Generation of APOE knock-down SK-N-SH human neuroblastoma cells using CRISPR/Cas9: a novel cellular model relevant to Alzheimer’s disease research

APOE ε4 is the major genetic risk factor for Alzheimer’s disease (AD). A precise role for apolipoprotein E (apoE) in the pathogenesis of the disease remains unclear in part due to its expression in multiple cell types of the brain. APOE is highly expressed in astrocytes and microglia, however its expression can also be induced in neurons under various conditions. The neuron-like cell line SK-N-SH is a useful model in the study of the cellular and molecular effects of apoE as it can be differentiated with retinoic acid to express and secrete high levels of apoE and it also shows the same apoE fragmentation patterns observed in the human brain. We previously found that apoE is cleaved into a 25-kDa fragment by high temperature-requirement serine protease A1 (HtrA1) in SK-N-SH cells. To further understand the endogenous functions of apoE, we used CRISPR/Cas9 to generate SK-N-SH cell lines with APOE expression knocked-down (KD). APOE KD cells showed lower APOE and HTRA1 expression than parental SK-N-SH cells but no overt differences in neuritogenesis or cell proliferation compared with the CRISPR/Cas9 control cells. This research shows that the loss of apoE and HtrA1 has a negligible effect on neuritogenesis and cell survival in SK-N-SH neuron-like cells.     

HtrA3 is regulated by 15-deoxy-Δ12,14-prostaglandin J2 independently of PPARγ in clear cell renal cell carcinomas

Peroxisome proliferator-activated receptor gamma (PPARγ) ligands have been shown to possess anti-proliferative effects in many types of cancer. In clear cell renal cell carcinoma (CCRCC), the targets involved in these effects are not known. In this study, we demonstrated that, in CCRCC cell lines, the endogenous PPARγ ligand 15-deoxy-Δ12,14-prostaglandin J2 (15dPGJ2) induces the expression, both at the mRNA and the protein levels, of the HtrA3 gene. This gene belongs to the High-Temperature Requirement Factor A family of serine proteases that repress signaling by TGF-β family members and inhibit cell migration. Rosiglitazone or ciglitazone, synthetic PPARγ agonists, did not induce HtrA3 expression, and the PPARγ antagonist GW9662 did not prevent 15dPGJ2 induction, suggesting that the up-regulation of HtrA3 by 15dPGJ2 is independent of PPARγ. The MEK/ERK inhibitor PD98059 dramatically repressed HtrA3 induction. Altogether, these data indicate that 15dPGJ2 is able to stimulate the expression of HtrA3 through an indirect mechanism involving the MEK/ERK pathway but independent of PPARγ. Our results provide a better understanding of the mechanisms involved in the regulation of HtrA3, a potential tumor suppressor gene.     

Design and synthesis of new substrates of HtrA2 protease

HtrA2 belongs to the HtrA (high temperature requirement A) family of ATP-independent serine proteases. The primary function of HtrA2 includes maintaining the mitochondria homeostasis, cell death (by apoptosis, necrosis, or anoikis), and contribution to the cell signaling. Several recent reports have shown involvement of HtrA2 in development of cancer and neurodegenerative disorders. Here, we describe the profiling of HtrA2 protease substrate specificity via the combinatorial chemistry approach that led to the selection of novel intramolecularly quenched substrates. For all synthesized compounds, the highest HtrA2-mediated hydrolysis efficiency and selectivity among tested HtrA family members was observed for ABZ-Ile-Met-Thr-Abu-Tyr-Met-Phe-Tyr(3-NO₂)-NH₂, which displayed a specificity constant k_cat/K_M value of 14,535 M⁻¹ s⁻¹.     

Osmoregulation of the HtrA (DegP) protease of Escherichia coli: an Hha-H-NS complex represses HtrA expression at low osmolarity

The HtrA protein of Escherichia coli is a heat-shock inducible periplasmic protease, essential for bacterial survival at high temperatures. Expression of htrA gene depends on the alternative factor sigmaE and on the two-component regulatory system Cpx. These regulators systems respond, among others factors, to overproduction of misfolded proteins in the periplasm or to high level synthesis of various extracytoplasmic proteins. We describe in this report the osmoregulation of the expression of htrA gene. Low osmolarity conditions result in htrA repression. We report, as well, the role of the nucleoid associated proteins H-NS and Hha in the repression of htrA expression at low osmolarity.     

The HtrA Protease from Streptococcus pneumoniae Digests Both Denatured Proteins and the Competence-stimulating Peptide

The HtrA protease of Streptococcus pneumoniae functions both in a general stress response role and as an error sensor that specifically represses genetic competence when the overall level of biosynthetic errors in cellular proteins is low. However, the mechanism through which HtrA inhibits development of competence has been unknown. We found that HtrA digested the pneumococcal competence-stimulating peptide (CSP) and constituted the primary extracytoplasmic CSP-degrading activity in cultures of S. pneumoniae. Mass spectrometry demonstrated that cleavage predominantly followed residue Phe-8 of the CSP-1 isoform of the peptide within its central hydrophobic patch, and in competition assays, both CSP-1 and CSP-2 interacted with HtrA with similar efficiencies. More generally, analysis of β-casein digestion and of digestion within HtrA itself revealed a preference for substrates with non-polar residues at the P1 site. Consistent with a specificity for exposed hydrophobic residues, competition from native BSA only weakly inhibited digestion of CSP, but denaturation converted BSA into a strong competitive inhibitor of such proteolysis. Together these findings support a model in which digestion of CSP by HtrA is reduced in the presence of other unfolded proteins that serve as alternative targets for degradation. Such competition may provide a mechanism by which HtrA functions in a quality control capacity to monitor the frequency of biosynthetic errors that result in protein misfolding.     

Nur77 在缺氧/ 复氧诱导的心肌细胞凋亡中的作用及其机制的研究

目的:观察Nur77通过线粒体转位对缺氧/复氧(H/R)诱导的心肌细胞凋亡的影响。方法:原代培养l-2天SD大鼠心肌细胞,建立H/R模型。随机分为正常对照组、H/R组、Nur77组,采用免疫荧光检测横纹肌肌动蛋白(α-actin)鉴定心肌细胞;采用TUNEL染色法及Caspase-3酶活性检测心肌细胞凋亡情况;采用Western blot检测细胞核及线粒体Nur77蛋白表达、线粒体及胞浆Omi/HtrA2蛋白表达。结果:H/R组细胞核中Nur77蛋白表达明显低于正常对照组;而在线粒体中则相反。Nur77组线粒体中的Omi/HtrA2蛋白表达明显低于正常对照组;而在胞浆中则相反。结论:在心肌细胞H/R损伤时,Nur77线粒体转位促使Omi/HtrA2蛋白从线粒体释放入胞浆,从而导致心肌细胞凋亡。     

Helicobacter pylori HtrA is a new secreted virulence factor that cleaves E‐cadherin to disrupt intercellular adhesion

Mammalian and prokaryotic high‐temperature requirement A (HtrA) proteins are chaperones and serine proteases with important roles in protein quality control. Here, we describe an entirely new function of HtrA and identify it as a new secreted virulence factor from Helicobacter pylori, which cleaves the ectodomain of the cell‐adhesion protein E‐cadherin. E‐cadherin shedding disrupts epithelial barrier functions allowing H. pylori designed to access the intercellular space. We then designed a small‐molecule inhibitor that efficiently blocks HtrA activity, E‐cadherin cleavage and intercellular entry of H. pylori.     

The structural basis of mode of activation and functional diversity: a case study with HtrA family of serine proteases

HtrA (High temperature requirement protease A) proteins that are primarily involved in protein quality control belong to a family of serine proteases conserved from bacteria to humans. HtrAs are oligomeric proteins that share a common trimeric pyramidal architecture where each monomer comprises a serine protease domain and one or two PDZ domains. Although the overall structural integrity is well maintained and they exhibit similar mechanism of activation, subtle conformational changes and structural plasticity especially in the flexible loop regions and domain interfaces lead to differences in their active site conformation and hence in their specificity and functions.     

Syk activation of phosphatidylinositol 3-kinase/Akt prevents HtrA2-dependent loss of X-linked inhibitor of apoptosis protein (XIAP) to promote survival of Epstein-Barr virus+ (EBV+) B cell lymphomas

B cell lymphoma survival requires tonic or ligand-independent signals through activation of Syk by the B cell receptor. The Epstein-Barr virus (EBV) protein latent membrane 2a (LMP2a), a mimic of the B cell receptor, provides constitutive survival signals for latently infected cells through Syk activation; however, the precise downstream mechanisms coordinating this survival response in EBV+ B cell lymphomas remain to be elucidated. Herein, we assess the mechanism of Syk survival signaling in EBV+ B cell lymphomas from post-transplant lymphoproliferative disorder (PTLD) to discover virally controlled therapeutic targets involved in lymphomagenesis and tumor progression. Using small molecule inhibition and siRNA strategies, we show that Syk inhibition reduces proliferation and induces apoptosis of PTLD-derived EBV+ B cell lines. Syk inhibition also reduces autocrine IL-10 production. Although Syk inhibition attenuates signaling through both the PI3K/Akt and Erk pathways, only PI3K/Akt inhibition causes apoptosis of PTLD-derived cell lines. Loss of the endogenous caspase inhibitor XIAP is observed after Syk or PI3K/Akt inhibition. The loss of XIAP and apoptosis that results from Syk or PI3K/Akt inhibition is reversed by inhibition of the mitochondrial protease HtrA2. Thus, Syk drives EBV+ B cell lymphoma survival through PI3K/Akt activation, which prevents the HtrA2-dependent loss of XIAP. Syk, Akt, and XIAP antagonists may present potential new therapeutic strategies for PTLD through targeting of EBV-driven survival signals.