An Intrinsically Disordered Peptide Tag that Confers an Unusual Solubility to Aggregation-Prone Proteins

There is a high demand for the production of recombinant proteins in Escherichia coli for biotechnological applications but their production is still limited by their insolubility. Fusion tags have been successfully used to enhance the solubility of aggregation-prone proteins; however, smaller and more powerful tags are desired for increasing the yield and quality of target proteins. Herein, NEXT tag, a 53 amino acid-length solubility enhancer, is described. The NEXT tag showed outstanding ability to improve both in vivo and in vitro solubilities with minimal effect on passenger proteins. The C-terminal region of the tag was mostly responsible for in vitro solubility, while the N-terminal region was essential for in vivo soluble expression. The NEXT tag appeared to be intrinsically disordered and seemed to exclude neighboring molecules and prevent protein aggregation by acting as an entropic bristle. This novel peptide tag should have general use as a fusion partner to increase the yield and quality of difficult-to-express proteins.

Decorating the surface of Escherichia coli with bacterial lipoproteins: a comparative analysis of different display systems

Background The display of recombinant proteins on cell surfaces has a plethora of applications including vaccine development, screening of peptide libraries, whole-cell biocatalysts and biosensor development for diagnostic, industrial or environmental purposes. In the last decades, a wide variety of surface display systems have been developed for the exposure of recombinant proteins on the surface of Escherichia coli, such as autotransporters and outer membrane proteins. Results In this study, we assess three approaches for the surface display of a panel of heterologous and homologous mature lipoproteins in E. coli: four from Neisseria meningitidis and four from the host strain that are known to be localised in the inner leaflet of the outer membrane. Constructs were made carrying the sequences coding for eight mature lipoproteins, each fused to the delivery portion of three different systems: the autotransporter adhesin involved in diffuse adherence-I (AIDA-I) from enteropathogenic E. coli, the Lpp’OmpA chimaera and a truncated form of the ice nucleation protein (INP), InaK-NC (N-terminal domain fused with C-terminal one) from Pseudomonas syringae. In contrast to what was observed for the INP constructs, when fused to the AIDA-I or Lpp’OmpA, most of the mature lipoproteins were displayed on the bacterial surface both at 37 and 25 °C as demonstrated by FACS analysis, confocal and transmission electron microscopy. Conclusions To our knowledge this is the first study that compares surface display systems using a number of passenger proteins. We have shown that the experimental conditions, including the choice of the carrier protein and the growth temperature, play an important role in the translocation of mature lipoproteins onto the bacterial surface. Despite all the optimization steps performed with the InaK-NC anchor motif, surface exposure of the passenger proteins used in this study was not achieved. For our experimental conditions, Lpp’OmpA chimaera has proved to be an efficient surface display system for the homologous passenger proteins although cell lysis and phenotype heterogeneity were observed. Finally, AIDA-I was found to be the best surface display system for mature lipoproteins (especially heterologous ones) in the E. coli host strain with no inhibition of growth and only limited phenotype heterogeneity.

Decorating the Surface of Escherichia Coli with Bacterial Lipoproteins: A Comparative Analysis of Different Display Systems

Background: The display of recombinant proteins on cell surfaces has a plethora of applications including vaccine development, screening of peptide libraries, whole-cell biocatalysts and biosensor development for diagnostic, industrial or environmental purposes. In the last decades, a wide variety of surface display systems have been developed for the exposure of recombinant proteins on the surface of Escherichia coli, such as autotransporters and outer membrane proteins.Results: In this study, we assess three approaches for the surface display of a panel of heterologous and homologous mature lipoproteins in E. coli: four from Neisseria meningitidis and four from the host strain that are known to be localised in the inner leaflet of the outer membrane. Constructs were made carrying the sequences coding for eight mature lipoproteins, each fused to the delivery portion of three different systems: the autotransporter adhesin involved in diffuse adherence-I (AIDA-I) from enteropathogenic E. coli, the Lpp’OmpA chimaera and a truncated form of the ice nucleation protein (INP), InaK-NC (N-terminal domain fused with C-terminal one) from Pseudomonas syringae. In contrast to what was observed for the INP constructs, when fused to the AIDA-I or Lpp’OmpA, most of the mature lipoproteins were displayed on the bacterial surface both at 37°C and 25°C as demonstrated by FACS analysis, confocal and transmission electron microscopy.Conclusions: To our knowledge this is the first study that compares surface display systems using a number of passenger proteins. We have shown that the experimental conditions, including the choice of the carrier protein and the growth temperature, play an important role in the translocation of mature lipoproteins onto the bacterial surface. Despite all the optimization steps performed with the InaK-NC anchor motif, surface exposure of the passenger proteins used in this study was not achieved. For our experimental conditions, Lpp’OmpA chimaera has proved to be an efficient surface display system for the homologous passenger proteins although cell lysis and phenotype heterogeneity were observed. Finally, , AIDA-I was found to be the best surface display system for mature lipoproteins (especially heterologous ones) in the E. coli host strain with no inhibition of growth and only limited phenotype heterogeneity.

A cross-kingdom conserved ER-phagy receptor maintains endoplasmic reticulum homeostasis during stress

Eukaryotes have evolved various quality control mechanisms to promote proteostasis in the endoplasmic reticulum (ER). Selective removal of certain ER domains via autophagy (termed as ER-phagy) has emerged as a major quality control mechanism. However, the degree to which ER-phagy is employed by other branches of ER-quality control remains largely elusive. Here, we identify a cytosolic protein, C53, that is specifically recruited to autophagosomes during ER-stress, in both plant and mammalian cells. C53 interacts with ATG8 via a distinct binding epitope, featuring a shuffled ATG8 interacting motif (sAIM). C53 senses proteotoxic stress in the ER lumen by forming a tripartite receptor complex with the ER-associated ufmylation ligase UFL1 and its membrane adaptor DDRGK1. The C53/UFL1/DDRGK1 receptor complex is activated by stalled ribosomes and induces the degradation of internal or passenger proteins in the ER. Consistently, the C53 receptor complex and ufmylation mutants are highly susceptible to ER stress. Thus, C53 forms an ancient quality control pathway that bridges selective autophagy with ribosome-associated quality control in the ER.

Identification of a Cytopathogenic Toxin from Sneathia amnii

ABSTRACT Sneathia amnii is a poorly characterized emerging pathogen that has been implicated in amnionitis and urethritis. We found that S. amnii damages fetal membranes, and we identified and purified a cytotoxic exotoxin that lyses human red blood cells and damages cells from fetal membranes. The gene appears to be cotranscribed with a second gene that encodes a protein with identity to two-partner system transporters, suggesting that it is the “A,” or secreted component of a type Vb system. The toxin is 1,881 amino acids with a molecular weight of approximately 200 kDa. It binds to red blood cell membranes and forms pores with a diameter of 2.0 to 3.0 nm, resulting in osmolysis. Because it appears to be the “A” or passenger component of a two-partner system, we propose to name this novel cytotoxin/hemolysin CptA for cytopathogenic toxin component A. IMPORTANCE Sneathia amnii is a very poorly characterized emerging pathogen that can affect pregnancy outcome and cause urethritis and other infections. To date, nothing is known about its virulence factors or pathogenesis. We have identified and isolated a cytotoxin, named CptA for cytopathogenic toxin, component A, that is produced by S. amnii. CptA is capable of permeabilizing chorionic trophoblasts and lysing human red blood cells and, thus, may play a role in virulence. Except for small domains conserved among two-partner secretion system passenger proteins, the cytotoxin exhibits little amino acid sequence homology to known toxins. In this study, we demonstrate the pore-forming activity of this novel toxin.

A cross-kingdom conserved ER-phagy receptor maintains endoplasmic reticulum homeostasis during stress

SummaryEukaryotes have evolved various quality control mechanisms to promote proteostasis in the ER. Selective removal of certain ER domains via autophagy (termed as ER-phagy) has emerged as a major quality control mechanism. However, the degree to which ER-phagy is employed by other branches of ER-quality control remains largely elusive. Here, we identify a cytosolic protein, C53, that is specifically recruited to autophagosomes during ER-stress, in both plant and mammalian cells. C53 interacts with ATG8 via a distinct binding epitope, featuring a shuffled ATG8 interacting motif (sAIM). C53 senses proteotoxic stress in the ER lumen by forming a tripartite receptor complex with the ER-associated ufmylation ligase UFL1 and its membrane adaptor DDRGK1. The C53/UFL1/DDRGK1 receptor complex is activated by stalled ribosomes and induces the degradation of internal or passenger proteins in the ER. Consistently, the C53 receptor complex and ufmylation mutants are highly susceptible to ER stress. Thus, C53 forms an ancient quality control pathway that bridges selective autophagy with ribosome-associated quality control at the ER.

Basal-Type Breast Cancer Stem Cells Over-Express Chromosomal Passenger Complex Proteins

(1) Aim: In the present paper we analyzed the transcriptome of CSCs (Cancer Stem Cells), in order to find defining molecular processes of breast cancer. (2) Methods: We performed RNA-Seq from CSCs isolated from the basal cell line MDA-MB-468. Enriched processes and networks were studied using the IPA (Ingenuity Pathway Analysis) tool. Validation was performed with qRT-PCR and the analysis of relevant genes was evaluated by overexpression, flow cytometry and in vivo zebrafish studies. Finally, the clinical relevance of these results was assessed using reported cohorts. (3) Results: We found that CSCs presented marked differences from the non-CSCs, including enrichment in transduction cascades related to stemness, cellular growth, proliferation and apoptosis. Interestingly, CSCs overexpressed a module of co-regulated Chromosomal Passenger Proteins including BIRC5 (survivin), INCENP and AURKB. Overexpression of BIRC5 increased the number of CSCs, as assessed by in vitro and in vivo zebrafish xenotransplant analyses. Analysis of previously published cohorts showed that this co-regulated module was not only overexpressed in basal breast tumors but also associated with relapse-free and overall survival in these patients. (4) Conclusions: These results underline the importance of Cancer Stem Cells in breast cancer progression and point toward the possible use of chromosomal passenger proteins as prognostic factors.

Passenger sequences can promote interlaced dimers in a common variant of the maltose-binding protein

The maltose-binding protein (MBP) is one of the most frequently used protein tags due to its capacity to stabilize, solubilize and even crystallize recombinant proteins that are fused to it. Given that MBP is thought to be a highly stable monomeric protein with known characteristics, fused passenger proteins are often studied without being cleaved from MBP. Here we report that a commonly used engineered MBP version (mutated to lower its surface entropy) can form interlaced dimers when fused to short protein sequences derived from the focal adhesion kinase (FAK) or the homologous protein tyrosine kinase 2 (PYK2). These MBP dimers still bind maltose and can interconvert with monomeric forms in vitro under standard conditions despite a contact surface of more than 11,000 Å2. We demonstrate that both the mutations in MBP and the fused protein sequences were required for dimer formation. The FAK and PYK2 sequences are less than 40% identical, monomeric, and did not show specific interactions with MBP, suggesting that a variety of sequences can promote this MBP dimerization. MBP dimerization was abrogated by reverting two of the eight mutations introduced in the engineered MBP. Our results provide an extreme example for induced reversible domain-swapping, with implications for protein folding dynamics. Our observations caution that passenger-promoted MBP dimerization might mislead experimental characterization of the fused protein sequences, but also suggest a simple mutation to stop this phenomenon.