Characterization of a Novel Subtilisin-like Protease Myroicolsin from Deep Sea Bacterium Myroides profundi D25 and Molecular Insight into Its Collagenolytic Mechanism

Collagen is an insoluble protein that widely distributes in the extracellular matrix of marine animals. Collagen degradation is an important step in the marine nitrogen cycle. However, the mechanism of marine collagen degradation is still largely unknown. Here, a novel subtilisin-like collagenolytic protease, myroicolsin, which is secreted by the deep sea bacterium Myroides profundi D25, was purified and characterized, and its collagenolytic mechanism was studied. Myroicolsin displays low identity (<30%) to previously characterized subtilisin-like proteases, and it contains a novel domain structure. Protein truncation indicated that the Pro secretion system C-terminal sorting domain in the precursor protein is involved in the cleavage of the N-propeptide, and the linker is required for protein folding during myroicolsin maturation. The C-terminal β-jelly roll domain did not bind insoluble collagen fiber, suggesting that myroicolsin may degrade collagen without the assistance of a collagen-binding domain. Myroicolsin had broad specificity for various collagens, especially fish-insoluble collagen. The favored residue at the P1 site was basic arginine. Scanning electron microscopy and atomic force microscopy, together with biochemical analyses, confirmed that collagen fiber degradation by myroicolsin begins with the hydrolysis of proteoglycans and telopeptides in collagen fibers and fibrils. Myroicolsin showed strikingly different cleavage patterns between native and denatured collagens. A collagen degradation model of myroicolsin was proposed based on our results. Our study provides molecular insight into the collagen degradation mechanism and structural characterization of a subtilisin-like collagenolytic protease secreted by a deep sea bacterium, shedding light on the degradation mechanism of deep sea sedimentary organic nitrogen.     

Suppression of PPARγ through MKRN1-mediated ubiquitination and degradation prevents adipocyte differentiation

The central regulator of adipogenesis, PPARγ, is a nuclear receptor that is linked to obesity and metabolic diseases. Here we report that MKRN1 is an E3 ligase of PPARγ that induces its ubiquitination, followed by proteasome-dependent degradation. Furthermore, we identified two lysine sites at 184 and 185 that appear to be targeted for ubiquitination by MKRN1. Stable overexpression of MKRN1 reduced PPARγ protein levels and suppressed adipocyte differentiation in 3T3-L1 and C3H10T1/2 cells. In contrast, MKRN1 depletion stimulated adipocyte differentiation in these cells. Finally, MKRN1 knockout MEFs showed an increased capacity for adipocyte differentiation compared with wild-type MEFs, with a concomitant increase of PPARγ and adipogenic markers. Together, these data indicate that MKRN1 is an elusive PPARγ E3 ligase that targets PPARγ for proteasomal degradation by ubiquitin-dependent pathways, and further depict MKRN1 as a novel target for diseases involving PPARγ.     

Good correlation between changes in objective and subjective signs of inflammation in patients with short- but not long duration of axial spondyloarthritis treated with tumor necrosis factor-blockers

Introduction

The aim of this study was to investigate the influence of symptom duration on treatment response and on the correlation between improvements in patient reported outcomes (PRO) and objective inflammation in patients with axial spondylarthritis (SpA) treated with etanercept (ETA) or adalimumab (ADA).

Methods

Data from 112 patients with axial SpA originally enrolled in two randomized controlled clinical trials were pooled and analyzed after one year of treatment with ETA (n = 66) or ADA (n = 46). Patients with <4 years and ≥4 years of disease were compared for improvement in Bath Ankylosing Spondylitis Disease Activity Index (BASDAI), Bath Ankylosing Spondylitis Functional Index (BASFI), Ankylosing Spondylitis Disease Activity Score (ASDAS), C-reactive protein (CRP) and magnetic resonance imaging (MRI) score for sacroiliac joints (SIJ).

Results

Patients with <4 years of disease showed a significantly better improvement than longer diseased patients in BASDAI (3.2 (95% confidence interval (CI): 2.7 to 3.7) vs. 1.7 (1.1 to 2.2)), BASFI, BASMI and ASDAS (1.6 (1.4 to 1.8) vs. 0.9 (0.7 to 1.1)). The change in BASDAI showed a significant correlation with the change in SIJ score (Spearman’s rank correlation coefficient (rho) = 0.37, P = 0.01) and the change in CRP (rho = 0.45, P = 0.001) in patients with <4 years of disease. For long diseased patients this correlation was poor and did not achieve statistical significance (rho = 0.13, P = 0.46; rho = 0.22, P = 0.13 respectively).

Conclusion

The low correlation between change of PROs and change of objective signs of inflammation seen in axial SpA patients with longer symptom duration treated with tumor necrosis factor-blocker seems to indicate that inflammation is not the only cause of the patients’ symptoms, while inflammation seems to be the major cause in short diseased patients.

Trial registration

Clinical Trials.gov NCT00844142 (Trial 1); NCT00235105 (Trial 2)     

Modification of Sialylation Mediates the Invasive Properties and Chemosensitivity of Human Hepatocellular Carcinoma

Aberrant sialylation is closely associated with malignant phenotypes of tumor cells, including invasiveness and metastasis. This study investigated sialylation with regard to the modification of invasive properties and chemosensitivity in human hepatocellular carcinoma (HCC) cell lines and the association between the sialyltransferase gene family and clinicopathological characteristics in HCC patients. Using mass spectrometry analysis, we found that the composition profiling of sialylated N-glycans differed between MHCC97H and MHCC97L cells with different metastatic potential. The expressional profiles of 20 sialyltransferase genes showed differential expression in two cell lines, transitional and tumor tissues, from the same patients. Two genes, ST6GAL1 and ST8SIA2, were detected as overexpressed in MHCC97H and MHCC97L cells. The altered expression levels of ST6GAL1 and ST8SIA2 corresponded to a changed invasive phenotype and chemosensitivity of MHCC97H and MHCC97L cells both in vitro and in vivo. Further data indicated that manipulation of the expression of the two genes led to altered activity of the phosphoinositide-3 kinase (PI3K)/Akt signaling pathway. Targeting the PI3K/Akt pathway by its specific inhibitor wortmannin or by Akt RNA interference resulted in a reduced capacity for invasion and chemoresistance of MHCC97H cells. Our results imply that sialylation may function as an internal factor, regulating the invasion and chemosensitivity of HCC, probably through ST6GAL1 or ST8SIA2 regulation of the activity of the PI3K/Akt pathway.Metastasis of tumor cells and the development of resistance to antitumor therapies are the major causes of death in cancer patients. Specific changes in the glycosylation patterns of cell surface glycoproteins have been shown to enhance the metastatic efficiency of tumor cells, in particular that of terminal sialylation (1). It is well known that alterations in cell surface sialylated antigens affect many cellular properties—for example, cell–cell adhesion, cell–extracellular matrix adhesion, immune defense, cell metastasis, and invasion abilities (2–5). Sialyltransferases catalyze the transfer of sialic acid from cytidine 5′-monophospho-N-acetylneuraminic acid to terminal positions of glycoprotein and glycolipid carbohydrate groups (6).The sialyltransferase (ST)¹ family is a family of anabolic enzymes consisting of 20 members that are divided into three subfamilies (7). α-2,3-sialyltransferases mediate the transfer of sialic acid with an α-2,3-linkage to it with terminal Gal residues (ST3GalI-VI). α-2,6-sialyltransferases mediate the transfer of sialic acid with an α-2,6-linkage to it with terminal Gal (ST6GalI-II) (8, 9) or GalNAc residues (ST6GalNAcI-VI). α-2,8-sialyltransferases mediate the transfer of sialic acid with an α-2,8-linkage (ST8SiaI-VI). Changes in specific sialyltransferase expression in several tumors have been reported. ST3GalIII modulates pancreatic cancer cell motility and adhesion in vitro and enhances its metastatic potential in vivo (10). The high expression of ST3GalIV is associated with the malignant behavior of gastric cancer cells (11). ST6GalI is up-regulated in colon adenocarcinoma, and its expression is positively correlated with tumor cell invasiveness and metastasis (12–14). ST6GalNAcI expression is sufficient to enhance the tumorigenicity of MDA-MB-231 breast cancer cells (15). Overexpression of ST6GalNAcII has been correlated with poor patient survival (16). ST6GalNAcV has recently been reported to mediate brain metastasis of breast cancer cells (17). ST8Sia I is also overexpressed in neuroectoderm-derived malignant tumors such as melanoma, glioblastoma, and neuroblastoma, as well as in estrogen receptor negative breast cancer, where it plays a role in cell proliferation, migration, adhesion, and angiogenesis (18).The phosphoinositide 3 kinase (PI3K)/Akt pathway is involved in many cellular processes, including proliferation, differentiation, apoptosis, cell cycle progression, cell motility, tumorigenesis, tumor growth, and angiogenesis (19, 20). In addition, several reports highlight that the PI3K/Akt pathway is responsible for the proliferation, invasion, metastasis, and drug resistance of hepatocellular carcinoma (HCC), and targeting PI3K/AKT inhibits the proliferation and tumorigenesis of HCC cells (21, 22). MicroRNA-7 plays a substantial role in inhibiting the tumorigenesis and reversing the metastasis of HCC through the PI3K/Akt/mTOR signaling pathway in vitro and in vivo (23). The proliferation and invasion of HCC cells are inhibited by lipocalin 2 through the blockade of PI3K/Akt signaling (24). Activation of the PI3K/Akt pathway mediates rapamycin and sorafenib resistance in HCC cells (25, 26). However, little is known about the ST family and its signaling pathway in relation to malignant phenotypes of human HCC.Therefore, the aims of the present study were to determine sialylated oligosaccharide alteration and expression levels of ST genes among the MHCC97H and MHCC97L cell lines and HCC patient cells by using MS and real-time PCR. In addition, we investigated whether the ST gene family participates in the regulation of tumor invasion and chemosensitivity via the PI3K/Akt pathway and the possible mechanisms.     

A Novel Newborn Rat Kernicterus Model Created by Injecting a Bilirubin Solution into the Cisterna Magna

Background

Kernicterus still occurs around the world; however, the mechanism of bilirubin neurotoxicity remains unclear, and effective treatment strategies are lacking. To solve these problems, several kernicterus (or acute bilirubin encephalopathy) animal models have been established, but these models are difficult and expensive. Therefore, the present study was performed to establish a novel kernicterus model that is simple and affordable by injecting unconjugated bilirubin solution into the cisterna magna (CM) of ordinary newborn Sprague-Dawley (SD) rats.

Methods

On postnatal day 5, SD rat pups were randomly divided into bilirubin and control groups. Then, either bilirubin solution or ddH₂O (pH = 8.5) was injected into the CM at 10 µg/g (bodyweight). For model characterization, neurobehavioral outcomes were observed, mortality was calculated, and bodyweight was recorded after bilirubin injection and weaning. Apoptosis in the hippocampus was detected by H&E staining, TUNEL, flow cytometry and Western blotting. When the rats were 28 days old, learning and memory ability were evaluated using the Morris water maze test.

Results

The bilirubin-treated rats showed apparently abnormal neurological manifestations, such as clenched fists, opisthotonos and torsion spasms. Bodyweight gain in the bilirubin-treated rats was significantly lower than that in the controls (P<0.001). The early and late mortality of the bilirubin-treated rats were both dramatically higher than those of the controls (P = 0.004 and 0.017, respectively). Apoptosis and necrosis in the hippocampal nerve cells in the bilirubin-treated rats were observed. The bilirubin-treated rats performed worse than the controls on the Morris water maze test.

Conclusion

By injecting bilirubin into the CM, we successfully created a new kernicterus model using ordinary SD rats; the model mimics both the acute clinical manifestations and the chronic sequelae. In particular, CM injection is easy to perform; thus, more stable models for follow-up study are available.     

Comparative Genomics and Evolution of Avian Specialized Traits

Genomic data are important for understanding the origin and evolution of traits. Under the context of rapidly developing of sequencing technologies and more widely available genome sequences, researchers are able to study evolutionary mechanisms of traits via comparative genomic methods. Compared with other vertebrates, bird genomes are relatively small and exhibit conserved synteny with few repetitive elements, which makes them suitable for evolutionary studies. Increasing genomic progress has been reported on the evolution of powered flight, body size variation, beak morphology, plumage colouration, high-elevation colonization, migration, and vocalization. By summarizing previous studies, we demonstrate the genetic bases of trait evolution, highlighting the roles of small-scale sequence variation, genomic structural variation, and changes in gene interaction networks. We suggest that future studies should focus on improving the quality of reference genomes, exploring the evolution of regulatory elements and networks, and combining genomic data with morphological, ecological, behavioural, and developmental biology data.     

Developmental partitioning of SYK and ZAP70 prevents autoimmunity and cancer

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TIPE1‐mediated autophagy suppression promotes nasopharyngeal carcinoma cell proliferation via the AMPK/mTOR signalling pathway

Recent studies have shown that tumour necrosis factor‐α–induced protein 8 like‐1(TIPE1) plays distinct roles in different cancers. TIPE1 inhibits tumour proliferation and metastasis in a variety of tumours but acts as an oncogene in cervical cancer. The role of TIPE1 in nasopharyngeal carcinoma (NPC) remains unknown. Interestingly, TIPE1 expression was remarkably increased in NPC tissue samples compared to adjacent normal nasopharyngeal epithelial tissue samples in our study. TIPE1 expression was positively correlated with that of the proliferation marker Ki67 and negatively correlated with patient lifespan. In vitro, TIPE1 inhibited autophagy and induced cell proliferation in TIPE1‐overexpressing CNE‐1 and CNE‐2Z cells. In addition, knocking down TIPE1 expression promoted autophagy and decreased proliferation, whereas overexpressing TIPE1 increased the levels of pmTOR, pS6 and P62 and decreased the level of pAMPK and the LC3B. Furthermore, the decrease in autophagy was remarkably rescued in TIPE1‐overexpressing CNE‐1 and CNE‐2Z cells treated with the AMPK activator AICAR. In addition, TIPE1 promoted tumour growth in BALB/c nude mice. Taken together, results indicate that TIPE1 promotes NPC progression by inhibiting autophagy and inducing cell proliferation via the AMPK/mTOR signalling pathway. Thus, TIPE1 could potentially be used as a valuable diagnostic and prognostic biomarker for NPC.     

Streptomyces luteolifulvus sp. nov., a novel actinomycete isolated from soil in Nanjing,China

During the investigation of exploring potential sources of novel species and natural bioactives, a novel actinomycete, designated strain HIT-DPA4^T, was isolated from a soil sample, which was collected from Nanjing, Jiangsu Province, PR China and characterized using a polyphasic approach. On the basis of 16S rRNA gene sequence similarities and the result of phylogenetic analysis, strain HIT-DPA4^T was most closely related to Streptomyces cyaneus CGMCC 4.1671 ^T, and shared the highest sequence similarity of 98.76%. In addition, the cell walls of the species HIT-DPA4^T contained LL-diaminopimelic acid as the diagnostic diamino acid and the whole-cell hydrolysates were identified as glucose and ribose, and the principal phospholipids were found to be diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol mannoside and phosphatidylmonomethylethanolamine. MK-9(H₆) and MK-9(H₄) were predominant menaquinones; and C_16:0, anteiso-C_15:0 and C_15:0 as major cellular fatty acids of the organism HIT-DPA4^T. Gene Ontology database analysis and antiSMASH server predicted results displayed that strain HIT-DPA4^T was a promising classification units, which has various types of functions and contains multiple biosynthetic gene clusters with the similarity more than 80%. Multilocus sequence analysis (MLSA) of five housekeeping genes (atpD, gyrB, recA, rpoB and trpB) illustrated that Streptomyces luteolifulvus formed a separate branch in the genus Streptomyces. However, a combination of low level of DNA-DNA relatedness and physiological properties indicated that strain HIT-DPA4^T can be distinguished from its phylogenetically related species Streptomyces cyaneus CGMCC 4.1671 ^T. Moreover, gene synteny research could be further differed organism HIT-DPA4^T from similarity species. Therefore, the strain is concluded to represent a novel species of the genus Streptomyces, for which the name Streptomyces luteolifulvus sp. nov. is proposed. The type strain is HIT-DPA4^T (=?CGMCC 4.7558 ^T?=?TISTR 2751 ^T).