Simultaneous Quantification of Apolipoprotein C-III O-Glycoforms by Protein-MRM.

Apolipoprotein C-III (APOC-III) regulates triglyceride levels, associated with a risk of cardiovascular disease. One gene generates several proteoforms, each with a different molecular mass and a unique function. Unlike peptide multiple reaction monitoring (MRM), protein-MRM without digestion is required to analyze clinically relevant individual proteoforms. We developed a protein-MRM method without digestion to individually quantify APOC-III proteoforms in human serum. We optimized the protein-MRM method following 60% acetonitrile extraction with C18 filtration. Bovine serum and myoglobin served as supporting cushions and the internal standard during sample preparation, respectively. Furthermore, we evaluated the LOD, lower limit of quantification, linearity, accuracy, and precision. Good correlation compared with turbidimetric immunoassay (TIA) and peptide-MRM was observed using 30 clinical sera. Individual APOC-III O-glycoforms were identified by top-down proteomics and simultaneously quantified using the protein-MRM method. The sum abundance of APOC-III proteoforms was significantly correlated with TIA and peptide-MRM. Our protein-MRM method provides an affordable and rapid quantification of potential disease-specific proteoforms. Precise quantification of each proteoform allows investigators to identify novel biological roles potentially related to cardiovascular disease or novel biomarkers. We expect our protein-oriented method to be more clinically useful than antibody-based immunoassays and peptide-oriented MRM analysis, especially for quantification of a biomarker proteoform with certain post-translational modifications.

Improving mass accuracy in matrix-assisted laser desorption ionization time-of-flight mass spectrometry analysis of pathogenic proteins using 6xHIS-tagged internal calibration.

RATIONALE: Linear mode of matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOFMS) has been routinely used for bacterial identification in the clinic, depending on the pattern analysis of spectral libraries rather than accurate mass measurement of ribosomal proteins (10-15 kDa). However, a demand for more accurate mass analysis of pathogens (e.g. KPC-2 carbapenemase) has been recently increasing for diagnostic purposes. METHODS: We introduced a 6xHIS-tagged KPC-2 (i.e. hKPC-2) and used it as an internal mass calibrator for the mass calibration of target proteins. After internal mass calibration (In-Cal), we evaluated the observed mass of KPC-2 against the theoretical mass of hKPC-2, which has 823 Da mass difference from the target protein. We further assessed the accuracy and precision of our calibration method regarding the identification of KPC-2 and other pathogens in clinical isolates (n = 42). RESULTS: Among several candidates for internal mass calibrators, the In-Cal using a 6xHIS-tagged protein on the target showed the highest mass accuracy and precision in the detection of target proteins (e.g. KPC-2). The application of hKPC-2 as an internal calibrator showed substantial improvement of mass accuracy, mass precision and also quantification of KPC in linearity and repeatability for KPC detection in the clinical isolates. CONCLUSIONS: Our In-Cal method using 6xHIS-tagged protein in MALDI-TOFMS allows successful mass calibration (<3.5 Da) of pathogenic proteins (>20 kDa) and provides high mass accuracy as much as that of medium- and high-resolution mass spectrometry.

Clinical Performance of the Osmotic Shock-MALDI MS Method to Detect Klebsiella pneumoniae Carbapenemase in Clinical Isolates.

The World Health Organization recently highlighted the serious worldwide problem of the emergence of antibiotic-resistant or antibiotic multidrug-resistant bacteria. Carbapenem-resistant Enterobacterales, including carbapenemase-producing Enterobacterales (CPE), are major antibiotic-resistant bacteria that can be identified by various methods, including antibiotic susceptibility testing, PCR, and immunologic assays. However, there is a need for a faster, more accurate, low-cost, and easy method to detect CPE strains. We previously developed an osmotic shock matrix-assisted laser desorption/ionization mass spectrometry (OS-MALDI MS) method for directly detecting intact Klebsiella pneumoniae carbapenemase (KPC) using osmotic shock cell lysis. In this study, we evaluated the OS-MALDI MS method and compared it with two other methods (octyl-glucoside-aided direct KPC detection method [OG-MALDI MS] and Bruker's MBT subtyping module indirect method [MBT-SM MALDI MS]). We first completed an analytical performance evaluation of the OS-MALDI MS method according to Clinical and Laboratory Standards Institute guidelines. Clinical testing was performed with 437 clinical isolates, including 292 KPC-producing bacteria and 145 non-KPC-producing bacteria. The OS-MALDI MS method exhibited 95.9% sensitivity, 100.0% specificity, and 100.0% precision for detecting KPC. Accuracy of the OS-MALDI MS, OG-MALDI MS, and MBT-SM MALDI MS methods was 97.3%, 55.9%, and 50.2%, respectively. In conclusion, the OS-MALDI MS method clearly outperformed the other methods, exhibiting the highest accuracy and sensitivity of the three methods. We propose the OS-MALDI MS method as a practical, useful method for clinic environments, which may help guide appropriate antibiotic treatment and contribute to the prevention of the spread of CPE.

Targeting the interaction of AIMP2-DX2 with HSP70 suppresses cancer development.

A tumorigenic factor, AIMP2 lacking exon 2 (AIMP2-DX2), is often upregulated in many cancers. However, how its cellular level is determined is not understood. Here, we report heat-shock protein HSP70 as a critical determinant for the level of AIMP2-DX2. Interaction of the two factors was identified by interactome analysis and structurally determined by X-ray crystallography and NMR analyses. HSP70 recognizes the amino (N)-terminal flexible region, as well as the glutathione S-transferase domain of AIMP2-DX2, via its substrate-binding domain, thus blocking the Siah1-dependent ubiquitination of AIMP2-DX2. AIMP2-DX2-induced cell transformation and cancer progression in vivo was further augmented by HSP70. A positive correlation between HSP70 and AIMP2-DX2 levels was shown in various lung cancer cell lines and patient tissues. Chemical intervention in the AIMP2-DX2-HSP70 interaction suppressed cancer cell growth in vitro and in vivo. Thus, this work demonstrates the importance of the interaction between AIMP2-DX2 and HSP70 on tumor progression and its therapeutic potential against cancer.

Circadian modulation of the cardiac proteome underpins differential adaptation to morning and evening exercise training: an LC-MS/MS analysis.

All living beings on earth are influenced by the circadian rhythm, the rising and the setting of the sun. The ubiquitous effect of exercise is widely believed to maximize health benefits but has not been formally investigated for cardiac responses in the exercise-induced circadian rhythms. We hypothesized that the exercise-related proteome is differentially influenced by circadian rhythm and analyzed the differences between the effects of morning and evening exercise. Twenty-four Sprague-Dawley rats were randomly divided into four groups (n = 6 per group): morning control, morning exercise, evening control, and evening exercise groups. The exercise groups were subjected to 12-week treadmill exercise (5 days/week) performed either during daytime or nighttime. After 12 weeks, the physiological characteristics (e.g., body weight, heart weight, visceral fat, and blood metabolites), cardiovascular capacity (ejection fraction (%) and fractional shortening (%)), circadian gene expression levels (clock, ball1, per1, per2, cry1, and cry2), and the proteomic data were obtained and subjected to univariate and multivariate analysis. The mRNA levels of per1 and cry2 increased in the evening group compared with those in the morning group. We also found that per2 decreased and cry2 increased in the evening exercise groups. The evening exercise groups showed more decreased triacylglycerides and increased blood insulin levels than the morning exercise group. The principal component analysis, partial least squares discriminant analysis, and orthogonal partial least squares discriminant analysis indicated that the circadian rhythm differently influenced the protein networks of the exercise groups. In the morning exercise group, the transcription-translation feedback loop (TTFL) (clock, per1, per2, cry1, and cry2) formed a protein-protein interaction network with Nme2, Hint1, Ddt, Ndufb8, Ldha, and Eef1a2. In contrast, the TTFL group appeared close to Maoa, Hist2h4, and Macrod1 in the evening exercise group. Interestingly, the evening exercise group decreased the mRNA level of per2 but not per1. Per1 and Per2 are known to transport Cry1 and Cry2 into the nucleus. Taken together, we summarized the characteristics of enriched proteins in the aspect of their molecular function, cellular component, and biological process. Our results might provide a better understanding of the circadian effect on exercise-related proteins.

Common Repository of FBS Proteins (cRFP) To Be Added to a Search Database for Mass Spectrometric Analysis of Cell Secretome.

We propose to use cRFP (common Repository of FBS Proteins) in the MS (mass spectrometry) raw data search of cell secretomes. cRFP is a small supplementary sequence list of highly abundant fetal bovine serum proteins added to the reference database in use. The aim behind using cRFP is to prevent the contaminant FBS proteins from being misidentified as other proteins in the reference database, just as we would use cRAP (common Repository of Adventitious Proteins) to prevent contaminant proteins present either by accident or through unavoidable contacts from being misidentified as other proteins. We expect it to be widely used in experiments where the proteins are obtained from serum-free media after thorough washing of the cells, or from a complex media such as SILAC, or from extracellular vesicles directly.

Promiscuous methionyl-tRNA synthetase mediates adaptive mistranslation to protect cells against oxidative stress.

Aminoacyl-tRNA synthetases (ARSs) acylate transfer (t)RNAs with amino acids. Charging tRNAs with the right amino acids is the first step in translation; therefore, the accurate and error-free functioning of ARSs is an essential prerequisite for translational fidelity. A recent study found that methionine (Met) can be incorporated into non-Met residues of proteins through methionylation of non-cognate tRNAs under conditions of oxidative stress. However, it was not understood how this mis-methionylation is achieved. Here, we report that methionyl-tRNA synthetase (MRS) is phosphorylated at Ser209 and Ser825 by extracellular signal-related kinase (ERK1/2) under conditions of stress caused by reactive oxygen species (ROS), and that this phosphorylated MRS shows increased affinity for non-cognate tRNAs with lower affinity for tRNA(Met), leading to an increase in Met residues in cellular proteins. The expression of a mutant MRS containing the substitutions S209D and S825D, mimicking dual phosphorylation, reduced ROS levels and cell death. This controlled inaccuracy of MRS seems to serve as a defense mechanism against ROS-mediated damage at the cost of translational fidelity.

Chemical inhibition of prometastatic lysyl-tRNA synthetase-laminin receptor interaction.

Lysyl-tRNA synthetase (KRS), a protein synthesis enzyme in the cytosol, relocates to the plasma membrane after a laminin signal and stabilizes a 67-kDa laminin receptor (67LR) that is implicated in cancer metastasis; however, its potential as an antimetastatic therapeutic target has not been explored. We found that the small compound BC-K-YH16899, which binds KRS, impinged on the interaction of KRS with 67LR and suppressed metastasis in three different mouse models. The compound inhibited the KRS-67LR interaction in two ways. First, it directly blocked the association between KRS and 67LR. Second, it suppressed the dynamic movement of the N-terminal extension of KRS and reduced membrane localization of KRS. However, it did not affect the catalytic activity of KRS. Our results suggest that specific modulation of a cancer-related KRS-67LR interaction may offer a way to control metastasis while avoiding the toxicities associated with inhibition of the normal functions of KRS.

Integrated glycoproteomics demonstrates fucosylated serum paraoxonase 1 alterations in small cell lung cancer.

Small cell lung cancer (SCLC) is an aggressive type of lung cancer, and the detection of SCLCs at an early stage is necessary for successful therapy and for improving cancer survival rates. Fucosylation is one of the most common glycosylation-based modifications. Increased levels of fucosylation have been reported in a number of pathological conditions, including cancers. In this study, we aimed to identify and validate the aberrant and selective fucosylated glycoproteins in the sera of patients with SCLC. Fucosylated glycoproteins were enriched by the Aleuria aurantia lectin column after serum albumin and IgG depletion. In a narrowed down and comparative data analysis of both label-free proteomics and isobaric peptide-tagging chemistry iTRAQ approaches, the fucosylated glycoproteins were identified as up- or down-regulated in the sera of limited disease and extensive disease stage patients with SCLC. Verification was performed by multiple reaction monitoring-mass spectrometry to select reliable markers. Four fucosylated proteins, APCS, C9, SERPINA4, and PON1, were selected and subsequently validated by hybrid A. aurantia lectin ELISA (HLE) and Western blotting. Compared with Western blotting, the HLE analysis of these four proteins produced more optimal diagnostic values for SCLC. The PON1 protein levels were significantly reduced in the sera of patients with SCLC, whereas the fucosylation levels of PON1 were significantly increased. Fucosylated PON1 exhibited an area under curve of 0.91 for the extensive disease stage by HLE, whereas the PON1 protein levels produced an area under curve of 0.82 by Western blot. The glycan structural analysis of PON1 by MS/MS identified a biantennary fucosylated glycan modification consisting of a core + 2HexNAc + 1Fuc at increased levels in the sera of patients with SCLC. In addition, the PON1 levels were decreased in the sera of the Lewis lung carcinoma lung cancer mouse model that we examined. Our data suggest that fucosylated protein biomarkers, such as PON1, and their fucosylation levels and patterns can serve as diagnostic and prognostic serological markers for SCLC.

A Graphene-Coated Silicon Wafer Plate Improves the Sensitivity and Reproducibility of MALDI-TOF MS Analysis of Proteins.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is widely used to analyze small and large molecules. However, proteins are difficult to analyze with MALDI-TOF MS in clinical applications because of their low ionization efficiency and heterogeneous crystallization with the matrix on the sample spots. Here, we investigate the potential of a customized graphene-coated silicon wafer (G/SiO2) plate for MALDI-TOF MS analysis of a clinically important protein, KPC-2, in comparison with a conventional stainless steel (SUS) plate. Our results demonstrate that the G/SiO2 plate outperforms the SUS plate in terms of sensitivity, reproducibility, and mass accuracy/precision across a wide range of molecular weights, even with highly complex samples. Furthermore, a five-day robustness test confirms the practical applicability of the G/SiO2 plate for the reliable identification of target protein(s) in MALDI-TOF MS analysis. Overall, our findings suggest that the use of the G/SiO2 plate holds great potential for improving the sensitivity and reproducibility of MALDI-TOF MS analysis for the identification of proteins, making it a promising tool for clinical applications.

Fast and straightforward simultaneous quantification of multiple apolipoproteins in human serum on a high-throughput LC-MS/MS platform.

PURPOSE: Apolipoprotein monitoring is useful for diagnosing cardiovascular diseases, as they are risk factors of arteriosclerosis and other neutral fat-related diseases. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) is advantageous for simultaneous apolipoprotein quantification, differentiation, and standardization including their isoforms. However, fast and straightforward sample preparation that retains quantification accuracy remains challenging in clinical MS. EXPERIMENTAL DESIGN: We developed a simultaneous assay for serum apolipoprotein A-I (ApoA-I), apolipoprotein B100 family, and apolipoprotein C-III (ApoC-III) using a high-throughput LC-MS/MS platform coupled with a BRAVO system. The assay was simplified by using sodium deoxycholate and trypsin/lys-C without reduction and alkylation steps. RESULTS: Simple sample preparation reduced turnaround time by 1.5 h and neat goat serum was chosen as an optimal calibration matrix for accurate protein quantification. Assay precision, linearity, correlation, accuracy, limit of detection (LOD), limit of quantitation (LOQ), and carryover were validated according to CLSI guidelines over 41 days using more than 100 human serum samples. Good correlation compared with turbidimetric immunoassay (TIA) was observed by Deming regression for all analytes. CONCLUSIONS AND CLINICAL RELEVANCE: A high-throughput LC-MS/MS and BRAVO assay for simultaneous apolipoprotein analysis was validated using a simple preparation method with a human serum calibrator in goat serum matrix. The assay is readily expandable to include other target serum proteins and/or their isoforms.

Proteomic approach reveals FKBP4 and S100A9 as potential prediction markers of therapeutic response to neoadjuvant chemotherapy in patients with breast cancer.

Although doxorubicin (Doxo) and docetaxel (Docet) in combination are widely used in treatment regimens for a broad spectrum of breast cancer patients, a major obstacle has emerged in that some patients are intrinsically resistant to these chemotherapeutics. Our study aimed to discover potential prediction markers of drug resistance in needle-biopsied tissues of breast cancer patients prior to neoadjuvant chemotherapy. Tissues collected before chemotherapy were analyzed by mass spectrometry. A total of 2,331 proteins were identified and comparatively quantified between drug sensitive (DS) and drug resistant (DR) patient groups by spectral count. Of them, 298 proteins were differentially expressed by more than 1.5-fold. Some of the differentially expressed proteins (DEPs) were further confirmed by Western blotting. Bioinformatic analysis revealed that the DEPs were largely associated with drug metabolism, acute phase response signaling, and fatty acid elongation in mitochondria. Clinical validation of two selected proteins by immunohistochemistry found that FKBP4 and S100A9 might be putative prediction markers in discriminating the DR group from the DS group of breast cancer patients. The results demonstrate that a quantitative proteomics/bioinformatics approach is useful for discovering prediction markers of drug resistance, and possibly for the development of a new therapeutic strategy.

Functional unfolding of alpha1-antitrypsin probed by hydrogen-deuterium exchange coupled with mass spectrometry.

The native state of alpha(1)-antitrypsin (alpha(1)AT), a member of the serine protease inhibitor (serpin) family, is considered a kinetically trapped folding intermediate that converts to a more stable form upon complex formation with a target protease. Although previous structural and mutational studies of alpha(1)AT revealed the structural basis of the native strain and the kinetic trap, the mechanism of how the native molecule overcomes the kinetic barrier to reach the final stable conformation during complex formation remains unknown. We hypothesized that during complex formation, a substantial portion of the molecule undergoes unfolding, which we dubbed functional unfolding. Hydrogen-deuterium exchange coupled with ESI-MS was used to analyze this serpin in three forms: native, complexing, and complexed with bovine beta-trypsin. Comparing the deuterium content at the corresponding regions of these three samples, we probed the unfolding of alpha(1)AT during complex formation. A substantial portion of the alpha(1)AT molecule unfolded transiently during complex formation, including not only the regions expected from previous structural studies, such as the reactive site loop, helix F, and the following loop, but also regions not predicted previously, such as helix A, strand 6 of beta-sheet B, and the N terminus. Such unfolding of the native interactions may elevate the free energy level of the kinetically trapped native serpin sufficiently to cross the transition state during complex formation. In the current study, we provide evidence that protein unfolding has to accompany functional execution of the protein molecule.

Optimization of a lysis method to isolate periplasmic proteins from Gram-negative bacteria for clinical mass spectrometry.

PURPOSE: Clinical mass spectrometry requires a simple step process for sample preparation. This study aims to optimize the method for isolating periplasmic protein from Gram-negative bacteria and apply to clinical mass spectrometry. EXPERIMENTAL DESIGN: The Klebsiella pneumoniae carbapenemase (KPC)-producing E. coli standard cells were used for optimizing the osmotic shock (OS) lysis method. The supernatant from OS lysis was analysed by LC-MS/MS and MALDI-TOF MS. The effectiveness of the OS lysis method for KPC-2-producing Enterobacteriaceae clinical isolates were then confirmed by MALDI-TOF MS. RESULTS: The optimized OS lysis using KPC-2 producing E. coli standard cells showed a high yield of KPC-2 protein and enriches periplasmic proteins. Compared with other lysis methods, the detection sensitivity of KPC-2 protein significantly increased in MALDI-TOF MS analysis. Nineteen clinical isolates were validated by MALDI-TOF MS using the OS method, which also showed higher detection sensitivity compared to other lysis method (e.g., 1.5% n-octyl-ß-D-glucopyranoside) (p < 0.001). CONCLUSIONS AND CLINICAL RELEVANCE: This study provides a straightforward, rapid, affordable, and detergent-free method for the analysis of periplasmic proteins from Enterobacteriaceae clinical isolates. This approach may contribute to MS-based clinical diagnostics.

Fatty acid-bearing albumin induces VCAM-1 expression through c-Src kinase-AP-1/NF-kB pathways: effect of L-carnitine.

BACKGROUND: Fatty acid-bearing albumin [FA(+) albumin] exerts more deleterious effects in tubular cells than albumin alone. We investigated the effect of FA(+) albumin on the vascular cell adhesion molecule-1 (VCAM-1) expression and elucidated the underlying signaling pathways. We further examined the effect of L-carnitine, since it was known to modulate intracellular fatty acid concentration. METHODS: Activation of AP-1 and NF-kappaB was assessed by electrophoretic mobility shift assay. Phosphorylation of protein kinase was examined by Western blot analysis. VCAM-1 mRNA and protein expression were measured by Northern blot analysis and cell ELISA. RESULTS: FA(+) albumin induced VCAM-1 expression via activation of AP-1 and NF-kappaB, which was mediated through activation of c-Src kinase, followed by MAP kinases (p38, ERK 1/2, JNK-1) and IkappaB kinase and IkappaB-alpha, respectively. Inhibitors of protein kinase C and tyrosine kinase, anti-oxidants and intracellular calcium chelator suppressed the FA(+) albumin-induced activation of c-Src kinase. L-Carnitine suppressed the FA(+) albumin-induced VCAM-1 expression via inhibition of c-Src kinase. CONCLUSIONS: VCAM-1 expression with activation of c-Src kinase-AP-1/NFkappaB pathways might be one of the possible mechanisms that linked FA(+) albumin to tubulointerstitial injury. L-Carnitine might be beneficial in attenuating FA(+) albumin-induced tubular injury.

Extracellular vesicles derived from macrophages display glycyl-tRNA synthetase 1 and exhibit anti-cancer activity.

Glycyl-tRNA synthetase 1 (GARS1), a cytosolic enzyme secreted from macrophages, promotes apoptosis in cancer cells. However, the mechanism underlying GARS1 secretion has not been elucidated. Here, we report that GARS1 is secreted through unique extracellular vesicles (EVs) with a hydrodynamic diameter of 20-58 nm (mean diameter: 36.9 nm) and a buoyant density of 1.13-1.17 g/ml. GARS1 was anchored to the surface of these EVs through palmitoylated C390 residue. Proteomic analysis identified 164 proteins that were uniquely enriched in the GARS1-containing EVs (GARS1-EVs). Among the identified factors, insulin-like growth factor II receptor, and vimentin also contributed to the anti-cancer activity of GARS1-EVs. This study identified the unique secretory vesicles containing GARS1 and various intracellular factors that are involved in the immunological defence response against tumorigenesis.

Glucose-dependent control of leucine metabolism by leucyl-tRNA synthetase 1.

Despite the importance of glucose and amino acids for energy metabolism, interactions between the two nutrients are not well understood. We provide evidence for a role of leucyl-tRNA synthetase 1 (LARS1) in glucose-dependent control of leucine usage. Upon glucose starvation, LARS1 was phosphorylated by Unc-51 like autophagy activating kinase 1 (ULK1) at the residues crucial for leucine binding. The phosphorylated LARS1 showed decreased leucine binding, which may inhibit protein synthesis and help save energy. Leucine that is not used for anabolic processes may be available for catabolic pathway energy generation. The LARS1-mediated changes in leucine utilization might help support cell survival under glucose deprivation. Thus, depending on glucose availability, LARS1 may help regulate whether leucine is used for protein synthesis or energy production.

A threonyl-tRNA synthetase-mediated translation initiation machinery.

A fundamental question in biology is how vertebrates evolved and differ from invertebrates, and little is known about differences in the regulation of translation in the two systems. Herein, we identify a threonyl-tRNA synthetase (TRS)-mediated translation initiation machinery that specifically interacts with eIF4E homologous protein, and forms machinery that is structurally analogous to the eIF4F-mediated translation initiation machinery via the recruitment of other translation initiation components. Biochemical and RNA immunoprecipitation analyses coupled to sequencing suggest that this machinery emerged as a gain-of-function event in the vertebrate lineage, and it positively regulates the translation of mRNAs required for vertebrate development. Collectively, our findings demonstrate that TRS evolved to regulate vertebrate translation initiation via its dual role as a scaffold for the assembly of initiation components and as a selector of target mRNAs. This work highlights the functional significance of aminoacyl-tRNA synthetases in the emergence and control of higher order organisms.

The Possible Role of Neurobeachin in Extinction of Contextual Fear Memory.

Established fear memory becomes vulnerable to disruption after memory retrieval and extinction; this labile state is critical for inhibiting the return of fear memory. However, the labile state has a very narrow time window after retrieval, and underlying molecular mechanisms are not well known. To that end, we isolated the hippocampus immediately after fear memory retrieval and performed proteomics. We identified Neurobeachin (NBEA), an autism-related regulator of synaptic protein trafficking, to be upregulated after contextual fear memory retrieval. NBEA protein expression was rapid and transient after fear memory retrieval at the synapse. Nbea mRNA was enriched at the synapses, and the rapid induction of NBEA expression was blocked by inhibition of the mammalian target of rapamycin (mTOR)-dependent signaling pathway. Mice with cornu ammonis 1 (CA1)-specific Nbea shRNA knockdown showed normal fear acquisition and contextual fear memory but impaired extinction, suggesting an important role of Nbea in fear memory extinction processes. Consistently, Nbea heterozygotes showed normal fear acquisition and fear memory recall but showed impairment in extinction. Our data suggest that NBEA is necessary either for induction of memory lability or for the physiological process of memory extinction.

Production of recombinant human procollagen type I C-terminal propeptide and establishment of a sandwich ELISA for quantification.

Procollagen type I carboxy-terminal propeptide (PICP), derived from type I procollagen, has been identified as an indicator of type I collagen synthesis in bone matrix formation and skin recovery. PICP is a heterotrimeric glycoprotein consisting of two α1 chains (PICPα1) and one α2 chain (PICPα2). Here, we report the recombinant expression of human PICP using a mammalian expression system. Co-expression of PICPα1 and PICPα2 in HEK293F cells resulted in the production of functional PICP in the correctly assembled heterotrimeric form. Using the recombinant PICP as an antigen, we isolated PICP-specific human monoclonal antibodies from phage-displayed antibody libraries and raised rabbit polyclonal antibodies. Using those antibodies, we then developed a sandwich ELISA for PICP with a limit of detection of 1 ng/mL and a measurable range of 1-640 ng/mL. Both intra- and inter-assay imprecision values were <10%. For measuring PICP levels in human fibroblast cellular extracts and culture supernatants and a human serum, the developed ELISA kit displayed comparable performance to that of a commercialized kit. Our results provide an efficient production strategy for recombinant PICP, facilitating the generation of PICP-specific antibodies and development of PICP sandwich ELISA, with potential use in clinical diagnosis of serum samples and testing of cosmeceutical ingredients in fibroblast cell cultures.