Electrochemical aptasensor for human osteopontin detection using a DNA aptamer selected by SELEX

A DNA aptamer with affinity and specificity for human osteopontin (OPN), a potential breast cancer biomarker, was selected using the SELEX process, considering its homology rate and the stability of its secondary structures. This aptamer exhibited a satisfactory affinity towards OPN, showing dissociation constants lower than 2.5 nM. It was further used to develop a simple, label-free electrochemical aptasensor against OPN. The aptasensor showed good sensitivity towards OPN in standard solutions, being the square wave voltammetry (SWV), compared to the cyclic voltammetry, the most sensitive technique with detection and quantification limits of 1.4 ± 0.4 nM and 4.2 ± 1.1 nM, respectively. It showed good reproducibility and acceptable selectivity, exhibiting low signal interferences from other proteins, as thrombin, with 2.610 times lower current signals-off than for OPN. The aptasensor also successfully detected OPN in spiked synthetic human plasma. Using SWV, detection and quantification limits (1.3 ± 0.1 and 3.9 ± 0.4 nM) within the OPN plasma levels reported for patients with breast cancer (0.44.5 nM) or with metastatic or recurrent breast cancer (0.98.4 nM) were found. Moreover, preliminary assays, using a sample of human plasma, showed that the aptasensor and the standard ELISA method quantified similar OPN levels (2.2 ± 0.7 and 1.7 ± 0.1 nM, respectively). Thus, our aptasensor coupled with SWV represents a promising alternative for the detection of relevant breast cancer biomarkers.The authors acknowledge the financial support from the Strategic funding of UID/BIO/04469/2013 unit and COMPETE 2020 (POCI-01-0145-FEDER-006684), and from project BioTecNorte (project number NORTE-01-0145-FEDER-000004). This work was also financially supported by Project POCI-01–0145-FEDER-006984 – Associate Laboratory LSRE-LCM and by Project UID/QUI/00616/2013 – CQ-VR both funded by FEDER - Fundo Europeu de Desenvolvimento Regional through COMPETE2020 - Programa Operacional Competitividade e Internacionalização (POCI) – and by national funds through FCT - Fundação para a Ciência e a Tecnologia, Portugal. S. Meirinho also acknowledges the research grant provided by Project UID/EQU/50020/2013.info:eu-repo/semantics/publishedVersio

Identification of osteopontin as a novel marker for early hepatocellular carcinoma

The aim of this study was to identify a biomarker that could improve alpha‐fetoprotein (AFP) performance in hepatocellular carcinoma (HCC) surveillance among patients with cirrhosis. We performed proteomic profiling of plasma from patients with cirrhosis or HCC and validated selected candidate HCC biomarkers in two geographically distinct cohorts to include HCC of different etiologies. Mass spectrometry profiling of highly fractionated plasma from 18 cirrhosis and 17 HCC patients identified osteopontin (OPN) as significantly up‐regulated in HCC cases, compared to cirrhosis controls. OPN levels were subsequently measured in 312 plasma samples collected from 131 HCC patients, 76 cirrhosis patients, 52 chronic hepatitis C (CHC) and B (CHB) patients, and 53 healthy controls in two independent cohorts. OPN plasma levels were significantly elevated in HCC patients, compared to cirrhosis, CHC, CHB, or healthy controls, in both cohorts. OPN alone or in combination with AFP had significantly better area under the receiver operating characteristic curve, compared to AFP, in comparing cirrhosis and HCC in both cohorts. OPN overall performance remained higher than AFP in comparing cirrhosis and the following HCC groups: HCV‐related HCC, HBV‐associated HCC, and early HCC. OPN also had a good sensitivity in AFP‐negative HCC. In a pilot prospective study including 22 patients who developed HCC during follow‐up, OPN was already elevated 1 year before diagnosis. Conclusion : OPN was more sensitive than AFP for the diagnosis of HCC in all studied HCC groups. In addition, OPN performance remained intact in samples collected 1 year before diagnosis. (H EPATOLOGY 2012)Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/90204/1/24703_ftp.pd

Oligogenic inheritance of optineurin (OPTN) and C9ORF72 mutations in ALS highlights localisation of OPTN in the TDP-43-negative inclusions of C9ORF72-ALS.

Amyotrophic lateral sclerosis (ALS) is characterized by motor neurone loss resulting in muscle weakness, spasticity and ultimately death. 5-10% are caused by inherited mutations, most commonly C9ORF72, SOD1, TARDBP and FUS. Rarer genetic causes of ALS include mutation of optineurin (mt OPTN). Furthermore, optineurin protein has been localized to the ubiquitylated aggregates in several neurodegenerative diseases, including ALS. This study: (i) investigated the frequency of mt OPTN in ALS patients in England; (ii) characterized the clinical and neuropathological features of ALS associated with a mt OPTN; and (iii) investigated optineurin neuropathology in C9ORF72-related ALS (C9ORF72-ALS). We identified a heterozygous p.E322K missense mutation in exon 10 of OPTN in one familial ALS patient who additionally had a C9ORF72 mutation. This patient had bulbar, limb and respiratory disease without cognitive problems. Neuropathology revealed motor neurone loss, trans-activation response DNA protein 43 (TDP-43)-positive neuronal and glial cytoplasmic inclusions together with TDP-43-negative neuronal cytoplasmic inclusions in extra motor regions that are characteristic of C9ORF72-ALS. We have demonstrated that both TDP-43-positive and negative inclusion types had positive staining for optineurin by immunohistochemistry. We went on to show that optineurin was present in TDP-43-negative cytoplasmic extra motor inclusions in C9ORF72-ALS cases that do not carry mt OPTN. We conclude that: (i) OPTN mutations are associated with ALS; (ii) optineurin protein is present in a subset of the extramotor inclusions of C9ORF72-ALS; (iii) It is not uncommon for multiple ALS-causing mutations to occur in the same patient; and (iv) studies of optineurin are likely to provide useful dataregarding the pathophysiology of ALS and neurodegeneration

Functional role of the noncatalytic domains of elongation factor Tu in the interactions with ligands

Elongation factor (EF) Tu from Escherichia coli contains three domains, of which domain 1 (N-terminal domain) harbors the site for nucleotide binding and GTP hydrolysis. To analyze the function of domains 2 [middle (M) domain] and 3 [C-terminal (C) domain], EF-Tu(AM) and EF-Tu(δC) were engineered as GST-fused products and purified. Circular dichroism and thermostability showed that both constructs have conserved organized structures. Though inactive in poly(Phe) synthesis the two constructs could bind GDP and GTP with comparable micromolar affinities. Therefore, like the isolated N- terminal domain, they had lost a typical feature of EF-Tu, the ≤ 100 times stronger affinity for GDP than for GTP. EF-Tu(ΔM) and EF-Tu(AC) had an intrinsic GTPase activity comparable to that of wild-type EF-Tu. Ribosomes did not stimulate the GTPase activity of either factor, while kirromycin increased the GTPase activity of both constructs, particularly of EF-Tu(ΔC), to a level, however, much lower than that of the intact molecule. The interaction with aa-tRNA of both mutants was ≤90% reduced. As a major result, their GDP-bound form could efficiently respond to EF-Ts. All four EF- Tu-specific antibiotics [kirromycin, pulvomycin, GE2270 A (=MDL 62 879), and enacyloxin IIa] retarded significantly the dissociation of EF-Tu(ΔC)·GTP, showing the same kind of effect as on EF-Tu·GTP, but they were little active on EF-Tu(ΔM)·GTP. Like EF-Tu(ΔC)·GTP, EF-Tu(ΔM)·GTP was, however, able to bind efficiently kirromycin and enacyloxin IIa, as determined via competition with EF-Ts. Together, these results enlight selective functions of domains 2 and 3, particularly toward the interaction with EF-Ts and antibiotics, and emphasize their functional cooperativity for an efficient interaction of EF-Tu with ribosomes and aa-tRNA and for maintaining the differential affinity for GTP and GDP

NF-κB Mediates Tumor Necrosis Factor α-Induced Expression of Optineurin, a Negative Regulator of NF-κB

Optineurin is a ubiquitously expressed multifunctional cytoplasmic protein encoded by OPTN gene. The expression of optineurin is induced by various cytokines. Here we have investigated the molecular mechanisms which regulate optineurin gene expression and the relationship between optineurin and nuclear factor κB (NF-κB). We cloned and characterized human optineurin promoter. Optineurin promoter was activated upon treatment of HeLa and A549 cells with tumor necrosis factor α (TNFα). Mutation of a putative NF-κB-binding site present in the core promoter resulted in loss of basal as well as TNFα-induced activity. Overexpression of p65 subunit of NF-κB activated this promoter through NF-κB site. Oligonucleotides corresponding to this putative NF-κB-binding site showed binding to NF-κB. TNFα-induced optineurin promoter activity was inhibited by expression of inhibitor of NF-κB (IκBα) super-repressor. Blocking of NF-κB activation resulted in inhibition of TNFα-induced optineurin gene expression. Overexpressed optineurin partly inhibited TNFα-induced NF-κB activation in Hela cells. Downregulation of optineurin by shRNA resulted in an increase in TNFα-induced as well as basal NF-κB activity. These results show that optineurin promoter activity and gene expression are regulated by NF-κB pathway in response to TNFα. In addition these results suggest that there is a negative feedback loop in which TNFα-induced NF-κB activity mediates expression of optineurin, which itself functions as a negative regulator of NF-κB

Overexpression of optineurin E50K disrupts Rab8 interaction and leads to a progressive retinal degeneration in mice

Glaucoma is one of the leading causes of bilateral blindness affecting nearly 8 million people worldwide. Glaucoma is characterized by a progressive loss of retinal ganglion cells (RGCs) and is often associated with elevated intraocular pressure (IOP). However, patients with normal tension glaucoma (NTG), a subtype of primary open-angle glaucoma (POAG), develop the disease without IOP elevation. The molecular pathways leading to the pathology of NTG and POAG are still unclear. Here, we describe the phenotypic characteristics of transgenic mice overexpressing wild-type (Wt) or mutated optineurin (Optn). Mutations E50K, H486R and Optn with a deletion of the first (amino acids 153–174) or second (amino acids 426–461) leucine zipper were used for overexpression. After 16 months, histological abnormalities were exclusively observed in the retina of E50K mutant mice with loss of RGCs and connecting synapses in the peripheral retina leading to a thinning of the nerve fiber layer at the optic nerve head at normal IOP. E50K mice also showed massive apoptosis and degeneration of entire retina, leading to approximately a 28% reduction of the retina thickness. At the molecular level, introduction of the E50K mutation disrupts the interaction between Optn and Rab8 GTPase, a protein involved in the regulation of vesicle transport from Golgi to plasma membrane. Wt Optn and an active GTP-bound form of Rab8 complex were localized at the Golgi complex. These data suggest that alternation of the Optn sequence can initiate significant retinal degeneration in mice

Optineurin Is Required for CYLD-Dependent Inhibition of TNFα-Induced NF-κB Activation

The nuclear factor kappa B (NF-κB) regulates genes that function in diverse cellular processes like inflammation, immunity and cell survival. The activation of NF-κB is tightly controlled and the deubiquitinase CYLD has emerged as a key negative regulator of NF-κB signalling. Optineurin, mutated in certain glaucomas and amyotrophic lateral sclerosis, is also a negative regulator of NF-κB activation. It competes with NEMO (NF-κB essential modulator) for binding to ubiquitinated RIP (receptor interacting protein) to prevent NF-κB activation. Recently we identified CYLD as optineurin-interacting protein. Here we have analysed the functional significance of interaction of optineurin with CYLD. Our results show that a glaucoma-associated mutant of optineurin, H486R, is altered in its interaction with CYLD. Unlike wild-type optineurin, the H486R mutant did not inhibit tumour necrosis factor α (TNFα)-induced NF-κB activation. CYLD mediated inhibition of TNFα-induced NF-κB activation was abrogated by expression of the H486R mutant. Upon knockdown of optineurin, CYLD was unable to inhibit TNFα-induced NF-κB activation and showed drastically reduced interaction with ubiquitinated RIP. The level of ubiquitinated RIP was increased in optineurin knockdown cells. Deubiquitination of RIP by over-expressed CYLD was abrogated in optineurin knockdown cells. These results suggest that optineurin regulates NF-κB activation by mediating interaction of CYLD with ubiquitinated RIP thus facilitating deubiquitination of RIP

Osteopontin: A Novel Regulator at the Cross Roads of Inflammation, Obesity and Diabetes

Since its first description more than 20 years ago osteopontin has emerged as an active player in many physiological and pathological processes, including biomineralization, tissue remodeling and inflammation. As an extracellular matrix protein and proinflammatory cytokine osteopontin is thought to facilitate the recruitment of monocytes/macrophages and to mediate cytokine secretion in leukocytes. Modulation of immune cell response by osteopontin has been associated with various inflammatory diseases and may play a pivotal role in the development of adipose tissue inflammation and insulin resistance. Here we summarize recent findings on the role of osteopontin in metabolic disorders, particularly focusing on diabetes and obesity

Deletion of the thrombin cleavage domain of osteopontin mediates breast cancer cell adhesion, proteolytic activity, tumorgenicity, and metastasis

<p>Abstract</p> <p>Background</p> <p>Osteopontin (OPN) is a secreted phosphoprotein often overexpressed at high levels in the blood and primary tumors of breast cancer patients. OPN contains two integrin-binding sites and a thrombin cleavage domain located in close proximity to each other.</p> <p>Methods</p> <p>To study the role of the thrombin cleavage site of OPN, MDA-MB-468 human breast cancer cells were stably transfected with either wildtype OPN (468-OPN), mutant OPN lacking the thrombin cleavage domain (468-ΔTC) or an empty vector (468-CON) and assessed for <it>in vitro </it>and <it>in vivo </it>functional differences in malignant/metastatic behavior.</p> <p>Results</p> <p>All three cell lines were found to equivalently express thrombin, tissue factor, CD44, αvβ5 integrin and β1 integrin. Relative to 468-OPN and 468-CON cells, 468-ΔTC cells expressing OPN with a deleted thrombin cleavage domain demonstrated decreased cell adhesion (p < 0.001), decreased mRNA expression of MCAM, maspin and TRAIL (p < 0.01), and increased uPA expression and activity (p < 0.01) <it>in vitro</it>. Furthermore, injection of 468-ΔTC cells into the mammary fat pad of nude mice resulted in decreased primary tumor latency time (p < 0.01) and increased primary tumor growth and lymph node metastatic burden (p < 0.001) compared to 468-OPN and 468-CON cells.</p> <p>Conclusions</p> <p>The results presented here suggest that expression of thrombin-uncleavable OPN imparts an early tumor formation advantage as well as a metastatic advantage for breast cancer cells, possibly due to increased proteolytic activity and decreased adhesion and apoptosis. Clarification of the mechanisms responsible for these observations and the translation of this knowledge into the clinic could ultimately provide new therapeutic opportunities for combating breast cancer.</p