Quantifiable peptide library bridges the gap for proteomics based biomarker discovery and validation on breast cancer.

Mass spectrometry (MS) based proteomics is widely used for biomarker discovery. However, often, most biomarker candidates from discovery are discarded during the validation processes. Such discrepancies between biomarker discovery and validation are caused by several factors, mainly due to the differences in analytical methodology and experimental conditions. Here, we generated a peptide library which allows discovery of biomarkers in the equal settings as the validation process, thereby making the transition from discovery to validation more robust and efficient. The peptide library initiated with a list of 3393 proteins detectable in the blood from public databases. For each protein, surrogate peptides favorable for detection in mass spectrometry was selected and synthesized. A total of 4683 synthesized peptides were spiked into neat serum and plasma samples to check their quantifiability in a 10 min liquid chromatography-MS/MS run time. This led to the PepQuant library, which is composed of 852 quantifiable peptides that cover 452 human blood proteins. Using the PepQuant library, we discovered 30 candidate biomarkers for breast cancer. Among the 30 candidates, nine biomarkers, FN1, VWF, PRG4, MMP9, CLU, PRDX6, PPBP, APOC1, and CHL1 were validated. By combining the quantification values of these markers, we generated a machine learning model predicting breast cancer, showing an average area under the curve of 0.9105 for the receiver operating characteristic curve.

Structural insights into apoptotic regulation of human Bfk as a novel Bcl-2 family member.

Bcl-2 family kin (Bfk), also known as Bcl-2-like 15, plays an essential role in regulating apoptosis by eliciting weak pro-apoptotic responses in the gastrointestinal tract. Human Bfk is a novel Bcl-2 family protein owing to its unique domain composition involving BH2 and BH3. However, the molecular mechanism underlying the regulation of apoptosis by Bfk remains unclear. Here, we first report the crystal structure of human full-length Bfk. Surprisingly, the structure of Bfk adopts a canonical Bcl-2 fold but lacks the hydrophobic cleft, which could accommodate a BH3 domain from other Bcl-2 family proteins. Our biophysical interaction analysis proved that the full-length Bfk itself does not interact with multi-domain Bcl-2 family proteins or a BH3-containing peptide. Instead, Bfk is structurally and functionally reminiscent of Bid, a BH3-only protein in the Bcl-2 family, with similar conformations of helices α3-α5 and the specific motif in helix α5. Not only structural analyses of the full-length Bfk but also molecular dynamics simulation suggested that Bfk elicits its pro-apoptotic activity through a Bid-like apoptotic mechanism in which the BH3 domain is released upon caspase-mediated cleavage and a conformational change of the truncated form. Indeed, the BH3 peptide derived from Bfk exhibited in vitro interactions with Bcl-2, Bcl-XL, and Bak. These findings provide new insights into the molecular characteristics of Bfk and a valuable foundation for development of a new therapeutic target to control apoptosis.

Structural Analyses on the Deamidation of N-Terminal Asn in the Human N-Degron Pathway.

The N-degron pathway is a proteolytic system in which a single N-terminal amino acid acts as a determinant of protein degradation. Especially, degradation signaling of N-terminal asparagine (Nt-Asn) in eukaryotes is initiated from its deamidation by N-terminal asparagine amidohydrolase 1 (NTAN1) into aspartate. Here, we have elucidated structural principles of deamidation by human NTAN1. NTAN1 adopts the characteristic scaffold of CNF1/YfiH-like cysteine hydrolases that features an α-ß-ß sandwich structure and a catalytic triad comprising Cys, His, and Ser. In vitro deamidation assays using model peptide substrates with varying lengths and sequences showed that NTAN1 prefers hydrophobic residues at the second-position. The structures of NTAN1-peptide complexes further revealed that the recognition of Nt-Asn is sufficiently organized to produce high specificity, and the side chain of the second-position residue is accommodated in a hydrophobic pocket adjacent to the active site of NTAN1. Collectively, our structural and biochemical analyses of the substrate specificity of NTAN1 contribute to understanding the structural basis of all three amidases in the eukaryotic N-degron pathway.

Differential expression profiles and roles of inducible DUSPs and ERK1/2-specific constitutive DUSP6 and DUSP7 in microglia.

Dual-specificity phosphatases (DUSPs) show distinct substrate preferences for specific MAPKs. DUSPs sharing a substrate preference for ERK1/2 may be classified as inducible or constitutive. In contrast to the inducible DUSPs which also dephosphorylate p38 MAPK and JNK in the major inflammatory pathways, constitutive DUSP6 and DUSP7 are specific to ERK1/2 and have not been studied in microglia and other immune cells to date. In the present study, we differentiated mRNA expression profiles of inducible and constitutive DUSPs that dephosphorylate ERK1/2 in microglia. Lipopolysaccharide (LPS) at 1 ng/ml induced prompt phosphorylation of ERK1/2 with peak induction at 30 min. LPS induced expression of DUSP1, DUSP2, and DUSP5 within 60 min, whereas DUSP4 expression was induced more slowly. DUSP6 and DUSP7 exhibited constitutive basal expression, which decreased immediately after LPS stimulation but subsequently returned to basal levels. The expression of DUSP6 and DUSP7 was regulated inverse to the phosphorylation of ERK1/2 in LPS-stimulated microglia. Therefore, we next investigated the correlation between DUSP6 and DUSP7 expression and ERK1/2 phosphorylation in resting and LPS-stimulated microglia. Inhibition of the ERK1/2 pathway by PD98059 and FR180204 resulted in a decrease in DUSP6 and DUSP7 expression, both in resting and LPS-stimulated microglia. These inhibitors partially blocked the LPS-induced expression of DUSP1, DUSP2, and DUSP4, but had no effect on DUSP5. Finally, we examined the role of DUSP6 activity in the downregulation of ERK1/2 phosphorylation. BCI, an inhibitor of DUSP6, increased the phosphorylation of ERK1/2. However, pretreatment with BCI inhibited the LPS-induced phosphorylation of ERK1/2. These results demonstrate that constitutive DUPS6 and DUSP7 expression was downregulated inverse to the expression of inducible DUSPs and the phosphorylation of ERK1/2 in LPS-stimulated microglia. The expression of DUPS6 and DUSP7 was mediated by ERK1/2 activity both in resting and LPS-stimulated microglia. In turn, DUSP6 suppressed the basal phosphorylation of ERK1/2, but exerted no suppressive effect on LPS-induced phosphorylation. Although DUSP6 is acknowledged as a negative regulator of the ERK1/2 pathway, such roles of DUSP6 need to be examined further in activated microglia.

Retained topical delivery of 5-aminolevulinic acid using cationic ultradeformable liposomes for photodynamic therapy.

5-Aminolevulinic acid (5-ALA), inducing photodynamic protoporphyrin (PpIX), is a hydrophilic molecule, resulting in leashing the capacity to cross tissue barriers like stratum corneum (SC) of skin. Here, we aimed to develop 5-ALA loaded ultradeformable liposomes (UDL) with different surface charges, and to investigate their physicochemical characteristics and capability for the skin penetration and retention of 5-ALA for topical photodynamic therapy (PDT). The effects of surface charges of UDL on in vitro permeation of 5-ALA and in vivo accumulation of 5-ALA-induced PpIX in viable skin were determined and then compared with conventional neutral liposomes (nLiposome). All UDL showed smaller particle size and better deformability than nLiposome. However, entrapment efficiency of 5-ALA was similar to each vesicle. Among vesicles, the cationic UDL (cUDL) demonstrated higher stability and permeability, and could deliver 5-ALA into deep skin tissue by topical application. Moreover, the 5-ALA loaded in cUDL was long retained, and induced more amount of PpIX in viable skin than those in other UDL and nLiposome. Considering that the conversion of 5-ALA into PpIX occurs preferentially in epidermis, these results suggested that topical delivery of 5-ALA loaded in cUDL could be an interesting proposal to optimize PDT related to 5-ALA.

Analysis of carbamazepine and its active metabolite, carbamazepine-10,11-epoxide, in human plasma using high-performance liquid chromatography.

A sensitive method based on high-performance liquid chromatography (HPLC) with ultraviolet (UV) detection was developed for the determination of carbamazepine (CBZ) and one of its active metabolites, carbamazepine-10,11-epoxide (CBZ-E) in human plasma. CBZ, CBZ-E and the internal standard (IS) 10,11-dihydrocarbamazepine were extracted from human plasma into methyl tert-butyl ether. CBZ, CBZ-E and the IS were successfully separated on an RP C18 column with a mobile phase of acetonitrile:methanol:water (18:19:63, v/v/v) and monitored via UV detection at 210 nm. The calibration curves were linear over the concentration ranges of 0.01-10 microg/mL for CBZ and 0.005-5 microg/mL for CBZ-E in human plasma, respectively. The method displayed excellent sensitivity, precision and accuracy, and was successfully applied to the quantification of CBZ and CBZ-E in human plasma after oral administration of a single 200 mg CBZ CR tablet. This method is suitable for bioequivalence studies following single doses given to healthy volunteers.