Endoplasmic Reticulum Targeting Research Articles

Advance in endoplasmic reticulum-targeting nanodrugs

As an extremely important organelle in eukaryotic cells, endoplasmic reticulum (ER) plays a key role in the synthesis and processing of biomacromolecules, material transport, ion homeostasis maintenance, signal transduction, exchange of materials and signals between organelles. Many important human diseases, such as cancers, autoimmune diseases, pathogenic infections, neurodegenerative diseases and diabetes, are closely related to ER dysfunction. With the development of nanotechnology, the exploration and application of ER-targeted nanodrugs gradually become a research hotspot in the field of nanomedicine, bioengineering, material chemistry and other fields. In this paper, the relationship between ER dysfunction and disease occurrence, the principle of designing ER-targeted nanodrugs and their biomedical application are reviewed. ER-targeted nanodrugs are designed based on nanodrug carriers or self-assembly of bioactive molecules. These nanodrugs could target the ER in an active or passive manner and function by disrupting or maintaining the ER functions. The ER-targeting nanodrugs have a wide application prospect in cancer therapy, immune regulation, nervous system repairment, and so on.

Intrabodies targeting human papillomavirus 16 E6 and E7 oncoproteins for therapy of established HPV-associated tumors

BackgroundThe oncogenic activity of the high risk human papillomavirus type 16 (HPV16) is fully dependent on the E6 and E7 viral oncoproteins produced during viral infection. The oncoproteins interfere with cellular homeostasis by promoting proliferation, inhibiting apoptosis and blocking epithelial differentiation, driving the infected cells towards neoplastic progression. The causal relationship between expression of E6/E7 and cellular transformation allows inhibiting the oncogenic process by hindering the activity of the two oncoproteins. We previously developed and characterized some antibodies in single-chain format (scFvs) against the HPV16 E6 and E7 proteins, and demonstrated both in vitro and in vivo their antitumor activity consisting of protective efficacy against tumor progression of HPV16-positive cells.MethodsEnvisioning clinical application of the best characterized anti-HPV16 E6 and –HPV16 E7 scFvs, we verified their activity in the therapeutic setting, on already implanted tumors. Recombinant plasmids expressing the anti-HPV16 E6 scFvI7 with nuclear targeting sequence, or the anti-HPV16 E7 scFv43M2 with endoplasmic reticulum targeting sequence were delivered by injection followed by electroporation to three different preclinical models using C57/BL6 mice, and their effect on tumor growth was investigated. In the first model, the HPV16+ TC-1 Luc cells were used to implant tumors in mice, and tumor growth was measured by luciferase activity; in the second model, a fourfold number of TC-1 cells was used to obtain more aggressively growing tumors; in the third model, the HPV16+ C3 cells where used to rise tumors in mice. To highlight the scFv possible mechanism of action, H&E and caspase-3 staining of tumor section were performed.ResultsWe showed that both the anti-HPV16 E6 and HPV16 E7 scFvs tested were efficacious in delaying tumor progression in the three experimental models and that their antitumor activity seems to rely on driving tumor cells towards the apoptotic pathway.ConclusionBased on our study, two scFvs have been identified that could represent a safe and effective treatment for the therapy of HPV16-associated lesions. The mechanism underlying the scFv effectiveness appears to be leading cells towards death by apoptosis. Furthermore, the validity of electroporation, a methodology allowed for human treatment, to deliver scFvs to tumors was confirmed.

Pathogen Infection-Responsive Nanoplatform Targeting Macrophage Endoplasmic Reticulum for Alleviating Sepsis

Pathogen-induced sepsis represents the main cause of infection-related death, and is becoming a great threat to human health. In this study, we developed a pathogen infection-responsive and macrophage endoplasmic reticulum-targeting nanoplatform to alleviate sepsis. The nanoplatform is composed of large-pore mesoporous silica nanoparticles (MSNs) grafted by an endoplasmic reticulum-targeting peptide (ERP), and a pathogen infection-responsive cap (TPB) containing the reactive oxygen species (ROS)-cleavable boronobenzyl acid linker (TSPBA) and bovine serum albumin (BSA). The TPB-capped MSNs exhibited the capacity to highly load the antimicrobial peptide melittin (MEL), and to rapidly release the cargo triggered by H2O2 or the pathogen-macrophage interaction system. During the interaction between pathogenic C. albicans cells and macrophages, the MEL-loading nanoplatform MSNE+MEL+TPB strongly inhibited pathogen growth, survived macrophages, and suppressed endoplasmic reticulum stress and secretion of pro-inflammatory cytokines. In a systemic infection model, the nanoplatform efficiently protected the mice from death, prevented kidney dysfunction and alleviated sepsis symptoms. This study developed an efficient multifunctional nanoplatform for treatment of sepsis. Funding Information: This work was supported by National Natural Science Foundation of China (3217010793, 31870139), Natural Science Foundation of Tianjin (19JCZDJC33800), and Tianjin Synthetic Biotechnology Innovation Capacity Improvement Project (TSBICIP-KJGG-006). Declaration of Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Ethics Approval Statement: All animal experiments were approved by the Animal Care and Use Committee at Nankai University.

A solvent-free and efficient synthesis of bicyclic 2-pyridone derivatives for endoplasmic reticulum imaging

A solvent-free method was developed for the synthesis of bicyclic 2-pyridone (DHIP) derivatives, which demonstrated excellent endoplasmic reticulum (ER) targeting and antibacterial activity after a slight structure regulation.

The development of an endoplasmic reticulum-targeting fluorescent probe for the imaging of 1,4-dithiothreitol (DTT) in living cells.

1,4-Dithiothreitol (DTT) is a robust reducing agent that contributes significantly to the folding process of proteins and maintaining endoplasmic reticulum (ER) homeostasis. Abnormally high levels of DTT can lead to severe endoplasmic reticulum stress (ERS), which induces cell death. In addition, DTT can also hinder cell growth and enhance reactive oxygen species (ROS) production in the ER. Herein, an effective turn-on ER-targeting fluorescent probe, ER-DTT, was designed to image DTT for the first time. The probe ER-DTT was based upon naphthalimide as a fluorophore, p-toluenesulfonamide as an exceptional unit for ER-targeting, and sulfoxide as a response site for imaging DTT based on an intramolecular charge transfer (ICT) mechanism. Optical-response experiments showed that the probe ER-DTT had good selectivity and sensitivity for DTT. Furthermore, confocal microscopy indicated that ER-DTT was suitable for selectively targeting ER in living cells and could be implemented to recognize cellular DTT.

An endoplasmic reticulum-targeting fluorescent probe for discriminatory detection of Cys, Hcy and GSH in living cells

Endoplasmic reticulum, known to us as the ER, is the largest organelle in many kinds of eukaryotic cells and plays vital roles in maintaining the normal function of cells. Biothiols (Cys, Hcy, GSH) in secretory proteins will be modified as they enter the ER and are of great importance in balancing redox state of ER environments. In this article, we have developed the first endoplasmic reticulum-targeting fluorescent probe, ER-CP, for concurrent distinguishment of Cys, Hcy and GSH with favorable sensitivity and selectivity. ER-CP was successfully used in fluorescence imaging of Cys, Hcy and GSH in HeLa cells. In addition, ER-CP exhibited a good ER-targeting property (Pearson’s correlation coefficient = 0.90).

Endoplasmic Reticulum Targeting Reactive Oxygen Species Sensor Based on Dihydrofluorescein: Application of Endoplasmic Reticulum Stress

Endoplasmic reticulum (ER) has a unique redox environment, which plays critical roles in the organelle's function and in its pathological responses such as ER stress. In this work, we introduce an ER‐targeting fluorogenic reactive oxygen species (ROS) chemosensor (ER‐Flu) from copper(I)‐catalyzed alkyne‐azide cycloaddition of 3‐propargyl ester of 2′,7′‐dichlorodihydrofluorescein diacetate and N3‐glibenclamide, which were adopted as a fluorogenic ROS sensing module and an ER‐targeting module, respectively. Thereby, a series of confocal microscopic experiments of ER‐Flu demonstrated that the sensor localizes in ER of the live cells and that ROS are elevated in the cells by ER stress inducers such as thapsigargin, brefeldin A, and tunicamycin.

Secretory autophagy machinery and vesicular trafficking are involved in HMGB1 secretion

ABSTRACT Nuclear protein HMGB1 is secreted in response to various stimuli and functions as a danger-associated molecular pattern. Extracellular HMGB1 induces inflammation, cytokine production, and immune cell recruitment via activation of various receptors. As HMGB1 does not contain an endoplasmic reticulum-targeting signal peptide, HMGB1 is secreted via the endoplasmic reticulum-Golgi independently via an unconventional secretion pathway. However, the mechanism underlying HMGB1 secretion remains largely unknown. Here, we investigated the role of secretory autophagy machinery and vesicular trafficking in HMGB1 secretion. We observed that HSP90AA1 (heat shock protein 90 alpha family class A member 1), a stress-inducible protein, regulates the translocation of HMGB1 from the nucleus to the cytoplasm and its secretion through direct interaction. Additionally, geldanamycin, an HSP90AA1 inhibitor, reduced HMGB1 secretion. GORASP2/GRASP55 (golgi reassembly stacking protein 2), ARF1Q71L (ADP ribosylation factor 1), and SAR1AT39N (secretion associated Ras related GTPase 1A), which promoted unconventional protein secretion, increased HMGB1 secretion. HMGB1 secretion was inhibited by an early autophagy inhibitor and diminished in ATG5-deficient cells even when GORASP2 was overexpressed. In contrast, a late autophagy inhibitor increased HMGB1 secretion under the same conditions. The multivesicular body formation inhibitor GW4869 dramatically decreased HMGB1 secretion under HMGB1 secretion-inducing conditions. Thus, we demonstrated that secretory autophagy and multivesicular body formation mediate HMGB1 secretion.

A two-photon fluorescent probe for visualizing endoplasmic reticulum peroxynitrite in Parkinson's disease models

Endoplasmic reticulum (ER) stress is implicated in the pathogenesis of diseases such as Parkinson's disease (PD). Peroxynitrite is the pivotal causative factor of ER stress. However, fluorescent probes for imaging the peroxynitrite in the ER are still lacking. In the present paper, we reported the design and synthesis of a two-photon fluorescence probe (ER-PN) for visualizing the ER peroxynitrite in PD models of living cells and C. elegans. ER-PN showed a rapidly and highly selective “turn-on” response to peroxynitrite over other biological species in vitro. Moreover, ER-PN showed excellent ER targeting ability and worked well in living systems for the ER peroxynitrite imaging. Noteworthy, ER-PN has been successfully applied for imaging the peroxynitrite in PD models, indicating its potential application for revealing the fundamental roles of peroxynitrite in ER stress-related diseases.

Structural basis for the binding of SNAREs to the multisubunit tethering complex Dsl1

Multisubunit-tethering complexes (MTCs) are large (250 to >750 kDa), conserved macromolecular machines that are essential for soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE)-mediated membrane fusion in all eukaryotes. MTCs are thought to organize membrane trafficking by mediating the initial long-range interaction between a vesicle and its target membrane and promoting the formation of membrane-bridging SNARE complexes. Previously, we reported the structure of the yeast Dsl1 complex, the simplest known MTC, which is essential for coat protein I (COPI) mediated transport from the Golgi to the endoplasmic reticulum (ER). This structure suggests how the Dsl1 complex might tether a vesicle to its target membrane by binding at one end to the COPI coat and at the other to ER-associated SNAREs. Here, we used X-ray crystallography to investigate these Dsl1-SNARE interactions in greater detail. The Dsl1 complex comprises three subunits that together form a two-legged structure with a central hinge. We found that distal regions of each leg bind N-terminal Habc domains of the ER SNAREs Sec20 (a Qb-SNARE) and Use1 (a Qc-SNARE). The observed binding modes appear to anchor the Dsl1 complex to the ER target membrane while simultaneously ensuring that both SNAREs are in open conformations, with their SNARE motifs available for assembly. The proximity of the two SNARE motifs, and therefore their ability to enter the same SNARE complex, will depend on the relative orientation of the two Dsl1 legs. These results underscore the critical roles of SNARE N-terminal domains in mediating interactions with other elements of the vesicle docking and fusion machinery.

An Endoplasmic Reticulum-Targeted Ratiometric Fluorescent Probe for the Sensing of Hydrogen Sulfide in Living Cells and Zebrafish.

Hydrogen sulfide (H2S) is an endogenous gaseous signaling molecule in many physiological processes. Relevant investigations indicated that H2S plays a cytoprotective effect under endoplasmic reticulum stress. Currently, it is still a challenge to design effective methods for ratio detection of endoplasmic reticulum H2S. Herein we are the first to construct a ratiometric near-infrared fluorescent probe (M-H2S) for sensing H2S in the endoplasmic reticulum. M-H2S has high selectivity and sensitivity toward H2S (LOD = 39.1 nM). Additionally, M-H2S possessed an excellent endoplasmic reticulum targeting ability. Moreover, M-H2S was successfully utilized to visualize exogenous/endogenous H2S in HeLa cells and zebrafish. Particularly important, endogenously produced H2S was observed under endoplasmic reticulum stress induced by tunicamycin.

A highly sensitive endoplasmic reticulum-targeting fluorescent probe for the imaging of endogenous H2S in live cells.

Hydrogen sulfide (H2S) as an important signaling biomolecule participates in a series of complex physiological and pathological processes. In situ and rapid detection of H2S levels in endoplasmic reticulum (ER) is of great importance for the in-depth study of its virtual functional roles. However, the ER-targeting fluorescent probe for the detection of H2S in live cells is still quite rare. Herein, a new ER-targeting fluorescent probe (FER-H2S) for detecting H2S in live cells was characterized in the present study. This probe FER-H2S was built from the hybridization of three parts, including fluorescein-based skeleton, p-toluenesulfonamide as ER-specific group, and 2,4-nitrobenzene sulfonate as a response site for H2S. The response mechanism of the probe FER-H2S to H2S is on the basis of the ring-opening and ring-closing processes in fluorescein moiety. Moreover, the probe FER-H2S was successfully used for the imaging of exogenous and endogenous H2S in ER of live cells.

Cleavage of FNDC5 and insights into its maturation process

FNDC5 corresponds to an irisin precursor that increases with exercise. Studies suggest that irisin mediates beneficial effects in adipose tissues, skeletal muscle, bone, and brain. However, the cleavage and maturation processes of FNDC5 have not been clearly identified. This study aimed to show that the signal peptide and transmembrane domain of FNDC5 were associated with the secretion of its ectodomain. Localization studies identified the signal peptide that was responsible for endoplasmic reticulum targeting activity of nascent FNDC5 and showed that the FNDC5 ectodomain corresponding to irisin could be transported across the membrane by a transmembrane domain. Analysis of cleavage constructs revealed that the ectodomain of FNDC5 could be cleaved from its signal peptide and transmembrane attachment. Genetic ablation of the signal peptide cleavage site blocked N-glycosylation of FNDC5. Identification of the FNDC5 maturation process should facilitate our understanding of irisin secretion.

Endoplasmic Reticulum Targeting to Amplify Immunogenic Cell Death for Cancer Immunotherapy.

Immunogenic cell death (ICD) elicited by photodynamic therapy (PDT) is mediated through generation of reactive oxygen species (ROS) that induce endoplasmic reticulum (ER) stress. However, the half-life of ROS is very short and the intracellular diffusion depth is limited, which impairs ER localization and thus limits ER stress induction. To solve the problem, we synthesized reduction-sensitive Ds-sP NPs (PEG-s-s-1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-2000] nanoparticles) loaded with an efficient ER-targeting photosensitizer TCPP-TER (4,4',4″,4'″-(porphyrin-5,10,15,20-tetrayl)tetrakis(N-(2-((4-methylphenyl)sulfonamido)ethyl)benzamide). The resulting Ds-sP/TCPP-TER NPs could selectively accumulate in the ER and locally generate ROS under near-infrared (NIR) laser irradiation, which induced ER stress, amplified ICD, and activated immune cells, leading to augmented immunotherapy effect. This study presents a novel ICD amplifying, ER-targeting PDT strategy that can effectively eradicate primary tumors under NIR exposure, as well as distant tumors through an abscopal effect.

Small-molecule fluorescent dyes based on benzothiazole derivatives for targeting endoplasmic reticulum and tissue imaging

Small-molecule fluorescent dyes with excellent biocompatibility and deep tissue permeability are playing an important part in biological development. Herein, a series of D-A typesmall-molecules L1, L2 and L3 containing different terminal groups have been designed and synthesized, of which the structures were confirmed by single crystal X-ray diffraction analysis. Interestingly, it was found that different end groups within the molecules gave an obvious effect on the fluorescent emission properties while unconspicuous influence on the absorption properties. Moreover, these compounds were successfully applied to fluorescent imaging for staining cellular endoplasmic reticulum (ER) and tissue slice from mouse exhibiting negligible cytotoxicity as well as favourable permeability. These compounds may act as probes for ER targeting and alternative bio-imaging reagents.

An endoplasmic reticulum-targeting and ratiometric fluorescent probe for hypochlorous acid in living cells based on a 1,8-naphthalimide derivative

A novel reticulum-targeting and ratiometric fluorescent probe for determining hypochlorous acid has been developed.

An endoplasmic reticulum-targeting fluorescent probe for imaging ˙OH in living cells.

The hydroxyl radical (˙OH) in the endoplasmic reticulum has not been studied thoroughly due to the lack of proper methods. Here, an endoplasmic reticulum-targeting fluorescent probe for detecting ˙OH is reported. With this probe, the action of ˙OH in the endoplasmic reticulum has been imaged in living cells.

An endoplasmic reticulum-targeted two-photon fluorescent probe for bioimaging of HClO generated during sleep deprivation.

With the development of social society, sleep deprivation has become a serious and common issue. Previous studies documented that there is a correlation between sleep deprivation and oxidative stress. However, the information of sleep deprivation related ROS has rarely been obtained. Also, it has been demonstrated that sleep deprivation can induce endoplasmic reticulum (ER) stress. As such, for a better understanding of sleep deprivation as well as its related diseases, it is important to develop probes with ER-targeting ability for detecting ROS generated in this process. Herein, a novel two-photon fluorescent molecular probe, JX-1, was designed for sensing HClO in live cells and zebrafish. The investigation data showed that in addition to real-time response (about 150s), the probe also exhibited high sensitivity and selectivity. Moreover, the probe JX-1 demonstrated two-photon fluorescence, low cytotoxicity and ER targeting ability. These prominent properties enabled the utilization of the probe for monitoring exogenous and endogenous HClO in both live cells and zebrafish. Using this useful tool, it was found that sleep deprivation can induce the generation of HClO in zebrafish.

Cancer Cell Membrane-Camouflaged Nanorods with Endoplasmic Reticulum Targeting for Improved Antitumor Therapy.

Cell membrane-coated nanocarriers have been developed for drug delivery due to their enhanced blood circulation and tissue targeting capacities; however, previous works have generally focused on spherical nanoparticles and extracellular barriers. Many living organisms with different shapes, such as rod-shaped bacilli and rhabdovirus, display different functionalities regarding tissue penetration, cellular uptake, and intracellular distribution. Herein, we developed cancer cell membrane (CCM)-coated nanoparticles with spherical and rod shapes. CCM-coated nanorods (CRs) showed superior endocytosis efficiency compared with their spherical counterparts (CCM-coated nanospheres, CSs) due to the caveolin-mediated pathway. Moreover, CRs can effectively accumulate in the endoplasmic reticulum (ER) region and ship the loaded DOX to the nucleus at a considerable concentration, resulting in ER stress and subsequent apoptosis. After intravenous injection into human pancreatic adenocarcinoma cell (BxPC-3) and pancreatic stellate cell (HPSC) hybrid tumor-bearing nude mice, CRs exhibited improved immune escape ability, rapid extracellular matrix (ECM) penetration (8.2-fold higher than CSs), and enhanced tumor accumulation, further contributing to the enhanced antitumor efficacy. These findings may actually suggest the significance of shape design in improving current cell membrane-based drug delivery systems for effective subcellular targets and tumor therapy.

Expression of the Biologically Active Insulin Analog SCI-57 in Nicotiana Benthamiana.

Diabetes mellitus is a growing problem worldwide; however, only 23% of low-income countries have access to insulin, and ironically it costs higher in such countries than high-income ones. Therefore, new strategies for insulin and insulin analogs production are urgently required to improve low-cost access to therapeutic products, so as to contain the diabetes epidemic. SCI-57 is an insulin analog with a greater affinity for the insulin receptor and lower thermal degradation than native insulin. It also shows native mitogenicity and insulin-like biological activity. In this work, SCI-57 was transiently expressed in the Nicotiana benthamiana (Nb) plant, and we also evaluated some of its relevant biological effects. An expression plasmid was engineered to translate an N-terminal ubiquitin and C-terminal endoplasmic reticulum-targeting signal KDEL, in order to increase protein expression and stability. Likewise, the effect of co-expression of influenza M2 ion channel (M2) on the expression of insulin analog SCI-57 (SCI-57/M2) was evaluated. Although using M2 increases yield, it tends to alter the SCI-57 amino acid sequence, possibly promoting the formation of oligomers. Purification of SCI-57 was achieved by FPLC cation exchange and ultrafiltration of N. benthamiana leaf extract (NLE). SCI-57 exerts its anti-diabetic properties by stimulating glucose uptake in adipocytes, without affecting the lipid accumulation process. Expression of the insulin analog in agroinfiltrated plants was confirmed by SDS-PAGE, RP-HPLC, and MS. Proteome changes related to the expression of heterologous proteins on N. benthamiana were not observed; up-regulated proteins were related to the agroinfiltration process. Our results demonstrate the potential for producing a biologically active insulin analog, SCI-57, by transient expression in Nb.