Diabetic nephropathy (DN) has become the main cause of end-stage renal disease worldwide, causing significant health problems. Early diagnosis of the disease is quite inadequate. To screen urine biomarkers of DN and explore its potential mechanism, this study collected urine from 87 patients with type 2 diabetes mellitus (which will be classified into normal albuminuria, microalbuminuria, and macroalbuminuria groups) and 38 healthy subjects. Twelve individuals from each group were then randomly selected as the screening cohort for proteomics analysis and the rest as the validation cohort. The results showed that humoral immune response, complement activation, complement and coagulation cascades, renin-angiotensin system, and cell adhesion molecules were closely related to the progression of DN. Five overlapping proteins (KLK1, CSPG4, PLAU, SERPINA3, and ALB) were identified as potential biomarkers by machine learning methods. Among them, KLK1 and CSPG4 were positively correlated with the urinary albumin to creatinine ratio (UACR), and SERPINA3 was negatively correlated with the UACR, which were validated by enzyme-linked immunosorbent assay (ELISA). This study provides new insights into disease mechanisms and biomarkers for early diagnosis of DN.

It has been well-elaborated that KIN17 protein is closely related to the expression, development and prognosis of liver cancer; however, till date, there has been no study about detecting the KIN17 protein in serum, which is important to developing clinical applications. The objective of this work is to detect serum KIN17 protein by the ELISA method and to explore the diagnostic significance of the KIN17 protein in liver cancer. First, we verified the ELISA method for serum KIN17 measurement according to five aspects: accuracy, precision, specificity, stability and detection limit. Results illustrate that the recovery rate of the ELISA method can be controlled between 90% and 110%, the variation coefficient of intra-assay can be controlled within 16%, and the variation coefficient of inter-assay can be controlled within 10%. There is no non-specific reaction with common tumor markers, and the detection limit can reach 0.125 ng mL-1. The results show that the KIN17 protein can be detected by ELISA, and there is a significant rise in KIN17 concentration in a liver cancer group compared with a healthy group, whose average concentrations are 1.730 ng mL-1 and 0.3897 ng mL-1, respectively. On this basis, we hypothesize that the serum KIN17 protein can serve as a potential biomarker of liver cancer and be measurable with the verified ELISA system after specific ultrafiltration and centrifugation, which is of great significance for the diagnosis and treatment of liver cancer.

A comprehensive analysis of site-specific protein O-glycosylation is hindered by the absence of a consensus O-glycosylation motif, the diversity of O-glycan structures, and the lack of a universal enzyme that cleaves attached O-glycans. Here, we report the development of a robust O-glycoproteomic workflow for analyzing complex biological samples by combining four different strategies: removal of N-glycans, complementary digestion using O-glycoprotease (IMPa) with/without another protease, glycopeptide enrichment, and mass spectrometry with fragmentation of glycopeptides using stepped collision energy. Using this workflow, we cataloged 474 O-glycopeptides on 189 O-glycosites derived from 79 O-glycoproteins from human plasma. These data revealed O-glycosylation of several abundant proteins that have not been previously reported. Because many of the proteins that contained unannotated O-glycosylation sites have been extensively studied, we wished to confirm glycosylation at these sites in a targeted fashion. Thus, we analyzed selected purified proteins (kininogen-1, fetuin-A, fibrinogen, apolipoprotein E, and plasminogen) in independent experiments and validated the previously unknown O-glycosites.

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UNASSIGNED: Coagulation factor VIII (FVIII) and its carrier protein von Willebrand factor (VWF) are critical to coagulation and platelet aggregation. We leveraged whole-genome sequence data from the Trans-Omics for Precision Medicine (TOPMed) program along with TOPMed-based imputation of genotypes in additional samples to identify genetic associations with circulating FVIII and VWF levels in a single-variant meta-analysis, including up to 45 289 participants. Gene-based aggregate tests were implemented in TOPMed. We identified 3 candidate causal genes and tested their functional effect on FVIII release from human liver endothelial cells (HLECs) and VWF release from human umbilical vein endothelial cells. Mendelian randomization was also performed to provide evidence for causal associations of FVIII and VWF with thrombotic outcomes. We identified associations (P < 5 × 10-9) at 7 new loci for FVIII (ST3GAL4, CLEC4M, B3GNT2, ASGR1, F12, KNG1, and TREM1/NCR2) and 1 for VWF (B3GNT2). VWF, ABO, and STAB2 were associated with FVIII and VWF in gene-based analyses. Multiphenotype analysis of FVIII and VWF identified another 3 new loci, including PDIA3. Silencing of B3GNT2 and the previously reported CD36 gene decreased release of FVIII by HLECs, whereas silencing of B3GNT2, CD36, and PDIA3 decreased release of VWF by HVECs. Mendelian randomization supports causal association of higher FVIII and VWF with increased risk of thrombotic outcomes. Seven new loci were identified for FVIII and 1 for VWF, with evidence supporting causal associations of FVIII and VWF with thrombotic outcomes. B3GNT2, CD36, and PDIA3 modulate the release of FVIII and/or VWF in vitro.

UNASSIGNED: Hereditary angioedema (HAE) is associated with episodic kinin-induced swelling of the skin and mucosal membranes. Most patients with HAE have low plasma C1-inhibitor activity, leading to increased generation of the protease plasma kallikrein (PKa) and excessive release of the nanopeptide bradykinin from high-molecular-weight kininogen (HK). However, disease-causing mutations in at least 10% of patients with HAE appear to involve genes for proteins other than C1-inhibitor. A point mutation in the Kng1 gene encoding HK and low-molecular weight kininogen (LK) was identified recently in a family with HAE. The mutation changes a methionine (Met379) to lysine (Lys379) in both proteins. Met379 is adjacent to the Lys380-Arg381 cleavage site at the N-terminus of the bradykinin peptide. Recombinant wild-type (Met379) and variant (Lys379) versions of HK and LK were expressed in HEK293 cells. PKa-catalyzed kinin release from HK and LK was not affected by the Lys379 substitutions. However, kinin release from HK-Lys379 and LK-Lys379 catalyzed by the fibrinolytic protease plasmin was substantially greater than from wild-type HK-Met379 and LK-Met379. Increased kinin release was evident when fibrinolysis was induced in plasma containing HK-Lys379 or LK-Lys379 compared with plasma containing wild-type HK or LK. Mass spectrometry revealed that the kinin released from wild-type and variant kininogens by PKa is bradykinin. Plasmin also released bradykinin from wild-type kininogens but cleaved HK-Lys379 and LK-Lys379 after Lys379 rather than Lys380, releasing the decapeptide Lys-bradykinin (kallidin). The Met379Lys substitutions make HK and LK better plasmin substrates, reinforcing the relationship between fibrinolysis and kinin generation.

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BACKGROUND: Hepatitis B virus (HBV) infection is one of the main causes of hepatocellular carcinoma (HCC). The relationship between HBV infection and the host genome as well as their underlying mechanisms remain largely unknown.
METHODS: In this study, we performed a whole-genome exon sequencing analysis of 300 sib-pairs of Chinese HBV-infected families with the goal of identifying variants and genes involved in HBV infection. A site-direct mutant plasmid was used to investigate the function of SNP rs76438938 in KNG1. The functional and mechanical studies of KNG1 were conducted with in vitro liver cell lines and a hydrodynamic injection model in vivo. The impact of KNG1 on HBV infection therapy was determined in hepatocytes treated with IFN-α/λ1.
RESULTS: Our whole-exon association study of 300 families with hepatitis B infection found that SNP rs76438938 in KNG1 significantly increased the risk for HBV infection, and the rs76438938-T allele was found to promote HBV replication by increasing the stability of KNG1 mRNA. By competitively binding HSP90A with MAVS, KNG1 can inhibit the expression of types I and III IFNs by promoting MAVS lysosomal degradation. Such suppression of IFN expression and promotion of HBV replication by Kng1 were further demonstrated with an animal model in vivo. Lastly, we showed that the rs76438938-C allele can improve the therapeutic effect of IFN-α and -λ1 in HBV infection.
CONCLUSIONS: This study identified a SNP, rs76438938, in a newly discovered host gene, KNG1, for its involvement in HBV infection and treatment effect through modulating the cellular antiviral process.
BACKGROUND: This study was supported in part by the Independent Task of State Key Laboratory for Diagnosis and Treatment of Infectious Diseases of the First Affiliated Hospital of Zhejiang University, the China Precision Medicine Initiative (2016YFC0906300), and the Research Center for Air Pollution and Health of Zhejiang University.

High-molecular weight kininogen (HK) circulates in plasma as a complex with zymogen prekallikrein (PK). HK is both a substrate and a cofactor for activated plasma kallikrein, and the principal exosite interactions occur between PK N-terminal apple domains and the C-terminal D6 domain of HK.
To determine the structure of the complex formed between PK apple domains and an HKD6 fragment and compare this with the coagulation factor XI (FXI)-HK complex.
We produced recombinant FXI and PK heavy chains (HCs) spanning all 4 apple domains. We cocrystallized PKHC (and subsequently FXIHC) with a 31-amino acid synthetic peptide spanning HK residues Ser565-Lys595 and determined the crystal structure. We also analyzed the full-length FXI-HK complex in solution using hydrogen deuterium exchange mass spectrometry.
The 2.3Å PKHC-HK peptide crystal structure revealed that the HKD6 sequence WIPDIQ (Trp569-Gln574) binds to the apple 1 domain and HK FNPISDFPDT (Phe582-Thr591) binds to the apple 2 domain with a flexible intervening sequence resulting in a bent double conformation. A second 3.2Å FXIHC-HK peptide crystal structure revealed a similar interaction with the apple 2 domain but an alternate, straightened conformation of the HK peptide where residues LSFN (Leu579-Asn583) interacts with a unique pocket formed between the apple 2 and 3 domains. HDX-MS of full length FXI-HK complex in solution confirmed interactions with both apple 2 and apple 3.
The alternate conformations and exosite binding of the HKD6 peptide likely reflects the diverging relationship of HK to the functions of PK and FXI.

The epidermis is the outermost skin layer and is part of one of the largest organs in the body; it is supported by the dermis, a network of fibrils, blood vessels, pilosebaceous units, sweat glands, nerves, and cells. The skin as a whole is a protective shield against numerous noxious agents, including microorganisms and chemical and physical factors. These functions rely on the activity of multiple growth factors, peptide hormones, proteases, and specific signaling pathways that are triggered by the activation of distinct types of receptors sited in the cell membranes of the various cell types present in the skin. The human kallikrein family comprises a large group of 15 serine proteases synthesized and secreted by different types of epithelial cells throughout the body, including the skin. At this site, they initiate a proteolytic cascade that generates the active forms of the proteases, some of which regulate skin desquamation, activation of cytokines, and antimicrobial peptides. Kinin peptides are formed by the action of plasma and tissue kallikreins on kininogens, two plasma proteins produced in the liver and other organs. Although kinins are well known for their proinflammatory abilities, in the skin they are also considered important modulators of keratinocyte differentiation. In this review, we summarize the contributions of the kallikreins and kallikrein-related peptidases family and those of kinins and their receptors in skin homeostasis, with special emphasis on their pathophysiological role.