Due to the complexity of BC, the prediction of proteins involved in this disease is a trending topic in drug design. This work is proposing accurate prediction classifier for BC proteins using six sets of protein sequence descriptors and 13 machine-learning methods. After using a univariate feature selection for the mix of five descriptor families, the best classifier was obtained using multilayer perceptron method  and 300 features. The performance of the model is demonstrated by the area under the receiver operating characteristics (AUROC) of 0.980 ± 0.0037, and accuracy of 0.936 ± 0.0056 . Regarding the prediction of 4,504 cancer-associated proteins using this model, the best ranked cancer immunotherapy proteins related to BC were RPS27, SUPT4H1, CLPSL2, POLR2K, RPL38, AKT3, CDK3, RPS20, RASL11A and UBTD1; the best ranked metastasis driver proteins related to BC were S100A9, DDA1, TXN, PRNP, RPS27, S100A14, S100A7, MAPK1, AGR3 and NDUFA13; and the best ranked RNA-binding proteins related to BC were S100A9, TXN, RPS27L, RPS27, RPS27A, RPL38, MRPL54, PPAN, RPS20 and CSRP1. This powerful model predicts several BC-related proteins that should be deeply studied to find new biomarkers and better therapeutic targets. Scripts can be downloaded at  Over the last years, multi-omics studies, pharmacogenomics treatments and precision medicine strategies have evolved favorably; however, there are still biases such as the significant inclusion of minority populations in cancer research7. Nowadays, BC is the most commonly diagnosed cancer , and the leading cause of cancer-related deaths among women  worldwide8.The intricate interplay between several biological aspects such as environmental determinants, gene expression deregulation, genetic alterations, signaling pathway alterations and ethnicity causes the development of breast cancer (BC), a heterogeneous diseaseet al. developed the OncoOmics strategy to reveal essential genes in BC9. This strategy was a compendium of approaches that analyzed genomic alterations, protein expression, protein-protein interactome (PPi) network, dependency maps in cell lines and patient-derived xenografts of BC genes / proteins using relevant databases such as the Pan-Cancer Atlas project12, The Cancer Genome Atlas (TCGA)13, The Human Protein Atlas (HPA)16, the DepMap project19, and the OncoPPi network20.In our previous study, López-Cortés 21, the Pan-Cancer Atlas22, the Pharmacogenomics Knowledgebase (PharmGKB) 24, and the Cancer Genome Interpreter25. The Consensus Strategy, developed by López-Cortés et al., Tejera et al., and Cabrera-Andrade et al., was proved to be highly efficient in the recognition of genes associated with BC pathogenesis27. The Pan-Cancer Atlas reveals how genomic alterations, such as protein expression, copy number alterations (CNAs), mRNA expression, and putative mutations collaborate in BC progression32. PharmGKB is a comprehensive resource that collects the precise guidelines for the application of pharmacogenomics in clinical practice24. Lastly, the Cancer Genome Interpreter flags genomic biomarkers of drug response with different levels of clinical relevance25.Gene sets were taken from the Consensus StrategyRAC1, AKT1, CCND1, PIK3CA, ERBB2, CDH1, MAPK14, TP53, MAPK1, SRC, RAC3, BCL2, CTNNB1, EGFR, CDK2, GRB2, MED1, and GATA3 were significant in at least three OncoOmics approaches9. On the other hand, g:Profiler lets us know the enrichment map of the 140 essential genes in BC33. The most significant gene ontologies (GO) related to biological process and molecular function were the positive regulation of macromolecule metabolic process and the phosphatidylinositol 3-kinase activity, respectively. The most significant term, according to the Human Phenotype Ontology, was breast carcinoma34. Subsequently, the most relevant network interactions of the GO: biological process and the Reactome pathways were related to the immune system35, tyrosine kinase36, cell cycle37, DNA repair38, and RNA-binding proteins39. The Open Targets Platform has a largest number of drugs involved in clinical trials to treat BC with a direct focus on the OncoOmics BC essential genes were small molecules that correspond most likely to tyrosine kinases40. Hence, the essential proteins with signaling function are the interesting drug targets to modify any biological activity.The OncoOmics BC essential genes were rationally filtered to 140. 41. Different classification models have been published for prediction of protein activities: anti-oxidant42, lectins43, signaling44, anti-angiogenic45, anti-cancer46, and enzyme class47. Vilar et al. developed a QSAR model for alignment-free prediction of BC biomarkers using a linear discriminant analysis method, electrostatic potentials of protein pseudofolding HP-lattice networks as features, and 122 proteins related to BC and a control group of 200 proteins with classifications above 80%48. Our group proposed an improved multi-target classification model for human breast and colon cancer-related proteins by using a similar molecular graph theory for descriptors: star graph topological indices49. The accuracy of the models was 90.0% for a linear forward stepwise model. Both models presented linear relationships between graph-based protein sequence descriptors and BC, and unbalanced datasets. Thus, the aim of this study was to obtain an effective machine-learning classification model to predict BC-related proteins screening cancer immunotherapy proteins (CIPs), metastasis driver proteins (MDPs) and RNA-binding proteins (RBPs), using non-graph protein sequence descriptors and additional non-linear machine-learning techniques.Starting a screening applying theoretical methods could save economic resources and time. Therefore, machine-learning (ML) techniques could obtain classification models that links signaling activity to protein structure. ML encodes molecular features into invariant descriptors based on physical and chemical properties of the amino acids, 3D protein conformation, graph topology, and protein sequences. The classification model is a quantitative structure-activity relationship (QSAR) between the biological function and the protein structure50 molecular descriptors have been used: 20 amino acid composition (AC), 400 di-amino acid composition (DC), 8000 tri-amino acid composition (TC), 80 amphiphilic pseudo-amino acid composition (APAAC), and 240 normalized Moreau-Broto autocorrelation (MB). The six sets of descriptors were constructed by mixing all the five-descriptor families, resulting 8,708 total descriptors (Mix).Figure 51 were used to test 13 types of machine-learning classifiers for each set of descriptors, without feature selection, with univariate feature selection, or using principal component analysis (PCA)52. The classifiers were Gaussian Naive Bayes (NB)53, k-nearest neighbors algorithm (KNN)54, linear discriminant analysis (LDA)55, support vector machine (SVM) linear and non-linear based on radial basis functions (RBF), support vector classification (SVC) kernel = linear, and SVC kernel = RBF56, logistic regression (LR)57, multilayer perceptron (MLP) / neural network with 20 neurons in one hidden layer58, decision tree (DT)59, random forest (RF)60, XGBoost (XGB) is an optimized and distributed gradient boosting library61, Gradient Boosting for classification (GB)62, AdaBoost classifier (AdaB)63, and Bagging classifier (Bagging)64. The feature selection method was univariate filter such as SelectKBest , and the dimension reduction technique was PCA52.Jupyter notebooks with python/sklearn53. k-nearest neighbors algorithm assigns an unclassified sample using the nearest of k samples in the training set54. Linear discriminant analysis is a basic linear classifier55. SVM linear is using a higher dimensionality space to map the input features56. For non-linear problems, SVM uses Gaussian radial basis as non-linear kernels.Gaussian Naive Bayes is based on Bayes’ theorem and considers all the features are independent57. Multilayer perceptron represents a basic neural network with one hidden layer and with an ability to combine linear and nonlinear functions inside artificial neurons58. Decision tree represents a tree-type structure of decision rules obtained from the inputs59. Random forest is an ensemble method that combines parallel decision trees60. XGBoost uses sequential weak trees to improve the classification performance61. Gradient Boosting for classification is a basis boost method using sequential weak classifiers62. AdaBoost classifier is mixing different classifiers: it starts the fitting with a classifier based on the original dataset and adds additional copies of the original classifier with adjusted weights for the incorrectly classified instances63. Bagging classifier is a modified version of AdaB: the additional classifiers are based on subsets of the original dataset64.Logistics regression is another linear classifier that is able to calculate probability of a binary response using weightset al.9. On the other hand, the negative protein set was constructed as follows: non-cancer proteins from Piazza et al.65, without BC-related proteins, were reanalyzed using Piazza’s OncoScore algorithm , giving a final list of 233 non-cancer proteins. Supplementary Tables The machine-learning prediction model was constructed from two protein sets. On the one hand, the positive set named OncoOmics BC essential proteins was made up of 140 strongly associated proteins to BC pathogenesis, according to López-Cortés et al.,35 1,903 MDPs were taken from the Human Cancer Metastasis Database (HCMDB) (http://hcmdb.i-sanger.com/index)66, and 1,369 RBPs were taken from Hentze et al.,39  metrics68, and 3-fold cross-validation (CV) method.After the calculation of amino acid composition descriptors, the datasets contained 373 proteins. The BC class was labeled with 1 and non-cancer class with 0. Several preprocessing was done before any calculation: elimination of doubled examples, elimination of data with NA values, and elimination of features with zero variance. All feature values were normalized to values between 0 and 1 using MinMax scaler. A SMOTE filter was used to balance the datasethttps://github.com/muntisa/neural-networks-for-breast-cancer-proteins. The scaler, selected features and the best model were saved as files too. These are used to make predictions with another notebook for any new data (see 2-Predictions-BreastCancerPeptides.ipynb). We used these automatic scripts to predict the breast cancer activity for a 4,504 external proteins by using their molecular descriptors: 1,232 CIPs, 1,903 MDPs, and 1,369 RBPs.The best model to be used for predictions was chosen using criteria such as mean AUROC, standard deviation (SD) of AUROC, and the number of features. All the results obtained can be reproduced by using the scripts at https://www.cbioportal.org/)70, then, we downloaded and analyzed a matrix of CNAs (amplifications and deep deletions), putative mutations , mRNA alterations (mRNA high and mRNA down), and protein alterations (high and low expression) related to the 4,504 proteins queried in a cohort of 1,066 individuals according to the Pan-Cancer Atlas22. Lastly, a Mann-Whitney U test was performed to obtain significant differences (p < 0.001) on the amount of genomic alterations between CIPs related and non-related to BC, MDPs related and non-related to BC, and RBPs related and non-related to BC.After the screening of the 4,504 external proteins through the machine-learning model, complementary analyses were done to compare the amount of genomic alterations between BC related proteins (prediction 1) and BC non-related proteins (prediction 0). Firstly, we selected the study ‘Breast Invasive Carcinoma ’ from the cBioPortal , univariate filter as feature selection method, and PCA transformation of features. All the models used AUROC  to quantify the classification performance. Details about feature selection methods and parameters of machine-learning classifiers are included in the Supplementary_ML_Details.pdf.For the first models, we used the pool of features for the six sets of descriptors without any feature selection or dimension reduction with 12 machine-learning methods . Figure If the number of features increased to 100 (5 times from 20), better AUROC values are obtained in Fig. Figure In Fig. The best AUROC of 0.980 ± 0.0037 was obtained with MLP and Mix-Best300. The same AUROC value was generated by TC-Best300 and LR but with a double SD of 0.0077. In the best model with the mixed descriptors, between the 300 descriptors, seven DC  and two APAAC descriptors (Pc1.N and Pc1.M) were selected for BC function. The rest is TC descriptors without any MP descriptor selected  with the same MLP method (see CVs.ipynb for details). Thus, in the case of 5-fold CV, the mean AUROC was 0.9874 ± 0.0129 and the mean ACC was 0.9464 ± 0.0135. By increasing the number of folds to 10, the statistics showed a mean AUROC of 0.9831 ± 0.0158, and a mean ACC of 0.9401 ± 0.0226. All the models are saved into folder The 4,504 external proteins  were transformed into the molecular descriptors of the best model and were used to predict the breast cancer activity (see 2-Predictions-BreastCancerPeptides.ipynb): 1,232 CIPs, 1,903 MDPs and 1,369 RBPs. Thus, all these proteins were transformed into 300 selected descriptors of a Mix-300 set and were used with the saved MLP classifier. As a result, 608 cancer immunotherapy proteins, 971 metastasis driver proteins and 757 RNA binding proteins were predicted to be related to breast cancer . We choose, as the best classifier, the MLP classifier. As inputs, a mixture of 300 selected molecular descriptors has been used: DC, TC and APAAC. The model has a mean AUROC of 0.980 ± 0.0037 and a mean accuracy of 0.936 ± 0.0056 . 4,504 sequences of proteins related to cancer have been screened for breast cancer relation. Best predicted cancer immunotherapy proteins with BC were RPS27, SUPT4H1, CLPSL2, POLR2K and RPL38, and the most altered ones were POLR2K, ASH2L, MED30, NSL1 and RPRD2. Best predicted metastasis diver proteins with BC were S100A9, DDA1, TXN, PRNP and RPS27, and the most altered ones were YWHAZ, PTK2, SETDB1, EBAG9 and MTBP. Best predicted RNA-binding proteins with BC were S100A9, TXN, RPS27L, RPS27 and RPS27A, and the most altered ones were YWHAZ, DCAF13, TFB2M, PTDSS1 and NUCKS1. Finally, the association between the best-predicted BC proteins using powerful machine-learning methods and the amount of pathogenic genomic alterations in cancer immunotherapy proteins, metastasis driver proteins and RNA-binding proteins gives us candidate proteins that should be deeply studied to find novel therapeutic targets.Supplementary Information.Supplementary Dataset.


Pituitary apoplexy (PA) and posterior reversible encephalopathy syndrome (PRES) are rare neurologic diseases that show acute neuro-ophthalmologic symptoms such as headache, decreased visual acuity, and altered consciousness. These diseases are rarely found in patients with end-stage renal disease (ESRD) on hemodialysis, and simultaneous occurrence of these 2 diseases has not been reported.The patient was a 75-year-old man with a history of hypertension, diabetes mellitus, and non-functioning pituitary macroadenoma. He had been receiving hemodialysis for ESRD for 3 months before his presentation to the emergency room. The patient complained of headache, vomiting, and dizziness that started after the previous day's hemodialysis. The patient had voluntarily discontinued his antihypertensive medication 2 weeks before presentation and had high blood pressure with marked fluctuation during hemodialysis. Complete ptosis and ophthalmoplegia on the right side suggested 3rd, 4th, and 6th cranial nerve palsies.Magnetic resonance imaging of the brain revealed a pituitary tumor, intratumoral hemorrhage within the sella, and symmetric vasogenic edema in the subcortical white matter in the parieto-occipital lobes. Based on these findings, the patient was diagnosed with PA and PRES.Intravenous administration of hydrocortisone (50 mg every 6 hours after a bolus administration of 100 mg) was initiated. Although surgical decompression was recommended based on the PA score (5/10), the patient declined surgery.Headache and ocular palsy gradually improved after supportive management. The patient was discharged on the 14th day of hospitalization with no recurrence 5 months post-presentation. Current therapy includes antihypertensive agents, oral prednisolone (7.5 mg/day), and maintenance hemodialysis.Neurologic abnormalities developed in a patient with ESRD on hemodialysis, suggesting the importance of prompt diagnosis and treatment in similar instances. Posterior reversible encephalopathy syndrome (PRES) is a syndrome characterized by vasogenic edema on neuroimaging along with acute or subacute confusion, sudden headache, visual disturbances, and seizure. PA cases,3 or PRES cases–6 have been rarely reported in patients with end-stage renal disease (ESRD) undergoing dialysis therapy. Moreover, simultaneous occurrence of PA and PRES in ESRD patients has not been reported. Here, we report a case of PA accompanied by PRES in a 75-year-old man with ESRD on hemodialysis.Pituitary apoplexy (PA) is a rare clinical syndrome characterized by severe headache, visual impairment, ocular paresis, and an altered mental state due to hemorrhage or infarction in the pituitary gland.2+/K+/Cl−, 136/4.8/95 mEq/L; magnesium, 2.2 mg/dL; C-reactive protein, 3 mg/L; and a HbA1C level of 6.5%. Serologic test results for HBs Ag, anti-HCV Ab, anti-HIV Ab, anti-nuclear Ab, ds-DNA Ab, and Venereal Disease Research Laboratory (VDRL) test were all negative. Results from serologic tests were all negative. Since the clinical signs suggested central nervous system (CNS) disease, we performed brain computed tomography (CT) and brain magnetic resonance (MR) imaging. Initial brain MR imaging revealed a large pituitary tumor of 2.0 × 1.4 cm with internal hemorrhage within the sella, suggestive of PA  since being diagnosed with diabetes mellitus and hypertension 20 years earlier. Although the patient had been diagnosed with a non-functioning pituitary macroadenoma 2 years earlier, there were no specific symptoms, and no specific medications had been prescribed. The patient had received regular hemodialysis with systemic heparinization  for ESRD secondary to diabetic nephropathy that had developed 3 months before emergency room presentation. The usual range of interdialytic weight gain was 1.5 to 2.0 kg. The pre- and post-dialysis blood pressure (BP) measurements were 200/110 mmHg  and 120/70 mmHg , respectively, showing a marked fluctuation in BP during the previous hemodialysis. The patient had refused antihypertensive therapy during the 2 weeks before hospital presentation. At the time of admission, the vital signs were a BP of 110/70 mmHg, pulse rate of 101/minute, respiration rate of 20/minute, and body temperature of 35.8°C. The patient was lethargic  and complained of severe headache, particularly in the bi-frontal area and right retro-orbital region . Neurologic examination revealed a pupillary size of 3 mm/4 mm with normal light reflexes; complete ptosis and ophthalmoplegia were present on the right side. There were no occurrences of neck stiffness, focal neurologic deficits, or seizure. A peripheral blood test at admission showed the following: white blood cell count, 6,300/μL (neutrophils 59%); hemoglobin, 10.5 g/dL; platelet count, 142,000/μL; and an erythrocyte sedimentation rate of 20 mm/hour. Serum biochemical examination revealed the following: glucose, 126 mg/dL; blood urea nitrogen, 45.8 mg/dL; creatinine, 6.6 mg/dL; total protein, 5.2 g/dL; albumin, 3.1 g/dL; total cholesterol, 154 mg/dL; aspartate aminotransferase, 18 IU/L; alanine aminotransferase, 12 IU/L; calcium, 8.2 mg/dL; phosphorus, 4.3 mg/dL; uric acid, 5.4 mg/dL; Na3 PA occurs in 1.6% to 2.8% of patients with pituitary macroadenoma, with a male to female ratio of 2:1, and affects individuals in their 50s to 60s. Factors that predispose to PA, most commonly severe hypertension, are observed in 40% of cases. Other predisposing factors, including major surgery and anticoagulant administration, are also observed. The predisposing factors of the patient in this case were uncontrolled hypertension, diabetes mellitus, chronic kidney disease, and pituitary macroadenoma.,9 Liu et al analyzed 288 patients with pituitary adenoma and found pituitary hemorrhage in 81 (28%). ESRD was also reported to be a significant risk factor for pituitary hemorrhage along with macroadenoma (>10 mm), non-functioning adenoma, antihypertensive therapy, and dopamine agonist administration. Since sudden fluctuation of BP and use of anticoagulants, such as heparin, during hemodialysis are known major risk factors for pituitary hemorrhage or PA,,11 we hypothesized that the PA in this patient may have been primarily due to an underlying pituitary adenoma and dialysis therapy. The most common symptom of PA is severe headache in the retroorbital and bifrontal regions accompanied by nausea and vomiting.,12 In addition, as in the present case, optic abnormalities and multiple cranial nerve palsies are also common signs of PA.,13 Once an acute neurologic disorder is identified in patients with an underlying pituitary tumor, rapid performance of radiologic examinations such as brain CT and brain MR imaging is imperative to assess presence of pituitary hemorrhage and rule out more common CNS disorders. Higher incidences of dialysis-related neurologic complications accompany severe hemodynamic changes during hemodialysis and are associated with increased morbidity and mortality in these patients.,15 Like our patient, in case of PA accompanied by intratumoral hemorrhage, different intensities of brain MR imaging can be observed depending on the process of hemorrhagic degeneration. Within the first 7 days of PA, isointense or slightly hypointense signal in T1-weighted images (WI) and hypointense lesions in T2-WI are depicted. In the subacute stage, from 1 to 3 weeks, hyperintensity due to methemoglobin is visualized in T1-WI and T2-WI. After 3 weeks, strong hypointensity in both T1-WI and T2-WI, secondary to the accumulation of hemosiderin and ferritin, may be noted.PA is a disease in which the pituitary gland rapidly expands due to pituitary hemorrhage, infarction, and necrosis, causing compression of the proximal regions of suprasellar structures or signs of meningeal irritation.,16 Our patient's pituitary hormone levels, with the exception of decreased serum cortisol, were within normal limits at the time of presentation (Table  Corticosteroid administration is also indicated when signs of hemodynamic instability, altered consciousness, decreased visual acuity, and severe defects in the visual field are present.,17 Supportive management in addition to hormone replacement is recommended in the acute stage of PA. Elective surgery should be considered in cases of no clinical improvement, progression of visual field defects or vision loss, and aggravation of neurologic signs after 1 week of corticosteroid administration. Neurosurgical decompression has been reported to yield a favorable clinical outcome. We recommended surgical decompression on the 2nd day of hospitalization because the patient's PA score was 5 points.,7 However, the patient refused surgery. Fortunately, conservative therapy gradually alleviated the vomiting, headache, and progression of neuro-ophthalmic complications; and monitoring for signs and symptoms of relapse ensued.In most cases of PA, necrosis occurs in the anterior lobe of the pituitary gland and causes hormone deficiencies, particularly decreased prolactin production. Hormone deficiencies suggest a poor prognosis for complete recovery of pituitary function. PRES has a number of causes that, on rare occasions, includes chronic kidney disease or acute kidney injury that results in uremia, intravascular volume expansion, electrolyte disorders, and hypertensive crisis.–6,20 On brain MR imaging, PRES presents as cortical and/or subcortical lesions with iso-intense or low signal intensity in T1-WI and hyperintense signal in T2-WI and FLAIR sequences. In most cases of PRES, vasogenic edema gradually subsides after correction of risk factors and treatment of underlying disease. However, ESRD patients on dialysis are more likely to develop permanent neurologic sequelae or mortality due to increased vulnerability to microvascular brain injury. Thus, active treatment for risk factors, including hypertension, is required after prompt diagnosis. Considering this patient's clinical presentations and the evidence of radiologic resolution, all diagnostic criteria of PRES were satisfied. PA was diagnosed as the cause of this patient's ophthalmologic disorders, cranial nerve deficits, and decreased serum cortisol level. Marked variability of BP with systemic heparinization during hemodialysis is associated with PRES as well as PA. We surmised that the conservative treatment for PA of this patient also contributed to the improvement of PRES. However, the absence of previous reports on the pathogenic association between PA and PRES prevents the conclusion of their coincidence in this patient.PRES is a disorder in which vasogenic edema in the cerebral white matter causes seizures, headache, nausea, vomiting, visual disturbances, altered consciousness, and focal neurologic deficits.In summary, a 75-year-old male ESRD patient with an underlying pituitary macroadenoma was diagnosed with both PA and PRES during hemodialysis; these conditions improved with supportive therapy. This case suggests that ESRD patients on hemodialysis may be more susceptible to various neurologic diseases due to severe hypertension, systemic heparinization, and hemodynamic instability. Thus, this case demonstrates the importance of prompt diagnosis and treatment for ESRD with dialysis-related neurologic complications.Conceptualization: In Hee Lee.Data curation: Ho Kyun Kim, Dong Jik Ahn.Formal analysis: In Hee Lee.Methodology: In Hee Lee.Validation: Ho Kyun Kim, Dong Jik Ahn.Writing – original draft: In Hee Lee.Writing – review & editing: In Hee Lee.In Hee Lee orcid: 0000-0003-3562-7586.


NPR1-like gene family in cotton (Gossypium species). Therefore, to address the importance of the cotton NPR1-like gene family in the defense pathway, four Gossypium species were studied: two tetraploid species, G.hirsutum and G. barbadense, and their two potential ancestral diploids, G. raimondii and G. arboreum. In this study, 12 NPR1-like family genes in G. hirsutum were recognized, including six genes in the A-subgenome and six genes in the D-subgenome. Based on the phylogenetic analysis, gene and protein structural features, cotton NPR-like proteins were grouped into three different clades. Our analysis suggests the significance of cis-regulatory elements in the upstream region of cotton NPR1-like genes in hormonal signaling, biotic stress conditions, and developmental processes. The quantitative expression analysis for different developmental tissues and fiber stages (0 to 25 days post-anthesis), as well as salicylic acid induction, confirmed the distinct function of different cotton NPR genes in defense and fiber development. Altogether, this study presents specifications of conservation in the cotton NPR1-like gene family and their functional divergence for development of fiber and defense properties.The nonexpressor of pathogenesis-related 1 (NPR1) family plays diverse roles in gene regulation in the defense and development signaling pathways in plants. Less evidence is available regarding the significance of the  Arabidopsis mutants that are dysfunctional in mounting salicylic acid-mediated defense response leads to the identification of a master regulator of the SAR pathway, NPR1 (nonexpressor of pathogenesis-related 1) [The sessile nature of plants makes them susceptible to sporadicity in environmental and biotic stresses. To protect themselves against intruding pathogens, plants have evolved several barrier systems for defense. Systemic acquired resistance (SAR) is an induced immune mechanism activated against a broad spectrum of pathogens and provides long-lasting immunity to the distal uninfected tissues from the synchronized activation of various pathogenesis-related genes ,2. Saliclated 1) . Intereslated 1) ,7,8. In lated 1) . In uninlated 1) .NPR1-like gene family  in three different clades based on the phylogenetic analysis in Arabidopsis thaliana [AtNPR1 display improvised resistance against a broad spectrum of bacterial and fungal pathogens, for instance, wheat [Hepworth et al., 2005, classified six homologs of the thaliana . Many ree, wheat , citrus e, wheat ,14, ricee, wheat ,16, tomae, wheat , rapeseee, wheat , tobaccoe, wheat , carrot e, wheat ,22,23, de, wheat , papaya e, wheat , wheat [e, wheat , apple [e, wheat , and stre, wheat .NPR1-like gene family of the SAR pathway, which would be an essential advantage for providing resistance against pathogen infestation [NPR gene family is still unexplored. Recently, published draft genome sequences of Gossypium hirsutum, Gossypium barbadense, Gossypium arboreum, and Gossypium ramondii have opened up new avenues to explore genome-wide analysis of NPR family members after SA induction or developmental signaling in cotton [NPR1-like gene family in four Gossypium species—G. arboreum, G. barbadense, G. hirsutum, and G. ramondii—was done. Further, gene structure, protein domain, phylogenetic analysis, and synteny of the identified putative cotton NPR genes were also analyzed. This study will provide an essential source for utilizing the NPR1-like genes in cotton for developing inherent resistance or tolerance against pathogens, and will eventually help us in developing higher fiber yielding varieties, an important asset for the cotton industry.Cotton is an economically valuable industrial crop. All over the world, it is mostly grown for oil-rich seeds and fiber, which is a required raw material for the textile industry. Biotic stress is an important constraint that inhibits cotton growth and development and compromises its fiber quality and yield . Applicaestation . Howevern cotton ,32,33,34Arabidopsis NPR (AtNPR1-6) protein sequences were used as queries in the BLASTP protein database for genomes of two allotetraploid cotton species, G. hirsutum, and G. barbadense, and their two diploid ancestors, G. arboreum and G. ramondii  domains, which are indicative domains present in the members of Arabidopsis NPR and other plants’ NPR families  are present in the NPR1-like proteins of cotton species. The domains of the cotton NPR components share high similarity with their Arabidopsis counterparts.Six ramondii . Twelve ectively . The Ara% to 91% . To annomilarity . The idefamilies  [24,25,2A. thaliana, Persea americana, Carica papaya, Glycine max, Malus domestica, Populus trichocarpa, and Lycopersicon esculentum), monocot plants , bryophytes (Physcomitrella patens), and lycophytes (Selaginella moellendorffii)  . The phyd AtNPR6 . The app avocado ,25,27,35chocarpa .NPR1-like genes are mostly located on four chromosomes of the G. hirsutum A- and D-subgenomes , VDLNETP motif, and NIMIN1/2 binding site at the C-terminus. Cotton NPR1, 3, and 4 contain three out of five conserved basic amino acid motifs in their nuclear localization signal (NLS) except for GhNPR4A.1, GhNPR4D.1, GbNPR4A.1, GbNPR4D.1, GrNPR4.1, and GaNPR4, which have four substitutions in this region  score for cotton NPR1-like genes reflected their hydrophilic nature, revealing higher variability in hydrophilicity  in the promoter of the GhNPR homologs in defense hormonal or stress conditions or developmental stages of cotton were analyzed from the PLACE and PlantCare databases , 5 DPA, 10 DPA, 20 DPA, and 25 DPA, from the available transcriptome data [GhNPR1A/1D genes in various fiber stages, except for the high expression of GhNPR1D at the 20 DPA fiber stage. GhNPR6A and GhNPR6D were highly expressed throughout the 0 DPA to 25 DPA fiber stages, except for GhNPR6A at 0 DPA and GhNPR6D at 10 DPA. GhNPR3A.2 and GhNPR3D.2 were highly expressed throughout the 0DPA to 20DPA fiber stages, except for GhNPR3A.2 in 10 DPA. The transcriptomic analysis of the fiber also suggests that GhNPR5A and GhNPR5D were highly expressed at the 5 DPA and 10 DPA fiber stages. We can broadly conclude from the in silico analysis of the transcriptomic data of the cotton fiber that a few cotton NPR1-like genes are probably involved in various stages of the fiber development.We further analyzed the expression profiles for putative cotton ome data   of fiber development  at 12 h and downregulated 2.12-fold at 24 h  contains only five NPR1-like genes, whereas G. raimondii possesses six NPR1-like genes. Twelve NPR1-like genes in both allotetraploid cotton species of upland cotton (GhNPR genes) and Pima or sea-land cotton (GbNPR genes) were identified in an attempt to better understand signaling in the defense and fiber-related developmental response, which probably better facilitates these processes. Instead, NPR1 and BOP1 were identified earlier in G. hirsutum and their role in defense was identified, yet information about their isoforms and the functions of other members of the family is still elusive [pathways ,40,41,42 elusive ,39. NPR1-like genes of all four cotton species, Arabidopsis and other known species , were clustered in three different clades/groups , G. barbadense (At and Dt- genome), G. raimondii (D-genome), and G. arboreum (A-genome)  , revealiPR1 gene expression and proteasome-mediated turnover of NPR1 , whereas GhNPR3A.2/D.2 and GhNPR6A/D have higher expression levels during the rapid elongation stage (10–20 DPA). Interestingly, in silico expression profiling in the fiber reasonably correlated with qRT-PCR and analysis of the promoter cis-elements for GhNPR3A.2/D.2, GhNPR5A/D, and GhNPR6A/D, indicating that these genes are preferentially expressed in fiber and could potentially function for the initiation and elongation stages of fiber development. Additionally, for the proper development of the plant, its resistance/tolerance should be strong enough for a different kind of biotic or abiotic stress. NPR genes are mostly involved in the defense signaling pathway and their expression patterns are also variable at different time periods of SA induction or pathogen infestation [GhNPR1D, GhNPR3A.1/D.1, and GhNPR4A/D were significantly activated after SA exogenous treatment. Although Zhang et al., 2008, emphasized GhNPR1 expression in SA induction at different time intervals, no information was provided about SA’s effect on GhNPR1 homologs and their functions in fiber development [GhNPR3A.2/D.2, GhNPR5A/D, and GhNPR6A/D were not expressed after exogenous SA treatment, which suggests no probable significant contribution in the defense pathway, and conversely showed participation in fiber development. Interestingly, a lower number of defense-regulated cis-elements were present in the upstream region of GhNPR3A.2/D.2, GhNPR5A/D, and GhNPR6A/D genes.In addition to the cotton seed, which is used for oil and protein, the natural cotton fiber is also a valuable resource as a raw material in industrial textile manufacturing, and is thereby a major contributor to a state’s economy. The in silico expression profiling of nine different tissues revealed that t levels ,61,62,63estation ,26,35,40elopment . NotablyNPR1-like genes were identified in G. hirsutum, including six genes in the A-subgenome and six genes in the D-subgenome. Based on the gene and protein structural characteristics and comparison with homologs from other cotton and plant species, the NPR-like proteins were grouped into three different clades, signifying evolutionary conservation and functional divergence. Consequently, in silico and quantitative expression profile analysis of GhNPR genes suggest the probable functions of different aspects of G. hirsutum for biotic stress tolerance and various fiber development stages. Therefore, the present study could be a useful resource in using the cotton NPR1-like gene family for the future challenges, such as developing varieties for pathogen tolerance and higher fiber yield.In conclusion, a set of homologs of cotton G. hirsutum was grown in a soil mixture under long-day conditions  in a controlled glasshouse environment. Arabidopsis thaliana seeds (Col-0 ecotype) were stratified on Soilrite for four days at 4 °C, after which seeds were allowed to grow under controlled environmental conditions . Leaves from G. hirsutum and Arabidopsis plants 8 weeks and 4 weeks post-sowing were taken for study, respectively.Arabidopsis Information Resource (TAIR) and Cotton Functional Genomics Database (CottonFGD) were used for the identification of the NPR1-like genes family in four Gossypium species—G. hirsutum, G. barbadense, G. ramondii, and G. arboreum. Protein sequences of all 6 Arabidopsis NPR family members AtNPR1 (At1G64280), AtNPR2 (At4G26120), AtNPR3 (At5G45110), AtNPR4 (At4G19660), AtNPR5 (At2G41370), and AtNPR6 (At3G57130) were utilized as queries to accomplish a protein–protein BLAST (BLASTP) program for the protein sequences of all 4 Gossypium species.The Arabidopsis NPR proteins with Clustal-X version 2 [Arabidopsis and cotton sequences were identified by BioEdit software.Multiple sequence alignment was performed using identified putative cotton NPR and ersion 2 . The locersion 2 ,65. The NPR genes—deduced amino acid length, molecular weight, theoretical pI, and grand average of hydropathy (GRAVY)—were obtained from the CottonFGD.The parameters of the identified putative cotton G. hirsutum NPR genes in A- and D-subgenomes to particular chromosomes, distinctly, was marked using Mapchart 2.3 software. For the gene structure analysis, coordinates of exon-intron for cotton and Arabidopsis NPR1 gene families were diagrammatically represented by the Gene Structure Display Server 2.0 [The chromosomal localization of putative rver 2.0 .NPR1-like gene family homologs from different plant species were identified by executing BLASTP search or from published research articles [Arabidopsis NPR1 family members as a query for generation of a phylogenetic tree. MCScanX software was used for the syntenic analysis with the default parameters [The protein sequences of NPR proteins from different plant species were retrieved and their multiple sequence alignment (MSA) were carried out with identified cotton NPR proteins using Clustal-X version 2 with default parameters. Subsequently, the obtained aligned protein sequences were used as inputs to construct unrooted phylogenetic trees following the neighbor-joining method with a Jones–Taylor–Thornton model and pairwise gap deletion by MEGA-X software with 1000 bootstrap replicates . Other Narticles ,25,27,28rameters . Circos rameters .GhNPR genes from CottonFGD and AtNPR genes through the TAIR database for identification of the cis-regulatory elements. The promoter cis-regulatory elements present in the NPR1-like genes were identified by PLACE and PlantCare [Sequences from the 2 kb upstream region of translation sites were retrieved for lantCare ,71.G. hirsutum TM1 in different tissues and fiber development stages were downloaded from NCBI (PRJNA248163). The obtained raw reads were quality filtered and further aligned to the reference genome of G. hirsutum using TopHat2 with default parameters [The RNA-Seq data of rameters . The alirameters ,74.GhNPR gene expression in the different fiber development stages, the flower was tagged on the day of flowering. The fiber tissues were harvested at 0 DPA (ovule), 5 DPA, 10 DPA, 15 DPA, 20 DPA, and 25 DPA. Similarly, samples were also collected for RNA isolation from different tissues: young leaf (top leaves from 8-week-old plants), mature leaf (lowest leaves from 8-week-old plants), stem (8-week-old plants), flower (10-week-old plants), and root (8-week-old plants) of G. hirsutum. For the induction of salicylic acid, 2 mM of salicylic acid was sprayed on G. hirsutum leaf tissue and harvested at 12 h and 24 h after induction for RNA extraction. Similarly, 2 mM of salicylic acid was sprayed and Arabidopsis leaf tissue was harvested after 12 h from 30 DAS (days after sowing) for RNA extraction [-∆∆CT method was used for analyzing the relative expression of target genes. For the validation of all genes, gene-specific primers (forward and reverse) were made by Primer Express 3.0.1 software (Applied Biosystems)  [GhNPR and AtNPR genes, respectively.To evaluate the putative traction . RNA wassystems) . Similariquitin4  and AtUb4g05320)  were use


Here, we investigated the impact of oxidative stress on COX-2 and prostaglandin (PG) pathways in paw exudates, and we studied how this mechanism can be reversed by using antioxidants during hyperalgesia in a well-characterized model of inflammatory pain in rats. Our results reveal that during the inflammatory state, induced by intraplantar administration of carrageenan, the increase of PGE2 levels released in the paw exudates were associated with COX-2 nitration. Moreover, we showed that the inhibition of ROS with Mn (III) tetrakis (4-benzoic acid) porphyrin(MnTBAP) antioxidant prevented COX-2 nitration, restored the PGE2 levels, and blocked the development of thermal hyperalgesia.In clinical practice, inflammatory pain is an important, unresolved health problem, despite the utilization of non-steroidal anti-inflammatory drugs (NSAIDs). In the last decade, different studies have proven that reactive oxygen species (ROS) and reactive nitrogen species (RNS) are involved in the development and maintenance of inflammatory pain and hyperalgesia via the post-translation modification of key proteins, such as manganese superoxide dismutase (MnSOD). It is well-known that inducible cyclooxygenase 2 (COX-2) plays a crucial role at the beginning of the inflammatory response by converting arachidonic acid into proinflammatory prostaglandin PGE Inflammatory pain is considered a major health issue. Nowadays, non-steroidal anti-inflammatory drugs (NSAIDs) are the most common class of analgesics for moderate/severe inflammatory pain—in particular, non-selective cyclooxygenase 1/cyclooxygenase 2 (COX-1/COX-2) or more selective COX-2 inhibitors are the mostly used formulations, even if various side effects associated with their prolonged utilization are well-documented ,4.During acute/chronic inflammation, hyperalgesia is the result of a persistent state of peripheral afferent sensitization, which subsequently leads to spinal sensitization through the release of the excitatory amino acid glutamate and free radical production (reactive oxygen species (ROS) and reactive nitrogen species (RNS)) ,6,7,8.Free radical oxidative damage is strictly linked with oxidative stress, involved in several degenerative diseases, such as inflammatory, cardiovascular, and digestive diseases, as well as cancer, aging, and stroke ,11,12,13Free radical activity includes many proinflammatory effects—for example, endothelial cell damage and increased microvascular permeability, the recruitment of neutrophils at the sites of inflammation , the autThe concentration of nitroxidative species (ROS/RNS) can increase during stress conditions, inducing the activation of transcription factors  and nuclear factor kappa (NF-kB)) and mitogen-activated protein kinases (MAPKs), which can in turn activate COX enzymes and increase the production of prostaglandins (PGs) ,20,21.2 (PLA2), and then of arachidonic acid (AA) levels [Consequently, prostaglandins, through the activation of different nociceptors, lead to an increase of phospholipase A) levels ,22,23.Moreover, nitric oxide (NO) seems to interfere directly with the activity of COX-2, and therefore with the production of PGs; the metabolites of the arachidonic acid, similarly, would be able to modulate the biosynthesis of NO ,20,21,24Pharmacological removal of ROS/RNS by antioxidants ,26,27,28To date, the cross-talk mechanism through which free radicals and COXs pathway contribute to the development and maintenance of hyperalgesia needs to be better clarified . In the Our findings underline the pivotal role of nitroxidative stress in hyperalgesia pathway. Thus, we suggest that the chronicization of pain could depend on post-translational nitration of key enzymes and transporters linked to glutamatergic neuro-transmission.p < 0.05, as set forth by the International Society for the Study of Pain guidelines [Male Sprague–Dawley rats  were used following the Italian regulations for the protection of animals used for experimental and other scientific purposes (D.L. 26/2014); the European Economic Community regulations (2010/63/UE), with authorization number 577-2016-PR; and the National Institutes of Health (NIH) guidelines on laboratory animal welfare. The numbers of animals used are the minimum number necessary to achieve statistical significance at idelines . Rats (tn = 12) received an intraperitoneal (i.p.) injection of saline 15 min before intraplantar (i.pl.) injection of saline into the hindpaw;Vehicle group: animals (n = 12): 15 min before intraplantar injection of carrageenan  into the right hindpaw, rats received an intraperitoneal saline injection;Carr group (n = 12 for each dose) received an intraperitoneal injection of different doses of MnTBAP  15 min before intraplantar injection of carrageenan  into the right hindpaw.Drugs groups: animals  quantification, paw tissues were centrifuged at 250× g for 20 min, as previously described [The dose and the timing of MnTBAP administration were chosen according to the literature ,28,31. Fescribed ,33, and Hyperalgesic responses were detected following Hargreaves’s protocol . Twenty 2 released in the paw exudates was measured by enzyme-linked immunosorbent assay (ELISA), as described previously [g for 20 min in order to recover a sample of the oedematous fluid, and the volume of fluid recovered from each paw was measured. Results were expressed in pg/paw, normalizing values to the amount of exudates recovered from each paw. All determinations were performed in triplicate.PGEeviously ,21,32,33g for 20 min, was measured. The levels of LDH in the paw exudates  were used as an indicator of cell toxicity and cell death, and were spectrophotometrically measured (at an absorbance of 340 nm) using an LDH assay kit (Sigma). All determinations were performed in triplicate.Paw exudate, recovered through a centrifugation of 250× g at 4 °C. The MDA–TBA adduct was fluorometrically measured at an excitation wavelength of 530 nm and emission wavelength of 550 nm using an Infinite 200 microplate fluorometer (Tecan). All determinations were performed in triplicate.MDA quantification was measured in the paw soft tissues through a thiobarbituric acid (TBA) reactive substance (TBARS) assay, as previously described . Tissue v/v). Extracts were sonicated for 5 min (Fisher Scientific Sonicator) and incubated for 10 min on ice, and then were centrifuged at 12,500× g for 30 min at 4 °C. The obtained supernatants were stored immediately at −80 °C and used to evaluate immunoprecipitation and for Western blot analyses. Protein concentrations were determined using the bicinchoninic acid (BCA) protein assay (Pierce).Paw soft tissue homogenization was performed in lysis buffer ) with the addition of 1% protease inhibitor cocktail  washed in PBS (pH 7.4). The bead–antibodies and binding proteins were resuspended in 50 µL of sample buffer , 2.5% glycerol/0.5% SDS/200 mM 2-mercaptoethanol/0.001% bromophenol blue) and then boiled for 5 min at 95 °C. the immunoprecipitated proteins were resolved in 12% SDS-PAGE mini, and the proteins were transferred to nitrocellulose membranes. The membranes were blocked for 1 h at room temperature in 1% Bovine serum albumin (BSA), 0.1% thimerosal in 50 mM Tris-HCl (pH 7.4), and 150 mM NaCl, 0.01% Tween-20 (TBS/T), followed by incubation with rabbit polyclonal antibodies for MnSOD , COX-2 , and nitrotyrosine . Membranes were then washed with TBS/T and incubated with a secondary antibody conjugated to horseradish peroxidise  for 1 h at room temperature. After washes, the proteins were visualized by enhanced chemiluminescence . No difference in monoclonal β-actin  was detected among the lanes. All the densitometry units were normalized against actin for each lane, and are expressed as the ratio of nitrated to unnitrated proteins. Protein bands were quantified by densitometry using Image Quant 5.2 software (Molecular Dynamics).g.MnSOD activity was measured as previously described ,35,36. B2 to reduce nitrobluetetrazolium (NTB) to blue tetrazolium salt, was performed as previously described [To determinate MnSOD activity, a competitive inhibition assay that used xanthine–xantine oxidase-generated Oescribed ,35,36. T4)2CO3 at 56 °C for 30 min, and then cooled and alkylated in 55 mM Iodoacetamide (IAA) and 25 mM (NH4)2CO3 for 30 min in the dark at room temperature. Gel pieces were washed in 50 mM (NH4)HCO3/CH3CN 1:1 for 15 min and covered by acetonitrile until gel pieces shrunk. Under the vacuum, centrifugation allowed acetonitrile removal and gel particles drying. In-gel digestion was performed overnight at 37 °C under stirring, by adding 12.5 ng/μL of trypsin in 25 mM (NH4)2CO3. MALDI mass spectra were recorded directly from the overlay.Proteins of paw soft tissue, obtained as previously described through cytosolic extraction, were solubilized in NuPage LDS Sample Buffer (invitrogene), heated at 70 °C for 10 min, separated on precast 4–20% Bis-Tris gels (Invitrogen), and stained with colloidal Coomassie Blue. After visualization, each protein spot was cut and each gel slice was de-stained in 50 mM ammonium hydrogen carbonate/acetonitrile 1:1, and covered with acetonitrile for the reduction of gel pieces. Under a vacuum, centrifugation was performed to remove acetonitrile and to try gel particles. Proteins were suspended in 10 mM dithiothreitol (DTT) and 25 mM (NHv/v) was applied to the sample and dried again. MALDI mass spectra were recorded using a Voyager-DE STR Applied Biosystems, (United States), a MALDI time-of-flight (MALDI-ToF) mass spectrometer, using the reflectron mode of operation. Ionization was performed with a 337 nm pulsed nitrogen laser. Mass calibration was internally performed using the molecular ions from the trypsin autodigestion. Raw data were analysed using Data Explorer software provided by the manufacturer, and reported as monoisotopic masses. MALDI mass spectra were compared to identify signals with a mass difference of 45 kDa (aminoacidic nitration).Samples (1 μL) were applied to the target and air-dried. Subsequently, 1 μL of α-cyano-4- hydroxycinnamic acid (10 mg/mL) in 50 % acetonitrile and 0.1 % Trifluoroacetic acid (TFA) .The Kolmogorov–Smirnov test was used for analysis of the data distribution. After confirmation of normal data, differences between groups were compared by analysis of variance (ANOVA). The results are expressed as mean ± SEM. Two-way repeated measures ANOVA with Bonferroni comparisons were used for data obtained from each time point. Other data were analyzed via one-way ANOVA followed by the Newman–Keuls test. The level of statistical significance was fixed at 2, and MDA accumulation in paw exudates (Intraplantar (i.pl.) injection of carrageenan in rats determined a time-dependent development of thermal hyperalgesia, which showed up 2 h after the administration of carrageenan A. The deexudates .2, and MDA levels in paw exudates  injection of a different dose of MnTBAP  reduced the thermal hyperalgesia and led to a significant improvement in tissue damage and inflammation, characterized by the inhibition of oedema . Furtherexudates , observe2− dismutase (MnSOD), as evidenced by immunoprecipitation analysis  pretreatment (intermediate dose), intraperitoneally administered 15 min before carrageenan, blocked protein nitration A,B and r2 , but also different proteins expressed during inflammation, including inducible COX-2 in paw tissues, as shown by immunoprecipitation . This en2 , a hormo2 . This da2  was unde2 . The nitInflammatory pain is a serious health problem. Its treatment, despite the use of NSAIDs, remains inaccurate, expensive, and sometimes useless. Clinical trials have shown that free radicals, responsible for the onset of oxidative stress, are implicated in the development of numerous diseases, including acute and chronic inflammation ,41. HereHigh levels of ROS/RNS species and oxidative stress promote the oxidation of biological molecules, including DNA, lipids, and proteins in plasma and mitochondrial membranes . This pr2 (PGE2) levels released in paw exudates, thus suggesting their pivotal role in the generation of hyperalgesia and inflammation. These effects were significantly attenuated by pre-treatment with MnTBAP, an inhibitor of lipid peroxidation.Our results strongly suggest that during the well-known model of carrageenan-induced inflammation and hyperalgesia in rats, the MDA level increases in association with an increment of lactate dehydrogenase (LDH) and prostaglandin EIt is well known that prostaglandins (PGs) are the main lipid mediators, with important functions during inflammatory processes. They are synthesized from arachidonic acid by two isoforms of cyclooxygenases, known as COXs (COX-1 or COX-2).Specifically, COX-1, expressed in many tissues, plays a protective role, providing PG involvement in homeostatic functions ,46; COX-2 (PGE2), the most abundant PG, is involved in many biological functions, but its deregulation or degradation has been associated with several pathological conditions, including the development of acute and chronic inflammation [Prostaglandin Eammation ,47,48.2 production has been observed in mice and rats during carrageenan-induced inflammatory pain. In particular, it was demonstrated that the weight and mouse’s age are critical elements for oedema and correlated enzyme  development [2 production has been observed in the first oedema’s phase (during the first 6 h) due to COX-1, and during the following phase due to COX-2. Conversely, PGE2 production in rats is constant during all the inflammatory phase [In recent years, a different mechanism of PGEelopment . Differeelopment . In the elopment , PGE2 prry phase ,50,51. TIn addition, previous studies have demonstrated that NO and PGs released during inflammation is responsible of the activation and deliver of an inducible isoform of nitric oxide synthase (iNOS) and COX-2 ,22,52, wIn these conditions, tissue inflammation is characterized also by superoxide production that, interacting with NO, leads to the peroxynitrite formation .Our previous work has already documented the critical role of nitroxidative species, including peroxynitrite, in the development of peripheral and central sensitization associated with pain of diverse etiologies, such as inflammatory and neuropathic pain ,29,54,55In particular, peroxynitrite, a pro-inflammatory and cytotoxic agent, is responsible for protein nitration, including MnSOD, a mitochondrial enzyme able to desmute and remove high levels of superoxide . In the The excess of free radicals during the pain state is generally inactivated by antioxidants, which inhibit the oxidation of other molecules by directly removing free radicals (pro-oxidants) and providing maximal protection for biological sites ,37.It is well-known that antioxidants are classified based on their chemical nature or mode of function ,60,61,62In conclusion, these results reinforce the notion that free radicals and peroxynitrite are important mediators of the induction of hyperalgesia and inflammation. Moreover, peroxynitrite, through the nitration of the tyrosine residue of MnSOD and COX-2, contributes to the formation of free radicals and to the biosynthesis of prostaglandins in rat paw exudates, thus playing a key role as pro-inflammatory and pro-nociceptive agents in a novel mechanism of peripheral sensitization. This mechanism can be pharmacologically restored through the use of MnTBAP, most probably due to its ability to remove peroxynitrite.In this contest, where the complexity of pain requires new therapeutic approaches, the identification of innovative drugs is crucial to improve patient’s rehabilitation and life quality.


Indeed, high levels of T helper (Th) cells and related cytokines and chemokines have been found in CNS lesions and in cerebrospinal fluid (CSF) of MS patients, thus contributing to the breakdown of the blood–brain barrier (BBB), the activation of resident astrocytes and microglia, and finally the outcome of neuroinflammation. To date, several types of Th cells have been discovered and designated according to the secreted lineage-defining cytokines. Interestingly, Th1, Th17, Th1-like Th17, Th9, and Th22 have been associated with MS. In this review, we discuss the role and interplay of different Th cell subpopulations and their lineage-defining cytokines in modulating the inflammatory responses in MS and the approved as well as the novel therapeutic approaches targeting T lymphocytes in the treatment of the disease.Multiple sclerosis (MS) is a chronic neurodegenerative disease characterized by the progressive loss of axonal myelin in several areas of the central nervous system (CNS) that is responsible for clinical symptoms such as muscle spasms, optic neuritis, and paralysis. The progress made in more than one decade of research in animal models of MS for clarifying the pathophysiology of MS disease validated the concept that MS is an autoimmune inflammatory disorder caused by the recruitment in the CNS of self-reactive lymphocytes, mainly CD4 Multiple sclerosis (MS) is a chronic inflammatory autoimmune disorder of the central nervous system (CNS) affecting about 2–3 million people worldwide that is triggered by both environmental and genetic factors ,2. About+ T cells. Autoreactive CD4+ T cells likely activated in the peripheral lymph nodes migrate into the CNS [+ T cells [+ T helper (Th) cell subsets in the pathophysiology of MS and the current therapeutic approaches targeting T-cell mediated responses. The role of regulatory T (Treg) cells in suppressing the functions of autoreactive Th cells in MS is also briefly discussed.The pathological hallmarks of MS are the breakdown of the blood–brain barrier (BBB), oligodendrocyte loss, demyelination, astrocytes gliosis, and axonal degeneration ,7. Infla the CNS ,12,13,14 the CNS . For ins T cells . The aim+ Th cells are central regulators of the adaptive immune response against a wide variety of microbes by helping B lymphocytes to produce antibodies (Ab) and by secreting specific cytokines that provide efficient protection against pathogens. Distinct Th cell subsets, producing one or more lineage-defining cytokines and expressing master transcription factors and homing receptors, differentiate from naïve CD4+ T cells in response to a specific class of pathogenic microorganisms and to the cytokine milieu. Naïve CD4+ T cells are activated in peripheral lymph nodes by mature dendritic cells that present pathogen-derived peptides associated to class II major histocompatibility complex (MHC) and together with costimulatory molecules promote T cell proliferation and produce polarizing cytokines, which in turn orchestrate T cell differentiation in distinct Th cell subsets, such as Th1, Th2, Th17, Th22, and Th9 [CD4 and Th9 . In addi+ T cells that orchestrate efficient adaptive immune responses against intracellular pathogens by secreting interferon (IFN)-γ that activates macrophages to kill intracellular microbes and promotes the production of opsonizing Abs [Th1 cells were identified in the late 1980s  as a subzing Abs . Th1 mayzing Abs . IL-12 azing Abs ,23. Initzing Abs ,25. IFN-zing Abs  and in azing Abs , and thezing Abs ,29. Theszing Abs ,31 suppozing Abs . Th1 celzing Abs ,34. MoreHowever, further observations that mice deficient in IL-12p35 subunit  or IL-12+ Th cells that mediates efficient immune responses against extracellular bacteria and fungi [Th17 cells have been identified in 2005  as an imnd fungi . Th17 cend fungi , the intnd fungi . In partnd fungi ,48. IL-2nd fungi , and IL-nd fungi . The resnd fungi . Notablynd fungi , where tnd fungi  and by tnd fungi ,54  of RRMS patients during relapses [During the past years, several studies in EAE mice ,41,63 torelapses ,66,67 anrelapses . Moreoverelapses  and withrelapses . IL-17A relapses  by alterrelapses ,73 and frelapses . For insrelapses .+ T cells to produce inflammatory cytokines and chemokines related to the Th17 cell phenotype [Several key transcription factors, the most reliable RORγτ  and STAThenotype ,96 and thenotype ,98.+ T cells as well as from peripheral blood Th17 cells of RRMS patients display distinct inflammatory features compared to healthy donors. In particular, Th17 cells from RRMS patients upregulate the expression of IL-1R and produce higher levels of IL-21, IL-2, and TNF-β thus leading to the acquisition of a more pathogenic profile [Th17 cells exhibit high plasticity and may adopt different functional profiles depending on the inflammatory or anti-inflammatory environments . Th17 ce profile . Consist profile . Th17 ce profile . Moreove profile  and incr profile . Th17 ce profile , a cytok profile ,108 and  profile ,110.Recently, a novel Th1 cell subset known as Th1-like Th17 cells that produce both IFN-γ and IL-17 has been identified in both human and mice ,112. TheThe participation of highly pathogenic Th1-like Th17 cells in neuroinflammation has been evidenced by studies in both EAE and MS . For ins+ Th cell subset, named Th22 [Discovered in 2000 , IL-22 imed Th22 ,126,127.med Th22 ,129, ILCmed Th22 . IL-22 imed Th22 .+ T cells resistant to corticoids have been associated with active brain lesions in MS patients [Although the role of IL-22 in MS has not yet been fully elucidated, emerging evidence suggest the involvement of IL-22 in MS immunopathogenesis. Initial studies from Kebir et al. evidenced the upregulation of IL-22R in the brains of MS patients and the role of IL-22 in synergy with IL-17A in disrupting the integrity of BBB tight junctions by reducing the expression of occludin in endothelial cells . More repatients . Moreovepatients , which mpatients . Interespatients ,137  [+ T cells under specific conditions of antigen-stimulation and in the presence of a particular cytokine milieu. iTreg comprises different subsets including FoxP3+ Treg, type 1 regulatory (Tr1) T cells producing high levels of IL-10 and Th3 cells producing TGF-β [The development of autoimmunity is finely regulated by specific subsets of T cells that modulate immune responses by keeping pathogenic Th cells under control. Treg cells were firstly identified in 1995 by Sakaguchi et al. as a CD4olerance . In the  (FoxP3)  and indung TGF-β . In humang TGF-β .Alterations in both number and suppressive functions of distinct Treg subsets have been reported in MS, especially in RRMS patients ,156, andThe relevant contribution of autoreactive inflammatory Th cell subsets in the pathogenesis of MS is supported by the fact that most of the approved disease-modifying therapy target T cells.IFN-β an antiviral cytokine with immunosuppressive and anti-proliferative effects, has been the first-line drug approved by the Food and Drug Administration (FDA) for the treatment of RRMS in 1993. The most relevant effects of IFN-β on T cells are the inhibition of T cell activation and secretion of inflammatory cytokines , the shiGlatiramer acetate (GA), a mixture of synthetic polypeptides containing only 4 amino acids, glutamate, lysine, alanine, and tyrosine, was approved by the FDA for the treatment of RRMS in 1996. Although the mechanisms of action of GA are not completely understood and elucidated, several studies suggest a pivotal role of GA on different antigen-presenting cells by promoting the differentiation of microglia, macrophages, and dendritic cells into the suppressive M2 phenotype producing anti-inflammatory cytokines, such as TGF-β and IL-10 ,162,163.+ T cells [Dimethyl fumarate (DMF), a fumaric acid ester with anti-inflammatory activity has been approved for the treatment of RRMS in 2013 . DMF exe T cells . DMF tre T cells . Finally T cells ,181.Fingolimod, a first orally therapeutic molecule approved for RRMS ,183, actNatalizumab is another disease-modifying drug that interferes with the migration of T cells approved for RRMS in 2004. It is a monoclonal antibody (mAb) that targets VLA-4 expressed by activated T and B cells  and inhi+ and CD8+ T cells by Ab-dependent (ADCC) and complement-dependent cytotoxicity (CDC) [+ T cell subset producing inflammatory cytokines [+ inflammatory T cells are present in the peripheral blood and brain of MS patients [FDA and EMA approved two humanized mAbs targeting T cells for RRMS. Alemtuzumab, approved in 2013, targets CD52 that is expressed at high levels on B and T cells [ty (CDC) . Daclizuty (CDC) . In contty (CDC) . Due to ty (CDC) . In Novety (CDC) . Interesty (CDC) ,207, wasytokines . Since Cpatients ,209, theIn addition to the currently approved T cell targeting therapies, several therapeutic approaches aimed at inhibiting autoreactive inflammatory T cells are currently tested in clinical trials for MS . Among t+ T cells, in initiating and perpetuating the inflammatory responses and the consequent neurodegeneration in MS. Accordingly, most of the approved therapeutic strategies are aimed to attenuate inflammatory T helper cells and emerging more selective T-cell-based therapeutic approaches are currently ongoing in clinical trials for MS.In the last two decades, our knowledge of the immunopathogenic mechanisms of MS has strongly improved, thus evidencing a fundamental role of encephalitogenic Th cells, in particular, Th1-Th17 like, Th17, Th22, and GM-CSF-producing CD4


On MRI DWI sequence, all subjects had abnormal hyperintensities in various parts of the cerebral cortex, striatum, or thalamus, while EEG detected periodic triphasic waves only in 11. CSF 14-3-3 protein and total τ-protein were abnormal in 17 of 23 cases. All patients died, median lifespan being 2 months (range: 19 days−14 months). In conclusion, a complex of clinical, radiological, and laboratory manifestations of stroke-like onset of CJD is outlined. The clinical relationships between CJD and stroke are considered, in an attempt to highlight this rare presentation of an uncommon disease.Only a few case reports of stroke-like onset of Creutzfeldt-Jakob disease (CJD) have previously been published. We aimed to analyze the neurological, imaging, electroencephalographic (EEG), and laboratory features of patients with this very rare phenomenon. Here, we review the clinical characteristics, onset features, and clinical course variants of stroke-like CJD in 23 such patients. The median age of the patients was 71 years (range: 56–84 years); 12 were women. In 20 patients, CJD was sporadic. Thirteen patients developed apoplexy-like onset of symptoms, whereas the others had prodromal non-specific complaints. Most often the patients manifested with pyramidal signs ( Sc), with their deposition in the cortex, striatum, and thalamus (Creutzfeldt-Jakob disease (CJD) is a rare neurodegenerative disease caused by accumulated misfolded proteins  clinical situation when the onset of CJD was initially misdiagnosed as a stroke; (2) subsequent workup indicated probable or definite CJD according to the international criteria ; or (3) Data were analyzed using a Microsoft Excel 2010 spreadsheet. Results were expressed as means with standard deviations (SD) or as median with interquartile range (IQR).Twenty-three clinical cases (cases 1–23) were analyzed. The mean age of the subjects was 70.1 years , of whom 12 (52.2%) were females. In 20 cases, sporadic CJD was diagnosed, in two cases, the disease was familial [cases 6 ] Table .A 71-year-old right-handed woman was evaluated in the emergency department with a 2-day history of asthenia, dizziness, gait instability, speech disorders, and periodic involuntary jerky movements in her left arm. Past medical history included mild well-controlled hypertension. On admission, she was disorientated, her cranial nerves were normal, she had mild weakness and jerky dystonia with hyperactive tendon reflexes, and suspected hemi-ataxia were seen in the left arm. Plantar responses were flexor. A non-enhanced CT scan revealed a left parietal hypodense focus and cerebellar calcifications, which could not explain ipsilateral symptoms. At this stage the patient was clinically diagnosed with possible ischemic stroke in the right hemisphere. However, over a 3-week hospitalization period, her condition progressively deteriorated. She became negativistic and non-compliant. A psychiatric examination revealed inappropriate affect with euphoria and visual hallucinations. Her MoCA  score d in the lnursing home nearly 3 weeks later. Autopsy has not been performed.CSF exhibited no cells; glucose was 72 mg/dl, protein 34 mg/dl. However, total τ-protein was markedly elevated up to 2,524 pg/ml , suggesting a diagnosis of probable sporadic CJD according to the updated diagnostic criteria (Thirteen patients (56.5%) in this series presented with sudden apoplectic-like or wake up symptoms and , motor disorders mono- or hemiparesis, dysmetria, dystonia, alien hand, or combined disorders . Ten othOther differential diagnoses were antibody-mediated autoimmune encephalitis [cases 4  and 11 ] treatedThe vast majority of patients, 20/23 87%), showed a rapid progressive deterioration after the acute onset in both motor and cognitive state, followed by unresponsiveness, blindness, akinetic mutism, stupor, ataxia, generalized rigidity, spasticity, hyperreflexia, and myoclonus. In other 3 patients (13%) SD = 4.1, median = 2, IQR: 1.1–5), range 19 days−14.2 months.The mean survival of the 23 patients from the stroke-like onset was 4.2 months (Brain MRI studies were performed in 22/23 patients (95.6%) and in 13 of them (59.1%) within 1–14 days from the onset of CJD symptoms. Four patients in this group had MRI not characteristic for CJD (18.2%). In four other patients, non-enhanced MRI images revealed unilateral or bilateral diffuse areas of abnormal high signal intensities with restriction in diffusion (ribbon sign) in various parts of the cerebral cortex (18.2%), and in the remaining five patients (22.7%), there was a combined lesion of the cortex and basal ganglia (hyperintensity of the head of caudate nucleus and putamen) on DWI sequence. In the remaining nine patients (40.9%), MRI images were performed later within two months from the onset of the apoplectic presentation. All of them were pathological, often with combined lesions of various parts of the cortex and basal ganglia  see .Along with the above mentioned MRI signs characteristic of CJD, in 4/22 cases 18.2%), T2 FLAIR in the first 3–4 days detected only scattered hyperintensities in the centrum semiovale .Single or repeated EEG were recorded in all 23 studies. During the first 2 weeks after the CJD symptoms onset, the initial recordings were performed in 10/23 patients (43.5%). In two subjects, the EEG was within the normal range, in six, non-specific changes were registered , while periodic sharp waves were observed in only two individuals. In a later period, on average 55 ± 4.5 days, in 6/23 patients (26.1%), the EEG changes remained non-specific, whereas in 6/23 cases, periodic triphasic waves were recorded (26.1%). In one patient, the EEG remained normal at 7 months after the onset of the disease shortly before death [case 8 ] Table .CSF 14-3-3 protein was studied in 16 patents and was positive in 11 (68.8%), τ-protein was measured in seven patients and was elevated on average to 1,924 ± 1,459 pg/ml in six cases . RT-QuIC assay was positive in three cases: 1 , 2 23),,23), andGenetic analysis was abnormal in case 7 ].Autopsy was performed in 9/23 cases (39.1%) and confirmed spongiform encephalopathy with diffuse synaptic pattern of prion protein immunoreactivity. None of them had changes consistent with an acute stroke. A brain biopsy, which verified the diagnosis, was performed in one patient. In general, these pathologically confirmed cases  were notIn our case, as in the other cases cited, the sudden onset of neurological disorders suggested an acute stroke, but further developments and tests revealed the diagnosis of CJD.The available data on the relationship between CJD and stroke, suggest several possibilities.The apoplectic onset, presumably caused by local neuronal shutdown induced by rapid prion propagation, was initially diagnosed as stroke, and only later progressive clinical worsening supplanted by MRI, EEG, or CSF data indicated prion disease. No evidence for acute vascular brain lesion was observed on initial CT or MRI scans. This situation occurred in the majority of the cited cases: 1 , 4 10),,10), 6 , 10 25)25), 12 , 13 (12)In our subject case 23), as in some others [cases 2 , as in s, 15 19)19), 16 , and 18 PRNP gene sequence analysis with 129 polymorphism valine homozygosity (VV2) [case 3 .YB contributed to the conception, execution, design, and preparation of the manuscript for publication. AK contributed to the conception, design, and review of the manuscript. NK contributed to the execution and preparation of the manuscript for publication. AE contributed to the execution and review of the manuscript. RG contributed to the conception, execution, and review of the manuscript. MA contributed to the design and review of the manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The 26S proteasome is a large (~2.5 MDa) protein complex consisting of at least 33 different subunits and many other components, which form the ubiquitin proteasomal system (UPS), an ATP-dependent protein degradation system in the cell. UPS serves as an essential component of the cellular protein surveillance machinery, and its dysfunction leads to cancer, neurodegenerative and immunological disorders. Importantly, the functions and regulations of proteins are governed by the combination of ordered regions, intrinsically disordered protein regions (IDPRs) and molecular recognition features (MoRFs). The structure–function relationships of UPS components have not been identified completely; therefore, in this study, we have carried out the functional intrinsic disorder and MoRF analysis for potential neurodegenerative disease and anti-cancer targets of this pathway. Our report represents the presence of significant intrinsic disorder and disorder-based binding regions in several UPS proteins, such as extraproteasomal polyubiquitin receptors (UBQLN1 and UBQLN2), proteasome-associated polyubiquitin receptors (ADRM1 and PSMD4), deubiquitinating enzymes (DUBs) (ATXN3 and USP14), and ubiquitinating enzymes (E2 (UBE2R2) and E3 (STUB1) enzyme). We believe this study will have implications for the conformation-specific roles of different regions of these proteins. This will lead to a better understanding of the molecular basis of UPS-associated diseases. Before 1970, lysosomes were thought of as exclusive cellular machinery to execute misfolded protein degradation. However, in 1977, work by Etlinger et al. reported the presence of a second intracellular ATP-dependent mechanism for degradation of proteins ,3,4. ThiUBB gene encoding ubiquitin-B and molecular misreading of this gene that introduces dinucleotide deletions  can generate mutated Ub forms, which are associated with human diseases. For example, the UBB+1 (Ubiquitin-B+1) form of Ub generated as a result of molecular misreading is linked to Alzheimer’s disease (AD), other tauopathies, and polyglutamine (PolyQ) diseases ) [UBA1 gene lead to X-linked spinal muscular atrophy (SMAX2), and reduced UBA1 levels affect UPS-mediated degradation of misfolded proteins leading to neurodegenerative diseases, such as AD [UBQLN1, UBQLN2, UBQLN3, and UBQLN4, which encode structurally related and conserved proteins. A fifth ubiquilin gene, UBQLNL, was later identified in humans. Although all ubiquilin family members are present in cytosol, they exert different tissue expression patterns [UBQLN2 was recently described in familial amyotrophic lateral sclerosis (ALS) [In UPS, the misfolded proteins get ubiquitinated by a cascade of enzymes, including ubiquitin-activating E1s as well as ubiquitin-conjugating E2s and E3 ubiquitin ligases . The ubise (HD)) ,17,18, wse (HD)) . These Use (HD)) . Missensch as AD . Ubiquitch as AD . Studiesch as AD . Ubiquitch as AD . Ubiquilch as AD . The humpatterns . Ubiquilpatterns . Ubiquilis (ALS) .Ubiquitination and deubiquitination are dynamic processes that involve transient protein–protein interactions. There are ~100 deubiquitinating enzymes (DUBs) in the human genome that control several cellular processes in a very dynamic and specific manner. Among these DUB-controlled processes are the progression of the cell cycle, degradation of proteins, apoptosis, activation of kinases, chromosome segregation, gene expression, protein localization, and DNA repair . In the The ubiquitin-specific protease (USP) family of DUBs, with more than 50 members, is the largest family among all DUBs . UbiquitIntrinsically disordered proteins (IDPs) and IDP regions (IDPRs) are the proteins or regions of proteins that lack well-defined, three-dimensional unique structures and show structural transition upon binding with their biological partners ,47,48,49The 15 proteins of UPS that play a crucial role in this pathway and have been identified in the pathogenesis of human diseases were selected for disorder analysis, which is summarized in ® VSL2 [® VL3 [® VLXT [® FIT [Several commonly used disorder predictors, such as PONDR® VSL2 , PONDR® 2 [® VL3 , PONDR®  [® VLXT , PONDR® T [® FIT , and IUPT [® FIT  were utiT [® FIT ,84,85,86The MoRFs are disorder binding motifs that are found in specific disordered regions. These regions were predicted using four different web servers, MoRFCHiBi_Web , ANCHOR The functioning of disordered regions from important proteins of UPS are explained in the current study. Furthermore, to explore the interaction among these proteins and with other proteins, they have been analyzed using the STRING v11 database . The datThe predicted IDP regions based on mean PPID value and MoRFCHiBi_Web-predicted MoRF regions were shown on available crystal or NMR structures using the Maestro GUI . All the color schemes for representing these regions are given in respective figure legends.1–7, β1–7, β1–7 and α1–7. This is 28 subunits in total. β1 has caspase-like, β2 has trypsin-like, and β5 has chymotrypsin-like peptidase activities [UPS is employed by the eukaryotic cell to get rid of excess, unnecessary, or misfolded short-lived regulatory proteins . Figure tivities . These stivities ,98,99.Here, we looked at the intrinsic disorder predisposition of 15 UPS proteins , which p® VLXT and PONDR® VSL2. The results of these analyses are summarized in PONDR VLXT vs. PPIDPONDR VSL2 plot. According to the overall levels of intrinsic disorder, proteins can be classified as highly ordered (PPID < 10%), moderately disordered (10% ≤ PPID < 30%) and highly disordered (PPID ≥ 30%) [To obtain a global overview, we looked at the predicted percentage of intrinsic disorder (PPIDs) in these proteins evaluated by PONDRD ≥ 30%) . From th2P2 database (http://d2p2.pro/) [2P2 is a database of predicted disorders for a large library of proteins from completely sequenced genomes. D2P2 uses outputs of IUPred [® VLXT [® VSL2 [2P2-generated functional disorder profiles of 15 UPS proteins are discussed below for individual proteins. Overall, this shows that all these proteins contain noticeable levels of intrinsic disorder, are heavily decorated with various PTMs, and many of them contain multiple MoRFs, suggesting that these proteins are expected to be characterized by high binding promiscuity. Additionally, the disorder-based protein-binding regions/MoRFs for individual proteins, identified by MoRFCHiBi_Web, MoRFpred, DISOPRED3, and ANCHOR, are listed in We also utilized Dp2.pro/)  to retrif IUPred , PONDR®  [® VLXT , PrDOS [ [® VLXT , PONDR®  [® VSL2 , PV2 [93 [® VSL2 . The outiN is also connected to every other node within the neighborhood, and is equal to 0 if no node that is connected to a given node iN connects to any other node that is connected to iN) is 0.895. Furthermore, since the expected number of interactions among proteins in a similar size set of proteins randomly selected from human proteome is equal to 5, the inter-set PPI network has significantly more interactions than expected, being characterized by a PPI enrichment p-value of <10−16.Next, we analyzed the inter-set intractability of 15 UPS proteins associated with human diseases using a publicly available computational platform, STRING, which integrates extensive information on protein–protein interactions (PPIs), complements it with computational predictions, and returns a PPI network showing all possible PPIs of a query protein(s) . The resp-value is <10−16).We also used STRING to study the engagement of the subunits of 15 UPS proteins in interactions with 500 proteins forming the first shell of the resulting interactome (note that the number of interactors in STRING is limited to 500). In this analysis, the highest confidence level of 0.9 was used. Most of the proteins associated with diseases, such as cancer, AD, PD, diabetes, and cardiovascular disease are either IDPs or contain long IDPRs, and misbehavior or mutations in the IDPs/IDPRs have broad involvement in the pathogenesis of these diseases ,47,48,49® VLXT, PONDR® VSL2, PONDR® VL3, PONDR® FIT, and IUPred, was 9.74% , the HECT (homologous to the E6AP carboxyl terminus), and the RBR (RING between RING) types . RING E32+ ions . A subse2+ ions . Some RI2+ ions . HECT E32+ ions . Similar2+ ions . FurtherUBA52 and RPS27A code for a single copy of Ub fused to the ribosomal proteins L40 and S27a, respectively, and where a polyubiquitin precursor with exact head-to-tail Ub repeats is encoded by the UBB and UBC genes, with the corresponding products, polyubiquitin-B and polyubiquitin-C, containing three and six Ub chains, respectively. Mature Ub exists either as a free protein or as a conjugated form that is covalently bound to various intracellular proteins, typically for their degradation through 26S proteasome [Ubiquitin (Ub) is a 76-amino-acid-long, highly conserved protein expressed in all eukaryotic cells. Ub is encoded by four different genes, where oteasome ,71. Howeoteasome . Importaoteasome . As a reoteasome . Increasoteasome ,124. Intoteasome . Accumuloteasome . More spoteasome . In thisUbiquilin 1 (UBQLN1) (UniProt ID: Q9UMX0) is a 589-residue-long extraproteasomal ubiquitin receptor  that plaUBQLN2 gene mutations were reported in frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS). Abnormal UBQLN2 inclusions have been observed in the cytosol of degenerating motor neurons of ALS patients [Ubiquilin-2 (UBQLN2) (UniProt ID: Q9UHD9) is a 624-residue-long protein present in cytosol, which is mostly expressed in the brain, liver, spleen, pancreas, heart, and other tissues . Like UBpatients . Furtherpatients . Furtherpatients . Hjerpe patients . Interespatients . Studiespatients . SimilarUCHL1 (UniProt ID: P09936) is a small, 223-residue-long protein, which is highly abundant in the brain  and is normally expressed exclusively in neurons and testis ,70. UCHLUCHL5 (UniProt ID: Q9Y5K5) ia also known as ubiquitin C-terminal hydrolase 37 (UCH37). It is a 329-residue-long DUB that binds to the 19S regulatory subunit of 26S proteasome and deubiquitinates polyubiquitinated proteins. It has a thiol-dependent Ub-specific cysteine protease activity. It is physically associated with a base component of 19S proteasome. It removes poly-Ub chain from the distal end. UCHL5 gets activated by binding of ADRM1, which interacts with the C-terminal tail of UCHL5, a region that is different from the UCH catalytic domain ,141. Ub-USP7 (UniProt ID: Q93009) is also known as ubiquitin carboxyl-terminal hydrolase 7 or herpesvirus-associated ubiquitin-specific protease (HAUSP), and is a member of the USP family of deubiquitylating enzymes; it cleaves ubiquitin from polyubiquitin chains of substrate protein ,145,146.USP14 (UniProt ID: P54578), is a proteasome-associated deubiquitinating enzyme, which is unique among known UBP enzymes ,151. FigATXN3 (UniProt ID: P54252) is a 42-kDa ubiquitously expressed deubiquitinating enzyme involved in the degradation of misfolded chaperone substrates, transcription, cytoskeleton regulation, and maintenance of protein homeostasis ,74,156. ADRM1  is a 407-residue-long protein, which, in addition to being a component of the 26S proteasome, also has a role in cell adhesion. It is a proteasomal receptor that recognizes K-48-linked poly-Ub chains on the target proteins. It also acts as a receptor for a deubiquitinase, UCHL37. ADRM1 has a Pleckstrin-like a receptor for the ubiquitin domain located at its N-terminal region (residues 22–130), which binds to Ub. From our analysis, this domain is the part of the ordered region in ADRM1 a. HusnjaPSMD2  is a 100-kDa protein composed of 909 amino acids. PSMD2 may have a role in presenting ubiquitinated substrates to the proteasome. Shi et al., demonstrated, in 2016, that PSMD2 has two binding sites, T1 and T2, in its toroid domain, which interact with Ub, UBL of shuttles, and the UBL domains of DUB and Ubp6, respectively . It has PSMD4 is a non-ATPase 19S base component of the 26S proteasome complex (UniProt ID: P55036). Apart from this, it is also present in substantial amounts in free form. This is a 41-kDa protein composed of 377 amino acids. It is also called RPN10 and is responsible for recognizing Ub moieties on proteins. It has an N-terminal von Willebrand factor A domain (VWA) (residues 5–188) and two C-terminal helical UIM (residues 211–230 and 282–301). From our analysis, we observed that the N-terminal VWA domain is located in an ordered region, and the C-terminal UIM is located in disordered regions of PSMD4  is made up of 310 amino acids present in the 19S cap and helps in substrate deubiquitination . It is aAccording to the multifactorial disorder analysis, human PSMD14 contains several IDPRs see c. Some oThe ubiquitin proteasome system plays a key role in the pathogenesis of various types of cancers and neurodegenerative diseases. The components of UPS actively participate in the process of protein degradation, and any disturbance in this system leads to the occurrence of aforementioned diseases. Our intrinsic disorder and MoRF analysis of disease-associated UPS proteins recognized numerous functional IDPRs and disorder-based binding regions. Using five different IDP prediction tools, we found that UBA1 and UCHL1 are highly ordered; USP14, PSMD14, UCHL5, PSMD2, USP7, and UBB are moderately disordered; and UBQLN1, UBQLN2, ADRM1, PSMD4, ATXN3, STUB1, and UBE2R2 are highly disordered proteins. We also documented multiple post-translational modifications (PTMs) sites in all the proteins except ataxin-3, and most of the identified PTMs are located in the disordered regions of proteins. Since these proteins have to interact with their biological partners for the normal functioning of protein homeostasis, disorder-based binding regions may have an important role due to their flexibility and dynamic nature. For example, the 19S regulatory particle (RP) of the 26S proteasome contains receptors (such as ADRM1 and PSMD4) for interaction with the polyubiquitin chain of the target protein. Interestingly, in this study, these receptors are found to be highly disordered. Moreover, the domains of extraproteasomal polyubiquitin receptors (UBQLN1 and UBQLN2), which are responsible for interaction with proteasome as well as polyubiquitinated proteins for their degradation, are found to be highly disordered. These IDPRs in the components of UPS may have functional roles in maintaining cellular protein homeostasis, normal functioning of UPS, and aberrant UPS functionality in the pathogenesis of the diseases mentioned above. Further studies are required for complete elucidation of the roles of these identified IDPRs and disorder-based binding regions in the pathogenesis of diseases.