C-terminal amino acids are essential for human heat shock protein 70 dimerization

The human inducible heat shock protein 70 (hHsp70), which is involved in several major pathologies, including neurodegenerative disorders and cancer, is a key molecular chaperone and contributes to the proper protein folding and maintenance of a large number of protein structures. Despite its role in disease, the current structural knowledge of hHsp70 is almost exclusively based on its Escherichia coli homolog, DnaK, even though these two proteins only share ~50 % amino acid identity. For the first time, we describe a complete heterologous production and purification strategy that allowed us to obtain a large amount of soluble, full-length, and non-tagged hHsp70. The protein displayed both an ATPase and a refolding activity when combined to the human Hsp40. Multi-angle light scattering and bio-layer interferometry analyses demonstrated the ability of hHsp70 to homodimerize. The role of the C-terminal part of hHsp70 was identified and confirmed by a study of a truncated version of hHsp70 that could neither dimerize nor present refolding activity

N-glycosylation of mouse TRAIL-R and human TRAIL-R1 enhances TRAIL-induced death.

APO2L/TRAIL (TNF-related apoptosis-inducing ligand) induces death of tumor cells through two agonist receptors, TRAIL-R1 and TRAIL-R2. We demonstrate here that N-linked glycosylation (N-glyc) plays also an important regulatory role for TRAIL-R1-mediated and mouse TRAIL receptor (mTRAIL-R)-mediated apoptosis, but not for TRAIL-R2, which is devoid of N-glycans. Cells expressing N-glyc-defective mutants of TRAIL-R1 and mouse TRAIL-R were less sensitive to TRAIL than their wild-type counterparts. Defective apoptotic signaling by N-glyc-deficient TRAIL receptors was associated with lower TRAIL receptor aggregation and reduced DISC formation, but not with reduced TRAIL-binding affinity. Our results also indicate that TRAIL receptor N-glyc impacts immune evasion strategies. The cytomegalovirus (CMV) UL141 protein, which restricts cell-surface expression of human TRAIL death receptors, binds with significant higher affinity TRAIL-R1 lacking N-glyc, suggesting that this sugar modification may have evolved as a counterstrategy to prevent receptor inhibition by UL141. Altogether our findings demonstrate that N-glyc of TRAIL-R1 promotes TRAIL signaling and restricts virus-mediated inhibition

Tumor Exosomes: Potential Biomarkers and Targets in Cancer

IF 2.06International audienc

Membrane-bound exosomal HSP70 as a biomarker for detection and monitoring of malignant solid tumours: a pilot study.

BACKGROUND: Cancer is the second leading cause of death globally. Early detection and disease management lead to a better survival rate. Consequently, discovery of novel methods in cancer early diagnosis is a field of active research. Minimally invasive liquid biopsies are generating growing interest. Circulating tumour cells (CTCs) have been identified in patients' blood; nevertheless, these cells are rare and heterogeneous. Exosomes are extracellular nanovesicles released into the extracellular environment via the endosomal vesicle pathway and found in different body fluids. Exosomes deliver bioactive cargo such as proteins, mRNA and miRNA to recipient cells in the tumour environment. We have recently shown that heat shock protein 70 (HSP70) is detected in the membrane of tumour-derived exosomes, in contrast to normal cells. One single cancer cell can release thousands of HSP70-exosomes, facilitating detection. The aim of the pilot study ExoDiag is to determine whether it is possible to detect and quantify HSP70-exosomes in blood in patients with solid cancers. METHODS: Bicentric pilot study that will include 60 adult patients with metastatic and non-metastatic solid tumours and 20 healthy volunteers. Exosomes will be isolated from blood and urine samples, and HSP70 concentration will be determined. Patients will be followed for 1 year. The study is sponsored by Georges-François Leclerc Centre and is currently ongoing. DISCUSSION: We expect to demonstrate that HSP70-exosomes could be a powerful tool to diagnose cancer and to guide clinicians in therapeutic decision-making, improving patient's care. TRIAL REGISTRATION: ClinicalTrials.gov identifier NCT02662621. Registered 20 January 2016, https://clinicaltrials.gov/ct2/show/study/NCT02662621?term=NCT02662621&rank=1

A self-inducible heterologous protein expression system in Escherichia coli

International audienceEscherichia coli is an important experimental, medical and industrial cell factory for recombinant protein production. The inducible lac promoter is one of the most commonly used promoters for heterologous protein expression in E. coli. Isopropyl-beta-D-thiogalactoside (IPTG) is currently the most efficient molecular inducer for regulating this promoter's transcriptional activity. However, limitations have been observed in large-scale and microplate production, including toxicity, cost and culture monitoring. Here, we report the novel SILEX (Self-InducibLe Expression) system, which is a convenient, cost-effective alternative that does not require cell density monitoring or IPTG induction. We demonstrate the broad utility of the presented self-inducible method for a panel of diverse proteins produced in large amounts. The SILEX system is compatible with all classical culture media and growth temperatures and allows protein expression modulation. Importantly, the SILEX system is proven to be efficient for protein expression screening on a microplate scale

Dose-dependent biphasic leptin-induced proliferation is caused by non-specific IL-6/NF-κB pathway activation in human myometrial cells.

BACKGROUND AND PURPOSE: Leptin, an adipokine synthesized by the placenta during pregnancy, has been proposed for the management of preterm labour (PTL), as it is able to prevent in vitro uterine contractility and remodelling associated with labour onset. Another common feature of labour onset is the phenotypic switch of myometrial smooth muscle cells from a proliferative to a hypertrophic state. As proliferative effects have been demonstrated for leptin in other tissues, we aimed to investigate its ability to induce myometrial proliferation and thus to maintain uterine quiescence. EXPERIMENTAL APPROACH: We stimulated human primary myometrial smooth muscle cells with leptin in the presence or absence of receptor antagonists or signalling pathway inhibitors. KEY RESULTS: Leptin induced myometrial cell proliferation in a biphasic manner. At 6.25 ng · mL(-1), leptin-induced proliferation was mediated by the leptin receptor and required the early activation of ERK1/2. At a concentration above 25 ng · mL(-1), leptin induced direct non-specific stimulation of the IL-6 receptor, leading to NF-κB activation, and exerted anti-proliferative effects. However, at 50 ng · mL(-1), leptin re-induces proliferation via IL-6 receptor stimulation that requires STAT3 and delayed ERK1/2 activation. CONCLUSIONS AND IMPLICATIONS: These data bring new insights into leptin signalling-induced myometrial proliferation and its interrelationship with the IL-6/IL-6 receptor axis. In the light of our previous work, the present study emphasizes the potential value of leptin in the pharmacological management of PTL and it also strengthens the hypothesis that leptin might be a contributory factor in the parturition-related disorders observed in obese women

Radiotheranostic Agents in Hematological Malignancies.

peer reviewedRadioimmunotherapy (RIT) is a cancer treatment that combines radiation therapy with tumor-directed monoclonal antibodies (Abs). Although RIT had been introduced for the treatment of CD20 positive non-Hodgkin lymphoma decades ago, it never found a broad clinical application. In recent years, researchers have developed theranostic agents based on Ab fragments or small Ab mimetics such as peptides, affibodies or single-chain Abs with improved tumor-targeting capacities. Theranostics combine diagnostic and therapeutic capabilities into a single pharmaceutical agent; this dual application can be easily achieved after conjugation to radionuclides. The past decade has seen a trend to increased specificity, fastened pharmacokinetics, and personalized medicine. In this review, we discuss the different strategies introduced for the noninvasive detection and treatment of hematological malignancies by radiopharmaceuticals. We also discuss the future applications of these radiotheranostic agents