Cell-penetrating peptides with intracellular organelle targeting

Introduction: One of the major limiting steps in order to have an effective drug is the passage through one or more cell membranes to reach its site of action. To reach the action-site, the specific macromolecules are required to be delivered specifically to the cell compartment/organelle in their (pre)active form.

Areas covered: In this review, we will discuss cell-penetrating peptides (CPPs) developed in the last decade to transport small RNA/DNA, plasmids, antibodies, and nanoparticles into specific sites of the cell. The article describes CPPs in complex with cargo molecules that target specific intracellular organelles and their potential for pharmacological or clinical use.

Expert opinion: Organelle targeting is the ultimate goal to ensure selective delivery to the site of action in the cells. CPP technologies represent an important strategy to address drug delivery to specific intracellular compartments by covalent conjugation to targeting sequences, potentially enabling strategies to combat genomic diseases as well as infections, cancer, neurodegenerative and hereditary diseases. They have proven to be successful in delivering various therapeutic agents into cells however, further in vivo experiments and clinical trials are required to demonstrate the efficacy of this technology.     

Endoplasmic reticulum stress participates in uric acid - induced phenotypic transformation of renal tubular epithelial cells

Objective To explore the effect of endoplasmic reticulum stress (ER stress) in uric acid-induced phenotypic change in renal tubular epithelial cells (HK-2). Methods (1) HK-2 cells were cultured with 0, 75, 150, 225, 300 mg/L uric acid for 24 h in vitro. (2) The cells were divided into normal control group, ER stress inhibitor 4-PBA (5 μmol/L) group, uric acid (150 mg/L) group and 4-PBA+uric acid group for 24 h. Morphological changes of HK-2 cells were observed under inverted microscope. MTT assay was used to detect the proliferation of HK-2 cells treated with 150 mg/L uric acid for 24, 48 and 72 h. The protein expressions of α-smooth muscle actin (α-SMA), vimentin, snail, glucose regulated protein 78 (GRP78) and the phosphorylation of eukaryotic initiation factor 2α (p-eIF2α) in HK-2 cells were measured by Western blotting. Results Compared with the control group, HK-2 cells in uric acid groups (150, 225, 300 mg/L) showed fibroblast-like appearance. The protein expressions of α-SMA, vimentin, snail, GRP78 and p-eIF2α in 150 mg/L and 225 mg/L uric acid groups were higher than those in the control group (all P＜0.05). The proliferation of HK-2 cells in 150 mg/L uric acid group was lower than that in control group at 48 and 72 h (all P＜0.01). Compared with the uric acid group, the cell morphology in 4-PBA+uric acid group was improved, and the protein expressions of α-SMA, vimentin, snail, GRP78 and p-eIF2α were decreased (all P＜0.05). Conclusions Uric acid may induce the phenotype transformation of renal tubular epithelial cell, and ER stress is involved. 4-PBA may inhibit the uric acid-induced ER stress response and phenotypic transformation, and may be beneficial in attenuating uric acid-induced renal tubular damage.     

Role of PERK/eIF2α/CHOP Endoplasmic Reticulum Stress Pathway in Oxidized Low-density Lipoprotein Mediated Induction of Endothelial Apoptosis

Objective PERK/eI F2α/CHOP is a major signaling pathway mediating endoplasmic reticulum(ER) stress related with atherosclerosis.Oxidized LDL(ox-LDL) also induces endothelial apoptosis and plays a vital role in the initiation and progression of atherosclerosis.The present study was conducted to explore the regulatory effect of ox-LDL on PERK/e IF2α/CHOP signaling pathway in vascular endothelial cells.Methods The effects of ox-LDL on PERK and p-e IF2α protein expression of primary human umbilical vein endothelial cells(HUVECs) were investigated by Western blot analysis.PERK gene silencing and selective eI F2α phosphatase inhibitor,salubrinal were used to inhibit the process of ox-LDL induced endothelial cell apoptosis,caspase-3 activity,and CHOP mR NA level.Results Ox-LDL treatment significantly increased the expression of PERK,PERK-mediated inactivation of e IF2α phosphorylation,and the expression of CHOP,as well as the caspase-3 activity and apoptosis.The effects of ox-LDL were markedly decreased by knocking down PERK with stable transduction of lentiviral sh RNA or by selective eI F2α phosphatase inhibitor,salubrinal.Conclusion This study provides the first evidence that ox-LDL induces apoptosis in vascular endothelial cells mediated largely via the PERK/eI F2α/CHOP ER-stress pathway.It adds new insights into the molecular mechanisms underlying the pathogenesis and progression of atherosclerosis.     

The effects of isoproterenol on abnormal electrical and contractile activity and diastolic calcium are attenuated in myocytes from aged Fischer 344 rats

We investigated whether the age-related decrease in sensitivity of the heart to catecholamines was accompanied by changes in Ca(2+) homeostasis and abnormal electrical and contractile activity caused by beta-adrenergic receptor (beta-AR) stimulation. Ventricular myocytes were isolated from young adult (3 months) and aged (24 months) male Fischer 344 rats. Unloaded cell shortening was measured in field-stimulated myocytes (2Hz, 37 degrees C); membrane currents and action potentials were measured with microelectrodes. Contractile responses to the non-selective beta-AR agonist, isoproterenol were significantly decreased in aged myocytes compared to younger myocytes and aged myocytes were less sensitive to isoproterenol. In contrast, Ca(2+) transients measured simultaneously with contractions were similar between groups. Isoproterenol increased sarcoplasmic reticulum Ca(2+) stores in both groups, but the increase was larger in aged cells. However, signs of Ca(2+) overload induced by isoproterenol were reduced with age. Diastolic Ca(2+) accumulation, contracture and the incidences of transient inward current, oscillatory afterpotentials (OAPs), aftertransients and aftercontractions induced by isoproterenol also were reduced with age. These results demonstrate that aged myocytes exhibit fewer signs of Ca(2+) overload in response to isoproterenol than young adult myocytes. These age-related changes in intracellular Ca(2+) may protect the aging heart against induction of arrhythmias initiated by OAPs.(1).     

Membrane insertion and topology of the p7B movement protein of Melon Necrotic Spot Virus (MNSV)

Cell-to-cell movement of the Melon Necrotic Spot Virus (MNSV) is controlled by two small proteins working in trans, an RNA-binding protein (p7A) and an integral membrane protein (p7B) separated by an amber stop codon. p7B contains a single hydrophobic region. Membrane integration of this region was observed when inserted into model proteins in the presence of microsomal membranes. Furthermore, we explored the topology and targeting mechanisms of full-length p7B. Here we present evidence that p7B integrates in vitro into the ER membrane cotranslationally and with an Nt-cytoplasmic/Ct-luminal orientation. The observed topology was monitored in vivo by fusing GFP to the Ct of p7B, enabling the overexpression in Escherichia coli cultures. Finally, the topology of a putative p14 movement protein was established by replacing the amber stop codon located between p7A and p7B.     

Novel approach to real-time flash photolysis and confocal [Ca2+] imaging

Flash photolysis of “caged” compounds using ultraviolet light is a powerful experimental technique for producing rapid changes in concentrations of bioactive signaling molecules. Studies that employ this technique have used diverse strategies for controlling the spatial and temporal application of light to the specimen. In this paper, we describe a new system for flash photolysis that delivers light from a pulsed, adjustable intensity laser through an optical fiber coupled into the epifluorescence port of a commercial confocal microscope. Photolysis is achieved with extremely brief (5 ns) pulses of ultraviolet light (355 nm) that can be synchronized with respect to confocal laser scanning. The system described also localizes the UV intensity spatially so that uncaging only occurs in defined subcellular regions; moreover, because the microscope optics are used in localization, the photolysis volume can be easily adjusted. Experiments performed on rat ventricular myocytes loaded with the Ca²⁺ indicator fluo-3 and the Ca²⁺ cage o-nitrophenyl ethylene glycol bis(2-aminoethyl ether)-N,N,N′N′-tetraacetic acid (NP–EGTA) demonstrate the system’s capabilities. Localized intracellular increases in [Ca²⁺] can trigger sarcoplasmic reticular Ca²⁺ release events such as Ca²⁺ sparks and, under certain conditions, regenerative Ca²⁺ waves. This relatively simple and inexpensive system is, therefore, a useful tool for examining local signaling in the heart and other tissues.     

内源性抗氧化剂PRDX4及其在生殖系统作用的研究进展

过氧化物还原酶4（PRDX4）是过氧化物还原酶家族成员,是一种细胞内源性抗氧化剂,可锚定在内质网调节蛋白质氧化折叠,也可分泌到细胞外基质发生抗氧化作用。在女（雌）性生殖系统,氧化应激影响卵巢的功能状态,定位在颗粒细胞及卵母细胞的体细胞型PRDX4（PRDX4s）通过抗氧化应激机制促进卵泡发育成熟、抑制卵母细胞老化,并参与多囊卵巢综合征（PCOS）和卵巢功能减退的病理过程。在睾丸组织内同时表达两种PRDX4亚型,PRDX4s和睾丸特异型PRDX4（PRDX4t）。PRDX4通过抑制内质网应激、清除胞质和细胞外基质活性氧簇（ROS）保护睾丸组织结构与细胞功能,参与调节精子发生和精子形成,抑制生精细胞凋亡。综述PRDX4的结构与功能,及其在调节配子发生中的作用。     

Thymoquinone protects rat liver after partial hepatectomy under ischaemia/reperfusion through oxidative stress and endoplasmic reticulum stress prevention

Ischaemia reperfusion (I/R) is associated with liver injury and impaired regeneration during partial hepatectomy (PH). The aim of this study was to investigate the effect of thymoquinone (TQ), the active compound of essential oil obtained from Nigella sativa seeds, on rat liver after PH. Male Wistar rats were divided equally into four groups (n = 6) receiving an oral administration of either vehicle solution (sham and PH groups) or TQ at 30 mg/kg (TQ and TQ + PH groups) for 10 consecutive days. Then, rats underwent PH (70%) with 60 minutes of ischaemia followed by 24 hours of reperfusion (PH and TQ + PH groups). Alanine aminotransferase (ALT) activity and histopathological damage were determined. Also, antioxidant parameters, liver regeneration index, hepatic adenosine triphosphate (ATP) content, endoplasmic reticulum (ER) stress and apoptosis were assessed. In response to PH under I/R, liver damage was significantly alleviated by TQ treatment as evidenced by the decrease in ALT activity (P < .01) and histological findings (P < .001). In parallel, TQ preconditioning increased hepatic antioxidant capacities. Moreover, TQ improved mitochondrial function (ATP, P < .05), attenuated ER stress parameters and repressed the expression of apoptotic effectors. Taken together, our results suggest that TQ preconditioning could be an effective strategy to reduce liver injury after PH under I/R. The protective effects were mediated by the increase of antioxidant capacities and the decrease of ER stress and apoptosis.     

非酒精性脂肪肝与钙稳态关系的研究进展

钙离子（Ca²⁺）是细胞内重要的第二信使,其主要储存在内质网和线粒体中,参与调控细胞多种生理功能及维持内质网、线粒体功能。内质网和线粒体对钙离子的摄取与释放直接影响胞内钙离子水平的变化,继而影响细胞正常生理功能。病理条件下,细胞内钙离子稳态失衡,可引发细胞生理功能异常并进一步影响内质网、线粒体功能。非酒精性脂肪肝（NAFLD）是临床常见病和多发病,其主要病理特征是：肝脏脂肪样变性、内质网应激、线粒体损伤和非特异性炎性反应等。其中持续的内质网应激及线粒体功能障碍是NAFLD发生发展的重要诱因。而细胞内钙稳态的变化可直接导致内质网及线粒体功能异常,继而影响NAFLD的发生发展。本文主要从钙稳态的主要影响因素以及钙稳态变化与NAFLD的关系两方面进行阐述,为寻求和研发防治NAFLD的药物提供新的方向。