Targeted Proteins Reveal Cathepsin D as a Novel Biomarker in Differentiating Hepatocellular Carcinoma from Cirrhosis and Other Liver Cancers

Objective: Hepatocellular carcinoma (HCC) represents a global health concern, particularly in Southeast Asia where hepatitis B virus (HBV) infection is common. In this study, we applied tissue-based proteomics to identify novel serological proteins for HCC and validated their performance in serum specimens. Methods: In a discovery set, liver tissue specimens of HBV-related HCC, intrahepatic cholangiocarcinoma (iCCA) and colorectal cancer with liver metastasis (CRLM) were analyzed using mass spectrometry (LTQ-Orbitrap-XL). A subset of proteins that showed highly expressed in HCC were then confirmed by Western blotting. Additionally, clinical significance of selected candidate proteins was tested in serum samples of 80 patients with HBV-related HCC, 50 patients with HBV-related liver cirrhosis and 30 healthy controls. Results: Based on LTQ-Orbitrap-XL mass spectrometer, various differentially expressed proteins (DEPs) between tumor and adjacent non-tumor tissues were identified. These included 77 DEPs for HCC, 77 DEPs for iCCA and 55 DEPs for CRLM. Among selected candidate proteins, annexin A2 and cathepsin D were confirmed to be overexpressed in HCC tissue by Western blot analysis. In a validate cohort, serum cathepsin D level, but not annexin A2, was significantly higher in HCC compared with the non-HCC groups. Serum cathepsin D level was also positively correlated with tumor size and tumor stage. Additionally, the combined assay of serum cathepsin D and alpha-fetoprotein had a high sensitivity in detecting early HCC (83%) and intermediate/advanced HCC (96%). Moreover, patients with low serum cathepsin D (<305 ng/mL) displayed significantly better overall survival than those whose serum levels were high (≥305 ng/mL). Conclusions: Proteomics and subsequent validation revealed cathepsin D as a novel biomarker for HCC. Apart from its diagnostic role, serum cathepsin D might also serve as a prognostic biomarker of HCC. Additional large-scale studies are needed to verify our finding

Mitochondria-derived vesicles and their potential roles in kidney stone disease

Abstract Recent evidence has shown significant roles of mitochondria-derived vesicles (MDVs) in mitochondrial quality control (MQC) system. Under mild stress condition, MDVs are formed to carry the malfunctioned mitochondrial components, such as mitochondrial DNA (mtDNA), peptides, proteins and lipids, to be eliminated to restore normal mitochondrial structure and functions. Under severe oxidative stress condition, mitochondrial dynamics (fission/fusion) and mitophagy are predominantly activated to rescue mitochondrial structure and functions. Additionally, MDVs generation can be also triggered as the major MQC machinery to cope with unhealthy mitochondria when mitophagy is unsuccessful for eliminating the damaged mitochondria or mitochondrial fission/fusion fail to recover the mitochondrial structure and functions. This review summarizes the current knowledge on MDVs and discuss their roles in physiologic and pathophysiologic conditions. In addition, the potential clinical relevance of MDVs in therapeutics and diagnostics of kidney stone disease (KSD) are emphasized

Defining and Systematic Analyses of Aggregation Indices to Evaluate Degree of Calcium Oxalate Crystal Aggregation

Crystal aggregation is one of the most crucial steps in kidney stone pathogenesis. However, previous studies of crystal aggregation were rarely done and quantitative analysis of aggregation degree was handicapped by a lack of the standard measurement. We thus performed an in vitro assay to generate aggregation of calcium oxalate monohydrate (COM) crystals with various concentrations (25–800 μg/ml) in saturated aggregation buffer. The crystal aggregates were analyzed by microscopic examination, UV-visible spectrophotometry, and GraphPad Prism6 software to define a total of 12 aggregation indices (including number of aggregates, aggregated mass index, optical density, aggregation coefficient, span, number of aggregates at plateau time-point, aggregated area index, aggregated diameter index, aggregated symmetry index, time constant, half-life, and rate constant). The data showed linear correlation between crystal concentration and almost all of these indices, except only for rate constant. Among these, number of aggregates provided the greatest regression coefficient (r = 0.997; p &lt; 0.001), whereas the equally second rank included aggregated mass index and optical density (r = 0.993; p &lt; 0.001 and r = −0.993; p &lt; 0.001, respectively) and the equally forth were aggregation coefficient and span (r = 0.991; p &lt; 0.001 for both). These five indices are thus recommended as the most appropriate indices for quantitative analysis of COM crystal aggregation in vitro

Calcineurin B inhibits calcium oxalate crystallization, growth and aggregation via its high calcium-affinity property

Calcineurin inhibitors (CNIs) are widely used in organ transplantation to suppress immunity and prevent allograft rejection. However, some transplant patients receiving CNIs have hypocitraturia, hyperoxaluria and kidney stone with unclear mechanism. We hypothesized that CNIs suppress activities of urinary calcineurin, which may serve as the stone inhibitor. This study aimed to investigate effects of calcineurin B (CNB) on calcium oxalate monohydrate (COM) stone formation. Sequence and structural analyses revealed that CNB contained four EF-hand (Ca2+-binding) domains, which are known to regulate Ca2+ homeostasis and likely to affect COM crystals. Various crystal assays revealed that CNB dramatically inhibited COM crystallization, crystal growth and crystal aggregation. At an equal amount, degrees of its inhibition against crystallization and crystal growth were slightly inferior to total urinary proteins (TUPs) from healthy subjects that are known to strongly inhibit COM stone formation. Surprisingly, its inhibitory effect against crystal aggregation was slightly superior to TUPs. While TUPs dramatically inhibited crystal-cell adhesion, CNB had no effect on this process. Ca2+-affinity assay revealed that CNB strongly bound Ca2+ at a comparable degree as of TUPs. These findings indicate that CNB serves as a novel inhibitor of COM crystallization, growth and aggregation via its high Ca2+-affinity property

Changes in Mitochondrial Proteome of Renal Tubular Cells Induced by Calcium Oxalate Monohydrate Crystal Adhesion and Internalization Are Related to Mitochondrial Dysfunction

Calcium oxalate monohydrate (COM) crystals, the major crystalline compound in kidney stones, have been suggested to induce oxidative stress by overproduction of reactive oxygen species (ROS) and renal tubular cell injury. Our present study aimed to examine changes in mitochondrial proteome in distal renal tubular cells induced by COM crystals (100 μg of crystals/mL of culture medium). Adhesion and internalization of COM crystals by MDCK cells were examined by fluorescent and laser-scanning confocal microscopy. Moreover, the internalized COM crystals were quantified by flow cytometry. Thereafter, mitochondria were isolated from controlled and COM-treated cells, and mitochondrial proteins were subjected to 2-DE-based comparative proteomic analysis, which revealed 15 differentially expressed proteins. These significantly altered proteins were identified by Q-TOF MS and MS/MS analyses, including those involved in several biological processes, e.g., cellular structure, carbohydrate metabolism, and energy metabolism. 2-D Western blot analysis confirmed the increase of ezrin and decrease of β-actin. Global protein network analysis was then performed to obtain additional functional significance of the identified proteins and to guide for subsequent functional analysis. The results implicated that the altered proteins were involved in energy production and might contribute to mitochondrial dysfunction. The loss of ROS regulation by mitochondria was finally confirmed by OxyBlot assay, which demonstrated markedly increased levels of the oxidatively modified mitochondrial proteins in the COM-treated cells in a dose-dependent manner. Our data may lead to a better understanding of molecular mechanisms of mitochondrial dysfunction underlying the overt oxidative stress induced by COM crystals in kidney stone disease

Representative proteome map of differentially expressed proteins from the mitochondrial fractions of fibroblasts from (A) LHON cases (n = 7) and (U) unaffected LHON individuals (n = 3).

<p>Equal amounts of proteins (100 µg) from each sample were resolved by 2-DE. The numbers indicate the spot IDs of proteins whose expression levels differ significantly between the LHON cases and their unaffected relatives.</p

Profiling the Mitochondrial Proteome of Leber’s Hereditary Optic Neuropathy (LHON) in Thailand: Down-Regulation of Bioenergetics and Mitochondrial Protein Quality Control Pathways in Fibroblasts with the 11778G>A Mutation

<div><p>Leber’s Hereditary Optic Neuropathy (LHON) is one of the commonest mitochondrial diseases. It causes total blindness, and predominantly affects young males. For the disease to develop, it is necessary for an individual to carry one of the primary mtDNA mutations 11778G>A, 14484T>C or 3460G>A. However these mutations are not sufficient to cause disease, and they do not explain the characteristic features of LHON such as the higher prevalence in males, incomplete penetrance, and relatively later age of onset. In order to explore the roles of nuclear encoded mitochondrial proteins in development of LHON, we applied a proteomic approach to samples from affected and unaffected individuals from 3 pedigrees and from 5 unrelated controls. Two-dimensional electrophoresis followed by MS/MS analysis in the mitochondrial lysate identified 17 proteins which were differentially expressed between LHON cases and unrelated controls, and 24 proteins which were differentially expressed between unaffected relatives and unrelated controls. The proteomic data were successfully validated by western blot analysis of 3 selected proteins. All of the proteins identified in the study were mitochondrial proteins and most of them were down regulated in 11778G>A mutant fibroblasts. These proteins included: subunits of OXPHOS enzyme complexes, proteins involved in intermediary metabolic processes, nucleoid related proteins, chaperones, cristae remodelling proteins and an anti-oxidant enzyme. The protein profiles of both the affected and unaffected 11778G>A carriers shared many features which differed from those of unrelated control group, revealing similar proteomic responses to 11778G>A mutation in both affected and unaffected individuals. Differentially expressed proteins revealed two broad groups: a cluster of bioenergetic pathway proteins and a cluster involved in protein quality control system. Defects in these systems are likely to impede the function of retinal ganglion cells, and may lead to the development of LHON in synergy with the primary mtDNA mutation.</p></div

Differentially expressed proteins which show similar trends of down-regulation in the mitochondrial proteomes of both LHON cases and unaffected relatives when compared with controls.

<p>Differentially expressed proteins which show similar trends of down-regulation in the mitochondrial proteomes of both LHON cases and unaffected relatives when compared with controls.</p

Assessment of the purity of fibroblasts from a cultured skin biopsy.

<p>Fibroblast surface protein (FSP) was used as a marker in immunofluorescence of the cultured fibroblasts obtained directly obtained from the skin biopsy. The green represents fibroblasts and the nucleus was stained with Hoechst-dye 33342 which shows blue.</p

Functional categories and sub-cellular localizations of proteins identified in the study based on the MitoMiner Database and Nextprot.

<p>M = Mitochondria, MI = Mitochondrial intermembrane space, ER = Endoplasmic Reticulum, C = Cytoplasm, N = Nucleus, CSK = cytoskeleton.</p><p>Functional categories and sub-cellular localizations of proteins identified in the study based on the MitoMiner Database and Nextprot.</p