Telomerase-associated Protein Research Articles

Exploring the role of vault complex in the nervous system: a literature review.

Vault RNAs (vtRNAs) are a novel group of non-coding RNAs that are involved in various signaling mechanisms. vtRNAs are joined by three proteins major vault protein (MVP), vault poly (ADP-ribose) polymerase (VPARP), and telomerase-associated protein 1 (TEP1) to form the vault complex. In humans, only four vtRNA including vtRNA 1-1, vtRNA 1-2, vtRNA 1-3, vtRNA 2-1) have been discovered. In nerve cells, vtRNA is involved in synapse formation through MAPK signaling. vtRNA travels to the distal area of neurites as a key unit in the vault complex. Moreover, tRNA is detached from the vault complex in the neurite via a mitotic kinase Aurora-A-reliant MVP phosphorylation. Several molecules contribute to the formation of vtRNAs. For instance, SRSF2 and NSUN2 and their attachment to vtRNA1-1 determines the production of small-vtRNAs. Through the same factors, vtRNAs could play a role in neurodevelopmental deficits. Addition the role of vtRNA expression and vault proteins has been recently studied in neurodegenerative disorders such as Alzheimer's disease (AD), Parkinson's disease (PD), multiple sclerosis (MS), Huntington's disease (HD), and amyotrophic lateral sclerosis (ALS) as well as brain cancers. While the mechanisms of vtRNA involvement in neurological disorders is not well-demonstrated, we believe this could be related to the impact of vtRNA regulation in autophagy, immunoregulation, RNA stability, cellular stress, apoptosis, and regulation of other epigenetic pathways. The present review captures the state-of-the-art regarding the role of vtRNAs in neurodevelopment, normal nervous system function, and neurological disorders.

Structure, Dynamics and Functional Implications of the Eukaryotic Vault Complex.

Vault ribonucleoprotein particles are naturally designed nanocages, widely found in the eukaryotic kingdom. Vaults consist of 78 copies of the major vault protein (MVP) that are organized in 2 symmetrical cup-shaped halves, of an approximate size of 70x40x40 nm, leaving a huge internal cavity which accommodates the vault poly(ADP-ribose) polymerase (vPARP), the telomerase-associated protein-1 (TEP1) and some small untranslated RNAs. Diverse hypotheses have been developed on possible functions of vaults, based on their unique capsular structure, their rapid movements and the distinct subcellular localization of the particles, implicating transport of cargo, but they are all pending confirmation. Vault particles also possess many attributes that can be exploited in nanobiotechnology, particularly in the creation of vehicles for the delivery of multiple molecular cargoes. Here we review what is known about the structure and dynamics of the vault complex and discuss a possible mechanism for the vault opening process. The recent findings in the characterization of the vaults in cells and in its natural microenvironment will be also discussed.

Pathogenic variants in the NLRP3 LRR domain at position 861 are responsible for a boost-dependent atypical CAPS phenotype

Cryopyrin-associated periodic syndrome (CAPS) is associated with NLRP3 pathogenic variants, mostly located in the NACHT (neuronal apoptosis inhibitor protein, MHC class 2 transcription activator, incompatibility locus protein from Podospora anserina, telomerase-associated protein) domain. Cold-induced urticarial rash is among the main clinical features. However, this study identified a series of 14 patients with pathogenic variants of the Y861 residue (p.Tyr861) of the LRR domain of NLRP3 and minimal prevalence of cold-induced urticarial rash. This study aimed to address a possible genotype/phenotype correlation for patients with CAPS and to investigate at the cellular levels the impact of the Y861C substitution (p.Tyr861Cys) on NLRP3 activation. Clinical features of 14 patients with CAPS and heterozygous substitution at position 861 in the LRR domain of NLRP3 were compared to clinical features of 48 patients with CAPS and pathogenic variants outside the LRR domain of NLRP3. IL-1β secretion by PBMCs and purified monocytes from patients and healthy donors was evaluated following LPS and monosodium urate crystal stimulation. Patients with substitution at position 861 of NLRP3 demonstrated a higher prevalence of sensorineural hearing loss while being less prone to skin urticarial. In contrast to patients with classical CAPS, cells from patients with a pathogenic variant at position 861 required an activation signal to secrete IL-1β but produced more IL-1β during the early and late phase of secretion than cells from healthy donors. Pathogenic variants of Y861 of NLRP3 drive a boost-dependent oversecretion of IL-1β associated with an atypical CAPS phenotype.

Identification of new telomere- and telomerase-associated autoantigens in systemic sclerosis

PurposeIn up to 20% of patients with systemic sclerosis (SSc) no known autoantibody specificity can be identified. Recently discovered autoantigens, such as telomeric repeat binding factor 1 (TERF1), as well as established autoantigens, like RuvBL1/2, are associated with telomere and telomerase biology. We aimed to identify new telomere- and telomerase-associated autoantigens in patients with SSc without known autoantibody specificity. MethodsUnlabelled protein immunoprecipitation combined with gel-free liquid chromatography-tandem mass spectrometry (IP-MS) was performed with sera of 106 patients with SSc from two tertiary referral centres that had a nuclear pattern on HEp-2 indirect immunofluorescence without previously identified autoantibody. Telomere- or telomerase-associated proteins or protein complexes precipitated by individual sera were identified. Candidate autoantigens were confirmed through immunoprecipitation-western blot (IP-WB). A custom Luminex xMAP assay for 5 proteins was evaluated with sera from persons with SSc (n = 467), other systemic autoimmune rheumatic diseases (n = 923), non-rheumatic disease controls (n = 187) and healthy controls (n = 199). ResultsEight telomere- and telomerase-associated autoantigens were identified in a total of 11 index patients, including the THO complex (n = 3, all with interstitial lung disease and two with cardiac involvement), telomeric repeat-binding factor 2 (TERF2, n = 1), homeobox-containing protein 1 (HMBOX1, n = 2), regulator of chromosome condensation 1 (RCC1, n = 1), nucleolar and coiled-body phosphoprotein 1 (NOLC1, n = 1), dyskerin (DKC1, n = 1), probable 28S rRNA (cytosine(4447)-C(5))-methyltransferase (NOP2, n = 1) and nuclear valosin-containing protein-like (NVL, n = 2). A Luminex xMAP assay for THO complex subunit 1 (THOC1), TERF2, NOLC1, NOP2 and NVL revealed high reactivity in all index patients, but also in other patients with SSc and disease controls. However, the reactivity by xMAP assay in these other patients was not confirmed by IP-WB. ConclusionIP-MS revealed key telomere- and telomerase-associated proteins and protein complexes as autoantigens in patients with SSc.

Ultrasound deep brain stimulation decelerates telomere shortening in Alzheimer's disease and aging mice

Telomere length is a reliable biomarker for health and longevity prediction in both humans and animals. The common neuromodulation techniques, including deep brain stimulation (DBS) and optogenetics, have excellent spatial resolution and depth penetration but require implementation of electrodes or optical fibers. Therefore, it is important to develop methods for noninvasive modulation of telomere length. Herein, we reported on a new method for decelerating telomere shortening using noninvasive ultrasound deep brain stimulation (UDBS). Firstly, we found that UDBS could activate the telomerase-associated proteins in normal mice. Then, in the Alzheimer's disease mice, UDBS was observed to decelerate telomere shortening of the cortex and myocardial tissue and to effectively improve spatial learning and memory abilities. Similarly, UDBS was found to significantly slow down telomere shortening of the cortex and peripheral blood, and improve motor and cognitive functions in aging mice. Finally, transcriptome analysis revealed that UDBS upregulated the neuroactive ligand–receptor interaction pathway. Overall, the present findings established the critical role of UDBS in delaying telomere shortening and indicated that ultrasound modulation of telomere length may constitute an effective therapeutic strategy for aging and aging-related diseases.

Telomere and Telomerase-Associated Proteins in Endometrial Carcinogenesis and Cancer-Associated Survival.

Risk of relapse of endometrial cancer (EC) after surgical treatment is 13% and recurrent disease carries a poor prognosis. Research into prognostic indicators is essential to improve EC management and outcome. “Immortality” of most cancer cells is dependent on telomerase, but the role of associated proteins in the endometrium is poorly understood. The Cancer Genome Atlas data highlighted telomere/telomerase associated genes (TTAGs) with prognostic relevance in the endometrium, and a recent in silico study identified a group of TTAGs and proteins as key regulators within a network of dysregulated genes in EC. We characterise relevant telomere/telomerase associated proteins (TTAPs) NOP10, NHP2, NOP56, TERF1, TERF2 and TERF2IP in the endometrium using quantitative polymerase chain reaction (qPCR) and immunohistochemistry (IHC). qPCR data demonstrated altered expression of multiple TTAPs; specifically, increased NOP10 (p = 0.03) and reduced NHP2 (p = 0.01), TERF2 (p = 0.01) and TERF2IP (p < 0.003) in EC relative to post-menopausal endometrium. Notably, we report reduced NHP2 in EC compared to post-menopausal endometrium in qPCR and IHC (p = 0.0001) data; with survival analysis indicating high immunoscore is favourable in EC (p = 0.0006). Our findings indicate a potential prognostic role for TTAPs in EC, particularly NHP2. Further evaluation of the prognostic and functional role of the examined TTAPs is warranted to develop novel treatment strategies.

MVP Expression Facilitates Tumor Cell Proliferation and Migration Supporting the Metastasis of Colorectal Cancer Cells.

Cancer cells show significant dysregulation of genes expression, which may favor their survival in the tumor environment. In this study, the cellular vault’s components MVP (major vault protein), TEP1 (telomerase-associated protein 1) and vPARP (vault poly(ADP-ribose) polymerase) were transiently or completely inhibited in U2OS cells (human bone osteosarcoma epithelial cells) to evaluate their impact on the cell proliferative and migratory capacity as well as on the development of their resistance to the drug vinorelbine. Comparative analysis of MVP protein expression level in normal colon tissue, primary colorectal tumor, and metastasis showed that the expression of this protein does not increase significantly in the primary tumor, but its expression increases in metastatic cells. Further comparative molecular analysis using the whole transcriptome microarrays for MVP-positive and MVP-negative cells showed that MVP is involved in regulating proliferation and migration of cancer cells. MVP may facilitate metastasis of colon cancer due to its impact on cell migration. Moreover, two vault proteins, MVP and TEP1, contribute the resistance to vinorelbine, while vPARP does not.

Genetic variants in telomerase-associated protein 1 are associated with telomere damage in PAH-exposed workers

Telomeres are functional complexes at the ends of linear chromosomes, and telomerase aids in their maintenance and replication. Additionally, accumulating evidence suggests that telomerase-associated protein 1 (TEP1) is a component of the telomerase ribonucleoprotein complex and is responsible for catalyzing the addition of new synthetic telomere sequences to chromosome ends. In our previous study, we found that genetic variants of the TERT gene participated in the regulation of telomere length. Exposure to particulate matter, environmental pollutants, oxidative stress, and pesticides is associated with shortening of telomere length. However, it is unknown whether genetic variants in the TEP1 gene may affect telomere length (TL) in polycyclic aromatic hydrocarbon (PAH)-exposed workers. Therefore, we measured the peripheral leukocyte TL and genotyped the polymorphism loci in the TEP1 gene among 544 PAH-exposed workers and 238 healthy controls. Covariance analysis showed that the individuals carrying TEP1 rs1760903 CC and TEP1 rs1760904 TT had longer TL in the control group (P < 0.05). In the generalized linear model, we found that rs1760903 CC was a protective factor against TL shortening, and PAH exposure could promote telomere shortening (P < 0.05). Thus, this study reinforces the roles of environmental factors and genetic variations in telomere damage, and provides a theoretical foundation for the early detection of susceptible populations and the establishment of occupational standards.

The structurally conserved TELR region on shelterin protein TPP1 is essential for telomerase processivity but not recruitment

The shelterin protein TPP1 is involved in both recruiting telomerase and stimulating telomerase processivity in human cells. Assessing the in vivo significance of the latter role of TPP1 has been difficult, because TPP1 mutations that perturb telomerase function tend to abolish both telomerase recruitment and processivity. The Saccharomyces cerevisiae telomerase-associated Est3 protein adopts a protein fold similar to the N-terminal region of TPP1. Interestingly, a previous structure-guided mutagenesis study of Est3 revealed a TELR surface region that regulates telomerase function via an unknown mechanism without affecting the interaction between Est3 and telomerase [T. Rao et al., Proc. Natl. Acad. Sci. U.S.A. 111, 214-218 (2014)]. Here, we show that mutations within the structurally conserved TELR region on human TPP1 impaired telomerase processivity while leaving telomerase recruitment unperturbed, hence uncoupling the two roles of TPP1 in regulating telomerase. Telomeres in cell lines containing homozygous TELR mutations progressively shortened to a critical length that caused cellular senescence, despite the presence of abundant telomerase in these cells. Our findings not only demonstrate that telomerase processivity can be regulated by TPP1 in a process separable from its role in recruiting telomerase, but also establish that the in vivo stimulation of telomerase processivity by TPP1 is critical for telomere length homeostasis and long-term viability of human cells.

SUMOylation- and GAR1-Dependent Regulation of Dyskerin Nuclear and Subnuclear Localization.

The nuclear and subnuclear compartmentalization of the telomerase-associated protein and H/ACA ribonucleoprotein component dyskerin is an important although incompletely understood aspect of H/ACA ribonucleoprotein function. Four SUMOylation sites were previously identified in the C-terminal nuclear/nucleolar localization signal (N/NoLS) of dyskerin. We found that a cytoplasmic localized C-terminal truncation variant of dyskerin lacking most of the C-terminal N/NoLS represents an under-SUMOylated variant of dyskerin compared to wild-type dyskerin. We demonstrate that mimicking constitutive SUMOylation of dyskerin using a SUMO3 fusion construct can drive nuclear accumulation of this variant and that the SUMO site K467 in this N/NoLS is particularly important for the subnuclear localization of dyskerin to the nucleolus in a mature H/ACA complex assembly- and SUMO-dependent manner. We also characterize a novel SUMO-interacting motif in the mature H/ACA complex component GAR1 that mediates the interaction between dyskerin and GAR1. Mislocalization of dyskerin, either in the cytoplasm or excluded from the nucleolus, disrupts dyskerin function and leads to reduced interaction of dyskerin with the telomerase RNA. These data indicate a role for dyskerin C-terminal N/NoLS SUMOylation in regulating the nuclear and subnuclear localization of dyskerin, which is essential for dyskerin function as both a telomerase-associated protein and as an H/ACA ribonucleoprotein.

The Vault Nanoparticle: A Gigantic Ribonucleoprotein Assembly Involved in Diverse Physiological and Pathological Phenomena and an Ideal Nanovector for Drug Delivery and Therapy

Simple SummaryIn recent decades, a molecular complex referred to as vault nanoparticle has attracted much attention by the scientific community, due to its unique properties. At the molecular scale, it is a huge assembly consisting of 78 97-kDa polypeptide chains enclosing an internal cavity, wherein enzymes involved in DNA integrity maintenance and some small noncoding RNAs are accommodated. Basically, two reasons justify this interest. On the one hand, this complex represents an ideal tool for the targeted delivery of drugs, provided it is suitably engineered, either chemically or genetically; on the other hand, it has been shown to be involved in several cellular pathways and mechanisms that most often result in multidrug resistance. It is therefore expected that a better understanding of the physiological roles of this ribonucleoproteic complex may help develop new therapeutic strategies capable of coping with cancer progression. Here, we provide a comprehensive review of the current knowledge.The vault nanoparticle is a eukaryotic ribonucleoprotein complex consisting of 78 individual 97 kDa-“major vault protein” (MVP) molecules that form two symmetrical, cup-shaped, hollow halves. It has a huge size (72.5 × 41 × 41 nm) and an internal cavity, wherein the vault poly(ADP-ribose) polymerase (vPARP), telomerase-associated protein-1 (TEP1), and some small untranslated RNAs are accommodated. Plenty of literature reports on the biological role(s) of this nanocomplex, as well as its involvement in diseases, mostly oncological ones. Nevertheless, much has still to be understood as to how vault participates in normal and pathological mechanisms. In this comprehensive review, current understanding of its biological roles is discussed. By different mechanisms, vault’s individual components are involved in major cellular phenomena, which result in protection against cellular stresses, such as DNA-damaging agents, irradiation, hypoxia, hyperosmotic, and oxidative conditions. These diverse cellular functions are accomplished by different mechanisms, mainly gene expression reprogramming, activation of proliferative/prosurvival signaling pathways, export from the nucleus of DNA-damaging drugs, and import of specific proteins. The cellular functions of this nanocomplex may also result in the onset of pathological conditions, mainly (but not exclusively) tumor proliferation and multidrug resistance. The current understanding of its biological roles in physiological and pathological processes should also provide new hints to extend the scope of its exploitation as a nanocarrier for drug delivery.

A new role for proteins subunits of RNase P: stabilization of the telomerase holoenzyme.

RNase P, an RNA-protein complex, is essential for processing tRNAs. Three of the ten protein subunits of Saccharomyces cerevisiae RNase P (and a related complex, RNase MRP) co-purify with yeast telomerase, another RNA-protein complex. The three telomerase-associated proteins, Pop1, 6 and 7, bind to TLC1, the RNA subunit of telomerase. In a recent study (Garcia et al. Nat Commun), we used temperature sensitive alleles of the essential POP genes to determine their role in telomerase biogenesis. At permissive temperature, pop mutant cells grow normally, and the abundance of most proteins, including protein subunits of telomerase, is similar to wild type (WT). However, telomeres are short, and the amount of the mature telomerase holoenzyme is low. Unlike the RNA subunit of RNase MRP, TLC1 is more abundant in pop cells and properly folded, except at the Cs2a/TeSS domain where the Pop proteins bind. These defects correlate with defective movement of TLC1 from the cytoplasm, where it associates with telomerase proteins, back to the nucleus where it lengthens telomeres. Thus, Pop proteins are needed for the stable association of telomerase proteins with TLC1, and their reduction sequesters mature telomerase in the cytoplasm, away from its nuclear substrates.

Optimized Detection of Protein-Protein and Protein-DNA Interactions, with Particular Application to Plant Telomeres.

Characterization of protein-protein and protein-DNA interactions is critical to understand mechanisms governing the biology of cells. Here we describe optimizedmethods and theirmutual combinations for this purpose: bimolecular fluorescence complementation (BiFC), co-immunoprecipitation (Co-IP), yeast two-hybrid systems (Y2H), and chromatin immunoprecipitation (ChIP). These improved protocols detect trimeric complexes in which two proteins of interest interact indirectly via a protein sandwiched between them.They alsoallow isolation of low-abundance chromatin proteins and confirmation that proteins of interest are associated with specific DNA sequences, for example telomeric tracts.Here we describe these methods and their application to map interactions of several telomere- and telomerase-associated proteins and to purify a sufficient amount of chromatin from Arabidopsis thaliana for further investigations (e.g., next-generation sequencing, hybridization).

The vault RNA of Trypanosoma brucei plays a role in the production of trans-spliced mRNA

The vault ribonucleoprotein (RNP), comprising vault RNA (vtRNA) and telomerase-associated protein 1 (TEP1), is found in many eukaryotes. However, previous studies of vtRNAs, for example in mammalian cells, have failed to reach a definitive conclusion about their function. vtRNAs are related to Y RNAs, which are complexed with Ro protein and influence Ro's function in noncoding RNA (ncRNA) quality control and processing. In Trypanosoma brucei, the small noncoding TBsRNA-10 was first described in a survey of the ncRNA repertoire in this organism. Here, we report that TBsRNA-10 in T. brucei is a vtRNA, based on its association with TEP1 and sequence similarity to those of other known and predicted vtRNAs. We observed that like vtRNAs in other species, TBsRNA-10 is transcribed by RNA polymerase III, which in trypanosomes also generates the spliceosomal U-rich small nuclear RNAs. In T. brucei, spliced leader (SL)-mediated trans-splicing of pre-mRNAs is an obligatory step in gene expression, and we found here that T. brucei's vtRNA is highly enriched in a non-nucleolar locus in the cell nucleus implicated in SL RNP biogenesis. Using a newly developed permeabilized cell system for the bloodstream form of T. brucei, we show that down-regulated vtRNA levels impair trans-spliced mRNA production, consistent with a role of vtRNA in trypanosome mRNA metabolism. Our results suggest a common theme for the functions of vtRNAs and Y RNAs. We conclude that by complexing with their protein-binding partners TEP1 and Ro, respectively, these two RNA species modulate the metabolism of various RNA classes.

NOD-like receptors: major players (and targets) in the interface between innate immunity and cancer.

Innate immunity comprises several inflammation-related modulatory pathways which receive signals from an array of membrane-bound and cytoplasmic pattern recognition receptors (PRRs). The NLRs (NACHT (NAIP (neuronal apoptosis inhibitor protein), C2TA (MHC class 2 transcription activator), HET-E (incompatibility locus protein from Podospora anserina) and TP1 (telomerase-associated protein) and Leucine-Rich Repeat (LRR) domain containing proteins) relate to a large family of cytosolic innate receptors, involved in detection of intracellular pathogens and endogenous byproducts of tissue injury. These receptors may recognize pathogen-associated molecular patterns (PAMPs) and/or danger-associated molecular patterns (DAMPs), activating host responses against pathogen infection and cellular stress. NLR-driven downstream signals trigger a number of signaling circuitries, which may either initiate the formation of inflammasomes and/or activate nuclear factor κB (NF-κB), stress kinases, interferon response factors (IRFs), inflammatory caspases and autophagy. Disruption of those signals may lead to a number of pro-inflammatory conditions, eventually promoting the onset of human malignancies. In this review, we describe the structures and functions of the most well-defined NLR proteins and highlight their association and biological impact on a diverse number of cancers.

The journey to the end of the chromosome: delivering active telomerase to telomeres in plants.

The journey to the end of the chromosome: delivering active telomerase to telomeres in plants.

Emerging immunotherapeutic strategies targeting telomerases in genitourinary tumors.

Telomerase activity and telomere length are essential for the pathogenesis of several human diseases, including genitourinary tumors. Telomerase constitutes a complex system that includes human telomerase reverse transcriptase (hTERT), human telomerase RNA component (hTR) and telomerase associated protein 1 (TEP1), which are overexpressed in tumor cells compared to normal cells and are involved in the carcinogenesis and progression of renal cell carcinoma (RCC), bladder (BC) and prostate cancer (PCa). In addition, telomerase degraded peptide fragments expressed on the surface of tumor cells lead to their recognition by immune cells. On this scenario, in vitro and in vivo studies have shown effective anti-tumor activity of hTERT-tailored strategies in genitourinary tumors, including active immunotherapy with hTERT-peptide vaccines and passive immunotherapy with hTERT-transduced T cell infusion. This review emphasizes the role of telomerase in the carcinogenesis and progression of genitourinary tumors, thus underlying the potential of emerging telomerase-tailored immunotherapies in these patients.

A fast and straightforward procedure for vault nanoparticle purification and the characterization of its endocytic uptake

BackgroundVaults are eukaryotic ribonucleoprotein particles composed of up 78 copies of the 97 kDa major vault protein that assembles into a barrel-like, “nanocapsule” enclosing poly(ADP-ribose) polymerase, telomerase-associated protein-1 and small untranslated RNAs. Overall, the molecular mass of vault particles amounts to about 13 MDa. Although it has been implicated in several cellular functions, its physiological roles remain poorly understood. Also, the possibility to exploit it as a nanovector for drug delivery is currently being explored in several laboratories. MethodsUsing the baculovirus expression system, vaults were expressed and purified by a dialysis step using a 1 MDa molecular weight cutoff membrane and a subsequent size exclusion chromatography. Purity was assessed by SDS-PAGE, transmission electron microscopy and dynamic light scattering. Particle's endocytic uptake was monitored by flow cytometry and confocal microscopy. ResultsThe purification protocol here reported is far simpler and faster than those currently available and lead to the production of authentic vault. We then demonstrated its clathrin-mediated endocytic uptake by normal fibroblast and glioblastoma, but not carcinoma cell lines. In contrast, no significant caveolin-mediated endocytosis was detected. ConclusionsThese results provide the first evidence for an intrinsic propensity of the vault complex to undergo endocytic uptake cultured eukaryotic cells. General significanceThe newly developed purification procedure will greatly facilitate any investigation based on the use of the vault particle as a natural nanocarrier. Its clathrin-mediated endocytic uptake observed in normal and in some tumor cell lines sheds light on its physiological role.

Epitope Mapping of Anti-Telomerase Reverse Transcriptase Monoclonal Antibodies.

Telomerase reverse transcriptase (TERT) is the main catalytic subunit of telomerase-associated protein machinery. Upregulation of TERT at the transcriptional level results in immortal cell phenotype associated with cancer. To date we have developed eight anti-TERT monoclonal antibodies (mAbs) (TMab-4, TMab-5, TMab-6, TMab-7, TMab-9, TMab-10, TMab-11, and TMab-12) by immunizing mice with synthetic peptides (302-QHHAGPPSTSRPPRPWDTPC-321) of TERT. We further characterized those epitopes using enzyme-linked immunosorbent assay, and here we discuss the critical epitope of an anti-TERT mAb, which is applicable for immunohistochemical analysis.

Molecular characterization and expression of NLRP10 in the antibacterial host defense of the sea cucumber (Apostichopus japonicus)

The nucleotide-binding oligomerization domain-like receptors (NOD-like receptors, NLRs) can regulate the innate immune process and is an important part of inflammatory body. In this study, we use transcriptome sequencing and the rapid amplification of cDNA ends approach to identify a novel NLRP gene in Apostichopus japonicus. We designated the gene as AjNLRP10. The full-length of AjNLRP10 is 4509 bp. The putative open reading frame comprising 3489 bp encodes a polypeptide with 1162 amino acid residues. The predicted molecular mass of AjNLRP10 is 132.87 kDa and its theoretical pI is 5.60. AjNLRP10 comprises a signal peptide with two Ig superfamily (IgSF) domains and a NACHT [NAIP (neuronal apoptosis inhibitory protein), CIITA (MHC class II transcription activator), HET-E (incompatibility locus protein from Podospora anserina) and TP1 (telomerase-associated protein)] domain. Spatial distribution expression analysis detected AjNLRP10 in all of the tissues tested, but with higher expression in the coelomocytes, medium expression in the intestine and respiratory tree, and slightly weaker expression in the body wall, tube feet, and longitudinal muscle. The expression levels of AjNLRP10 in the respiratory tree and intestines of sea cucumbers with skin ulceration syndrome were increased by 4-fold and 2.7-fold compared with those in healthy sea cucumbers, respectively. We investigated expression profiles of AjCasepase-1 (Cysteinyl aspartate specific proteinase-1) and AjMMP37 (mitochondrial protein-37) after AjNLRP10 knock-down and discovered that AjCasepase-1 was raised by 2.60-fold and AjMMP37 was raised by 3.84-fold. The study showed that AjNLRP10 has inhibitory effect in the immune process. In conclusion, this study showed that the AjNLRP10 protein found in the sea cucumber involved with the innate immune responses against bacterial infection. It has a similar structure and biological function to that in other organisms, where it appears to be involved with these results provide insights into the innate immune mechanism in the sea cucumber as well as suggesting new strategies for disease prevention, molecular therapy, and the development of novel drugs for sea cucumbers.