Blood Immunoproteasome Activity Is Regulated by Sex, Age and in Chronic Inflammatory Diseases: A First Population-Based Study.

Dysfunction of the immunoproteasome has been implicated in cardiovascular and pulmonary diseases. Its potential as a biomarker for predicting disease stages, however, has not been investigated so far and population-based analyses on the impact of sex and age are missing. We here analyzed the activity of all six catalytic sites of the proteasome in isolated peripheral blood mononuclear cells obtained from 873 study participants of the KORA FF4 study using activity-based probes. The activity of the immuno- and standard proteasome correlated clearly with elevated leukocyte counts of study participants. Unexpectedly, we observed a strong sex dimorphism for proteasome activity with significantly lower immunoproteasome activity in women. In aging, almost all catalytic activities of the proteasome were activated in aged women while maintained upon aging in men. We also noted distinct sex-related activation patterns of standard and immunoproteasome active sites in chronic inflammatory diseases such as diabetes, cardiovascular diseases, asthma, or chronic obstructive pulmonary disease as determined by multiple linear regression modeling. Our data thus provides a conceptual framework for future analysis of immunoproteasome function as a bio-marker for chronic inflammatory disease development and progression.

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.

Proteasome Activity in the Plasma as a Novel Biomarker in Mild Cognitive Impairment with Chronic Tinnitus.

BACKGROUND: Although the existence of proteasomes in human blood, termed circulating proteasomes (c-proteasomes), has been reported previously, their origin and pathophysiological functions remain largely unknown. OBJECTIVE: Given that c-proteasome activity was significantly reduced in Alzheimer's disease model mice and relatively high frequency of mild cognitive impairment (MCI) is accompanied by chronic tinnitus in aged patients, we examined whether c-proteasome activity in human plasma was associated with cognitive function in patients with chronic tinnitus. METHODS: c-Proteasome activity in the plasma of tinnitus patients (N = 55) was measured with fluorogenic reporter substrate, suc-LLVY-AMC. To assess MCI, the Montreal Cognitive Assessment was conducted with a cut-off score of 22/23. All patients underwent audiological and psychoacoustic analyses. Levels of c-proteasomes, Aß42, and Aß40 were measured using ELISA, and their association with c-proteasome activity was evaluated. RESULTS: The activity of circulating proteasomes was significantly lower in patients with chronic tinnitus and MCI (p = 0.042), whereas activities of other plasma enzymes showed little correlation. In addition, c-proteasome activity was negatively associated with the level of plasma Aß and was directly dependent on its own concentration in the plasma of patients with chronic tinnitus. CONCLUSION: Our current work provides a new perspective for understanding the potential relationship between circulating proteasomes in the plasma and cognitive dysfunction, suggesting a novel, non-invasive biomarker in the context of MCI diagnosis.

Circulating 20S proteasome for assessing protein energy wasting syndrome in hemodialysis patients.

Protein energy wasting (PEW) including muscle atrophy is a common complication in chronic hemodialysis patients. The ubiquitin proteasome system (UPS) is the main proteolytic system causing muscle atrophy in chronic kidney disease and proteasome 20S is the catalytic component of the UPS. Circulating proteasome 20S (c20S proteasome) is present in the blood and its level is related to disease severity and prognosis in several disorders. We hypothesized that c20S proteasome could be related with muscle mass, other PEW criteria and their evolution in hemodialysis patients. Stable hemodialysis patients treated at our center for more than 3 months were followed over 2 years. C20S proteasome assay was performed at baseline. Biological and clinical data were collected, muscle mass was assessed by multi-frequency bio-impedancemetry, and nutritional scores were calculated at baseline, 1 year and 2 years. Hospitalizations and mortality data were collected over the 2 years. Forty-nine patients were included. At baseline, the c20S proteasome level was 0.40[0.26-0.55] µg/ml. Low muscle mass as defined by a lean tissue index (LTI) < 10th in accordance with the International Society of Renal Nutrition and Metabolism guidelines was observed in 36% and PEW in 62%. Increased c20S proteasome levels were related with LTI at baseline (R = 0.43, p = 0.004) and with its 2 year-variation (R = -0.56, p = 0.003). Two-year survival rate was not different between higher and lower c20S proteasome values (78.9 vs 78.4%, p = 0.98 log-rank test). C20S proteasome is not a good marker for assessing nutritional status in hemodialysis patients and predicting patient outcomes.

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.

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.

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.

Proteasome Levels and Activity in Pregnancies Complicated by Severe Preeclampsia and Hemolysis, Elevated Liver Enzymes, and Thrombocytopenia (HELLP) Syndrome.

Excessive accumulation of misfolded proteins was recently demonstrated in preeclampsia. We examined levels and activity of circulatory proteasome and immunoproteasome (inflammatory subtype) in preeclampsia and hemolysis, elevated liver enzymes, and thrombocytopenia (HELLP) syndrome. We analyzed samples from women with hypertensive pregnancy disorders (n=115), including preeclampsia with severe features (sPE) and HELLP syndrome, and normotensive controls (n=45). Plasma proteasome and immunoproteasome immunoreactivity were determined by quantifying the α-subunit of the 20S core and ß5i (proteasome subunit beta 8 [PSMB8]), respectively. Plasma proteasome activity was analyzed with fluorogenic substrates. MG132, lactacystin, and ONX0914 were used to inhibit the circulating proteasome and immunoproteasome, respectively. Plasma cytokine profiles were evaluated by multiplex immunoassay. Placental expression of ß5 (constitutive proteasome) and ß5i (immunoproteasome) was interrogated by immunohistochemistry. Women with sPE had increased plasma 20S levels ( P<0.001) and elevated lytic activities (chymotrypsin-like 7-fold, caspase-like 4.2-fold, trypsin-like 2.2-fold; P <0.001 for all) compared with pregnant controls. Women with features of HELLP displayed the highest plasma proteasome levels and activity, which correlated with decreased IFN-γ (interferon-γ), and increased IL (interleukin)-8 and IL-10. In sPE and HELLP, chymotrypsin-like activity was suppressed by proteasome inhibitors including ONX0914. Compared with gestational age-matched controls, sPE placentas harbored increased ß5 and ß5i immunostaining in trophoblasts. ß5i signal was elevated in HELLP with predominant staining in villous core, extravillous trophoblasts in placental islands, and extracellular vesicles in intervillous spaces. Pregnancy represents a state of increased proteostatic stress. sPE and HELLP were characterized by significant upregulation in circulating levels and lytic activity of the proteasome that was partially explained by placental immunoproteasome upregulation.

The immunoproteasome catalytic ß5i subunit regulates cardiac hypertrophy by targeting the autophagy protein ATG5 for degradation.

Pathological cardiac hypertrophy eventually leads to heart failure without adequate treatment. The immunoproteasome is an inducible form of the proteasome that is intimately involved in inflammatory diseases. Here, we found that the expression and activity of immunoproteasome catalytic subunit ß5i were significantly up-regulated in angiotensin II (Ang II)-treated cardiomyocytes and in the hypertrophic hearts. Knockout of ß5i in cardiomyocytes and mice markedly attenuated the hypertrophic response, and this effect was aggravated by ß5i overexpression in cardiomyocytes and transgenic mice. Mechanistically, ß5i interacted with and promoted ATG5 degradation thereby leading to inhibition of autophagy and cardiac hypertrophy. Further, knockdown of ATG5 or inhibition of autophagy reversed the ß5i knockout-mediated reduction of cardiomyocyte hypertrophy induced by Ang II or pressure overload. Together, this study identifies a novel role for ß5i in the regulation of cardiac hypertrophy. The inhibition of ß5i activity may provide a new therapeutic approach for hypertrophic diseases.

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.

Novel autoantibodies against the proteasome subunit PSMA7 in amyotrophic lateral sclerosis.

OBJECTIVE: To identify autoantibodies using sera from ALS patients and elucidate their roles in disease pathology. METHODS: An immunological screening was performed with a phage expression library SEREX method using sera from 3 ALS patients to identify ALS-related autoantibodies. Levels of antibodies identified by SEREX were measured in 33 ALS patients and 30 normal controls (NCs) by AlphaLISA using recombinant non-full-length proteins. The results were then validated by ELISA using full-length proteins in 71 ALS patients, 30 NCs and 34 disease controls (DCs). The relationship between the titres and clinical profiles of ALS patients were examined. RESULTS: Four autoantibodies identified by SEREX were proteasome subunit alpha type 7 (PSMA7), vimentin, hydroxymethylbilane synthase and TBC1 domain family member 2 (TBC1D2). AlphaLISA revealed that only the anti-PSMA7 and anti-TBC1D2 levels were significantly different between the ALS and NCs groups. ELISA showed that only the levels of antibody against PSMA7, involved in protein degradation by the ubiquitin-proteasome pathway (UPP), were higher in the ALS group than both the NC (P < .01) and DC (P = .034) groups. Anti-PSMA7 levels tended to be negatively correlated with the logarithm of disease duration (P = .052) and were significantly positively correlated with the logarithm of creatine kinase levels (P = .011). The anti-PSMA7 antibody levels were different between patients with and without dysphagia (P < .01). CONCLUSIONS: Serum anti-PSMA7 antibody might be a disease-promoting factor in early-stage ALS and might be a biomarker of ALS. Anti-PSMA7 autoantibody might contribute to the pathogenesis of ALS, possibly via its role in the UPP.

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.

Potential involvement of ubiquitin-proteasome system dysfunction associated with oxidative stress in the pathogenesis of sickle cell disease.

The ubiquitin-proteasome system (UPS) is an important intracellular proteolytic pathway responsible for the degradation of proteins and oxidative damage; hence it plays a central role in maintaining homeostasis of red blood cells (RBCs). The present study investigated the levels of polyubiquitination, the function of proteasomes and effect of hydroxycarbamide (HC) therapy in RBCs from sickle cell disease (SCD) patients. Polyubiquitinated proteins were found to be elevated in untreated SCD (UT-SCD) patients compared to those in HC-treated SCD patients (HC-SCD) and controls. Activities of ß1 and ß2 subunits were a little higher in UT-SCD patients, and much higher proteolytic activities were observed in all three subunits (ß1, ß2 and ß5) of RBCs in HC-SCD patients compared to those of UT-SCD patients and controls, although the protein levels of these subunits remained approximately the same. It is notable that, despite HC therapy, some patients showed persistent complications and accumulation of polyubiquitinated proteins. The enhanced proteasomal activity among HC-treated patients might remove the polyubiquitinated protein and could be one of the important mechanisms of therapeutic action. These findings could be useful to understand the pathophysiology of SCD and its clinical heterogeneity and identify a suitable therapeutic target for the better management of these patients.

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).

Evaluation of serum insulin-like growth factor-1 and 26S proteasome concentrations in healthy dogs and dogs with chronic diseases depending on body condition score.

In patients suffering from chronic diseases, the objective assessment of metabolic states could be of interest for disease prognosis and therapeutic options. Therefore, the aim of this study was to assess insulin-like growth factor-1 (IGF-1) and 26S proteasome (26SP) in healthy dogs and dogs suffering from chronic diseases depending on their body condition score (BCS) and to examine their potential for objective assessment of anabolic and catabolic states. Serum concentrations of IGF-1, an anabolic hormone, and 26SP, a multiprotein complex which is part of the ubiquitin-proteasome pathway, by which the majority of endogenous proteins including the muscle proteins are degraded, were measured in 21 healthy dogs and 20 dogs with chronic diseases by canine ELISA. The concentrations of IGF-1, 26SP and their ratio (IGF-1/26SP) were set in relationship to the BCS of the dogs. When examining healthy and chronically diseased dogs separately, a positive correlation between IGF-1 and the BCS was observed in the healthy group and a negative correlation between 26SP and the BCS was noted in dogs with chronic diseases. Further, dogs suffering from chronic diseases showed higher 26SP concentrations and lower values for IGF-1/26SP than the healthy dogs. Overall, we detected a negative correlation between 26SP and the BCS and a positive correlation between IGF-1/26SP and the BCS. The results of our study indicate usability of IGF-1 for description of anabolic states, while 26SP could be useful for detection and description of catabolic states. Finally, the ratio IGF-1/26SP seems to be promising for assessment of metabolic states.

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.

Immunoproteasome in the Plasma of Pediatric Patients With Moderate and Major Burns, and Its Correlation With Proteasome and UCHL1 Measured by SPR Imaging Biosensors.

The aim of this study was to determine the immunoproteasome concentration in blood plasma of pediatric patients with moderate and major burns and its correlation with circulating proteasome and ubiquitin carboxyl-terminal hydrolase L1 (UCHL1) with surface plasmon resonance imaging biosensor. The study population comprised of 30 patients with moderate (n = 21) and severe burns (n = 9), aged 9 months to 14 years. The control group represented 18 healthy, age-matched patients, admitted for herniotomy. Exclusion criteria were as follows: admission to the hospital later than 6 hours after burn, cardiovascular or immunological diseases, and severe preexisting infections. Mean concentrations of immunoproteasome, 20S proteasome, and UCHL1 in the blood plasma of children with burns-4 to 6 hours, 12 hours, 3 days, 5 days, and 7 days after thermal injury-were above the levels measured in controls. The immunoproteasome, 20S proteasome, and UCHL1 concentrations in the blood plasma of their patients were highest 12 hours after burn, slowly decreased over time, and on the 5th day still were higher than in controls (P < .05). There was a strong correlation between immunoproteasome and 20S proteasome concentrations 6 hours and 5 days after burn, and moderate correlation 12 hours after burn (P < .05). The immunoproteasome concentration is elevated after burn injury and slowly reaches the normal range during the wound healing process. There is strong correlation between immunoproteasome and 20S proteasome concentrations in the serum of children with moderate and major burns. They did not find such correlation between immunoproteasome and UCHL1 concentrations. Immunoproteasome concentration do not correlate with age or sex.

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.

The immunoproteasome subunit LMP10 mediates angiotensin II-induced retinopathy in mice.

Inflammation has been implicated in a variety of retinal diseases. The immunoproteasome plays a critical role in controlling inflammatory responses, but whether activation of immunoproteasome contributes to angiotensin II (Ang II)-induced retinopathy remains unclear. Hypertensive retinopathy (HR) was induced by infusion of Ang II (3000 ng/kg/min) in wild-type (WT) and immunoproteasome subunit LMP10 knockout (KO) mice for 3 weeks. Changes in retinal morphology, vascular permeability, superoxide production and inflammation were examined by pathological staining. Our results showed that immunoproteasome subunit LMP10 expression and its trypsin-like activity were significantly upregulated in the retinas and serum of Ang II-infused mice and in the serum from patients with hypertensive retinopathy. Moreover, Ang II-infused WT mice showed an increase in the central retinal thickness, vascular permeability, reactive oxygen species (ROS) production and inflammation compared with saline controls, and these effects were significantly attenuated in LMP10 KO mice, but were aggravated in mice intravitreally injected with rAAV2-LMP10. Interestingly, administration of IKKß specific inhibitor IMD-0354 remarkably blocked an Ang II-induced increase in vascular permeability, oxidative stress and inflammation during retinopathy. Mechanistically, Ang II-induced upregulation of LMP10 promoted PTEN degradation and activation of AKT/IKK signaling, which induced IkBα phosphorylation and subsequent degradation ultimately leading to activation of NF-kB target genes in retinopathy. Therefore, this study provided novel evidence demonstrating that LMP10 is a positive regulator of NF-kB signaling, which contributes to Ang II-induced retinopathy. Strategies for inhibiting LMP10 or IKKß activity in the eye could serve as a novel therapeutic target for treating hypertensive retinopathy.