combination with immunomodulatory agents in newly diagnosed multiple myeloma patients; no biomarker exists to accurately predict response and clinical outcomes. We prospectively assessed the activity in peripheral blood of the chymotrypsin-like (CHYM), caspase-like (CASP) and trypsin-like (TRYP) proteolytic sites in 45 newly diagnosed multiple myeloma patients treated with eight cycles of carfilzomib, lenalidomide and dexamethasone (CRd) (NCT01402284). Samples were collected per protocol and proteasome activity measured through a fluorogenic assay. Median CHYM levels after one dose of carfilzomib decreased by >70%. CHYM and CASP activity decreased throughout treatment reaching a minimum after eight cycles of treatment. Higher levels of proteasome activity associated with higher disease burden (r > 0.30; p < 0.05) and higher disease stage (0.10 < p <0.20). No association was found with the probability of achieving a complete response, minimal residual disease negativity or time to best response. Further studies evaluating proteasome activity in malignant plasma cells may help elucidate how proteasome activity can be used as a biomarker in multiple myeloma.

Reaction of complement factors and proteasomes in experimental encephalitis.

Herpes simplex virus type 1 (HSV-1) encephalitis causes a deleterious inflammation and elevated intracranial pressure. As a step towards examining the origin of the inflammation, we here report the response of circulating proteasomes and complement factors in blood and cerebrospinal fluid (CSF) in rats infected with HSV-1. Infection was via the nasal route, with 1.1 × 104 plaque-forming units of HSV-1 strain 2762 given in one or both nostrils. A sandwich enzyme-linked immunosorbent assay was used to study the level of 26S proteasomes and their complex formation with complement factors 3 and 4. HSV-1 infection in the rat causes a complex formation between complement factors and proteasomes, which we designate compleasomes. In the first experiment, with HSV-1 given in both nostrils, compleasomes containing complement factors 3 and 4 increased significantly in both blood plasma and CSF. The concentration of proteasomes in plasma was similar in controls and infected rats (320 ± 163 vs. 333 ± 125 ng/ml). In the second experiment, with HSV-1 given in one nostril, CSF levels were 1 ± 1 ng/ml in controls and 56 ± 22 ng/ml in the HSV-1 group, whereas the total protein concentration in CSF remained the same in the two groups. The compleasome response was limited to CSF, with a highly significant difference between infected rats and controls (n = 11, p < 0.001). It was possible to mimic the reaction between proteasomes and complements 3 and 4 in vitro in the presence of ATP.

Early detection of hepatocellular carcinoma co-occurring with hepatitis C virus infection: A mathematical model.

AIM: To develop a mathematical model for the early detection of hepatocellular carcinoma (HCC) with a panel of serum proteins in combination with α-fetoprotein (AFP). METHODS: Serum levels of interleukin (IL)-8, soluble intercellular adhesion molecule-1 (sICAM-1), soluble tumor necrosis factor receptor II (sTNF-RII), proteasome, and ß-catenin were measured in 479 subjects categorized into four groups: (1) HCC concurrent with hepatitis C virus (HCV) infection (n = 192); (2) HCV related liver cirrhosis (LC) (n = 96); (3) Chronic hepatitis C (CHC) (n = 96); and (4) Healthy controls (n = 95). The R package and different modules for binary and multi-class classifiers based on generalized linear models were used to model the data. Predictive power was used to evaluate the performance of the model. Receiver operating characteristic curve analysis over pairs of groups was used to identify the best cutoffs differentiating the different groups. RESULTS: We revealed mathematical models, based on a binary classifier, made up of a unique panel of serum proteins that improved the individual performance of AFP in discriminating HCC patients from patients with chronic liver disease either with or without cirrhosis. We discriminated the HCC group from the cirrhotic liver group using a mathematical model (-11.3 + 7.38 × Prot + 0.00108 × sICAM + 0.2574 × ß-catenin + 0.01597 × AFP) with a cutoff of 0.6552, which achieved 98.8% specificity and 89.1% sensitivity. For the discrimination of the HCC group from the CHC group, we used a mathematical model [-10.40 + 1.416 × proteasome + 0.002024 × IL + 0.004096 × sICAM-1 + (4.251 × 10(-4)) × sTNF + 0.02567 × ß-catenin + 0.02442 × AFP] with a cutoff 0.744 and achieved 96.8% specificity and 89.7% sensitivity. Additionally, we derived an algorithm, based on a binary classifier, for resolving the multi-class classification problem by using three successive mathematical model predictions of liver disease status. CONCLUSION: Our proposed mathematical model may be a useful method for the early detection of different statuses of liver disease co-occurring with HCV infection.