Search

Proteins harboring intrinsically disordered regions (IDRs) lacking stable secondary or tertiary structures are abundant across the three domains of life. These regions have not been systematically studied in prokaryotes. Our genome-wide analysis identifies extracytoplasmic serine/threonine-rich IDRs in several biologically important membrane proteins in streptococci. We demonstrate that these IDRs are O -glycosylated with glucose by glycosyltransferases GtrB and PgtC2 in Streptococcus pyogenes and Streptococcus pneumoniae , and with N-acetylgalactosamine by a Pgf-dependent mechanism in Streptococcus mutans . Absence of glycosylation leads to a defect in biofilm formation under ethanol-stressed conditions in S. mutans . We link this phenotype to the C-terminal IDR of a post-translocation secretion chaperone PrsA. O -glycosylation of the IDR protects this region from proteolytic degradation. The IDR length attenuates the efficiency of glycosylation and, consequently, the expression level of PrsA. Taken together, our data reveal that O -glycosylation of IDRs functions as a dynamic switch of protein homeostasis in streptococci., Competing Interests: Competing interests The authors declare no competing interests.

Background: Staphylococcus aureus secretes a variety of proteins including virulence factors that cause diseases. PrsA, encoded by many Gram-positive bacteria, is a membrane-anchored lipoprotein that functions as a foldase to assist in post-translocational folding and helps maintain the stability of secreted proteins. Our earlier proteomic studies found that PrsA is required for the secretion of protein A, an immunoglobulin-binding protein that contributes to host immune evasion. This study aims to investigate how PrsA influences protein A secretion., Results: We found that in comparison with the parental strain HG001, the prsA-deletion mutant HG001ΔprsA secreted less protein A. Deleting prsA also decreased the stability of exported protein A. Pulldown assays indicated that PrsA interacts with protein A in vivo. The domains in PrsA that interact with protein A are mapped to both the N- and C-terminal regions (NC domains). Additionally, the NC domains are essential for promoting PrsA dimerization. Furthermore, an immunoglobulin-binding assay revealed that, compared to the parental strain HG001, fewer immunoglobulins bound to the surface of the mutant strain HG001ΔprsA., Conclusions: This study demonstrates that PrsA is critical for the folding and secretion of protein A. The information derived from this study provides a better understanding of virulent protein export pathways that are crucial to the pathogenicity of S. aureus., (© 2024. The Author(s).)

Hemolysis in red blood cells followed by hemoglobin degradation results in high hemin levels in the systemic circulation. Such a level of hemin is disastrous for cells and tissues and is considerably responsible for the pathologies of diseases like severe malaria. Hemin's hydrophobic chemical nature and structure allow it to bind several proteins leading to their functional modification. Such modifications in physiologically relevant proteins can have a high impact on various cellular processes. HSPA8 is a chaperone that has a protective role in oxidative stress by aiding protein refolding. Through ATPase activity assays we found that hemin can competitively inhibit ATP hydrolysis by the chaperone HSPA8. Hemin as such does not affect the structural integrity of the protein which is inferred from CD spectroscopy and Gel filtration but it hinders the ATP-dependent foldase function of the chaperone. HSPA8 was not able to cause the refolding of the model protein lysozyme in the presence of hemin. The loss in HSPA8 function was due to competition between hemin and ATP as the chaperone was able to regain the foldase function when the concentration of ATP was gradually increased with hemin present at the inhibitory concentration. In-silico studies to establish the competition for the specific binding site revealed that ATP was unable to replace hemin from the ATP binding pocket of HSPA8 and was forced to form a non-specific and unstable complex. In-vitro isothermal calorimetry revealed that the affinity of ATP for binding to HSPA8 was reduced 22 folds in the presence of hemin. The prevention of HSPA8's cytoprotective function by hemin can be a major factor contributing to the overall cellular damage during hemin accumulation in the case of severe malaria and other hemolytic diseases., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Brevibacillus offers great potential as a recombinant protein expression host because of its exceptional abilities to synthesize and excrete proteins and its low extracellular protease activity. Despite these strengths, effective recombinant expression strategies are still the key to achieving high-level expression of recombinant proteins in Brevibacillus due to individual differences among strains and target proteins. Many strategies have been developed to improve recombinant protein expression in Brevibacillus . This review begins by introducing the processes used to establish and apply the Brevibacillus expression system, and then critically discusses the strategies available for improving recombinant protein expression in Brevibacillus , including optimization of the host and the expression vector, co-expression of a fusion partner or foldase, and optimization of the fermentation process. Finally, the prospects for further improvement of recombinant protein expression based on Brevibacillus are also discussed. This review is intended to provide a strategic reference for scientists wanting to improve the expression of a specific recombinant protein in Brevibacillus or other expression systems.

Lipomax is a commercialized foldase-dependent Pseudomonas lipase that was previously expressed only in Pseudomonas strains. Here, using Pichia pastoris as the host, we report a new co-expression method that leads to the successful production of Lipomax. The active Lipomax is extracellularly co-expressed with its cognate foldase (LIM); and the purified enzyme mix has the optimum pH at pH 8.0 and an optimal temperature around 40 °C. N-glycosylation was observed for Pichia produced Lipomax, and its reduction was shown to increase the lipolytic activity. With different p-nitrophenyl esters as the substrates, the substrate profiling analyses further indicate that Lipomax prefers esters with middle-long chain fatty acids, showing the highest specific activity to p-nitrophenyl caprylate (C8). The extracellular co-expression of Lipomax and LIM in Pichia will not only increase our ability to investigate additional eukaryotic hosts for lipase expression, but also be of considerable value in analyzing other foldase-dependent lipases., (Copyright © 2020 Elsevier Inc. All rights reserved.)

LipMF3 is a new lipase isolated from a metagenomic library derived from a fat-contaminated soil. It belongs to the lipase subfamily I.1 and has identities of 68% and 67% with lipases of Chromobacterium violaceum and C. amazonense, respectively. Genes encoding LipMF3 and its cognate foldase, LifMF3, were cloned and co-expressed in Escherichia coli. The highest hydrolytic activity of purified Lip-LifMF3 was at 40 °C and pH 6.5. Under these conditions, the highest activity was against tributyrin (1650 U mg -1 ), but it also had high activity against olive oil (862 U mg -1 ). It was stable in hydrophilic organic solvents (25%, v/v in water) with residual activity around 100% after 24 h. It also showed stability over a wide pH range (5.5 to 11) with residual activity above 80% after 24 h. Lip-LifMF3 was immobilized by covalent bonding onto Immobead 150P and by adsorption onto Sepabeads FP-BU. The latter preparation gave the best results, producing 94% conversion after 5 h for the synthesis of ethyl oleate and a 90% enantiomeric excess of the product (R)‑1‑phenylethyl acetate for the kinetic resolution of (R,S)‑1‑phenyl‑1‑ethanol. The results obtained in this work provide a basis for the development of applications of Lip-LifMF3 in biocatalysis., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Lipases are industrial enzymes that catalyze both triglyceride hydrolysis and ester synthesis. The overexpression of lipase genes is considered one of the best approaches to increase the enzymatic production for industrial applications. Subfamily I.2. lipases require a chaperone or foldase in order to become a fully-activated enzyme. The goal of this research was to isolate, clone, and co-express genes that encode lipase and foldase from Burkholderia territorii GP3, a lipolytic bacterial isolate obtained from Mount Papandayan soil via growth on Soil Extract Rhodamine Agar. Genes that encode for lipase (lipBT) and foldase (lifBT) were successfully cloned from this isolate and co-expressed in the E. coli BL21 background. The highest expression was shown in E. coli BL21 (DE3) pLysS, using pET15b expression vector. LipBT was particulary unique as it showed highest activity with optimum temperature of 80°C at pH 11.0. The optimum substrate for enzyme activity was C 10 , which is highly stable in methanol solvent. The enzyme was strongly activated by Ca 2+ , Mg 2+ , and strongly inhibited by Fe 2+ and Zn 2+ . In addition, the enzyme was stable and compatible in non-ionic surfactant, and was strongly incompatible in ionic surfactant.

Mitochondria contain two aqueous subcompartments, the matrix and the intermembrane space (IMS). The matrix is enclosed by both the inner and outer mitochondrial membranes, whilst the IMS is sandwiched between the two. Proteins of the matrix are synthesized in the cytosol as preproteins, which contain amino-terminal matrix targeting sequences that mediate their translocation through translocases embedded in the outer and inner membrane. For these proteins, the translocation reaction is driven by the import motor which is part of the inner membrane translocase. The import motor employs matrix Hsp70 molecules and ATP hydrolysis to ratchet proteins into the mitochondrial matrix. Most IMS proteins lack presequences and instead utilize the IMS receptor Mia40, which facilitates their translocation across the outer membrane in a reaction that is coupled to the formation of disulfide bonds within the protein. This process requires neither ATP nor the mitochondrial membrane potential. Mia40 fulfills two roles: First, it acts as a holdase, which is crucial in the import of IMS proteins and second, it functions as a foldase, introducing disulfide bonds into newly imported proteins, which induces and stabilizes their natively folded state. For several Mia40 substrates, oxidative folding is an essential prerequisite for their assembly into oligomeric complexes. Interestingly, recent studies have shown that the two functions of Mia40 can be experimentally separated from each other by the use of specific mutants, hence providing a powerful new way to dissect the different physiological roles of Mia40. In this review we summarize the current knowledge relating to the mitochondrial matrix-targeting and the IMS-targeting/Mia40 pathway. Moreover, we discuss the mechanistic properties by which the mitochondrial import motor on the one hand and Mia40 on the other, drive the translocation of their substrates into the organelle. We propose that the lateral diffusion of Mia40 in the inner membrane and the oxidation-mediated folding of incoming polypeptides supports IMS import.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by a progressive upper and lower motor neuron degeneration. One of the peculiar clinical characteristics of ALS is the wide distribution in age of onset, which is probably caused by different combinations of intrinsic and exogenous factors. We investigated whether these modifying factors are converging into common pathogenic pathways leading either to an early or a late disease onset. This would imply the identification of phenotypic biomarkers, that can distinguish the two populations of ALS patients, and of relevant pathways to consider in a therapeutic intervention. Toward this aim a differential proteomic analysis was performed in peripheral blood mononuclear cells (PBMC) from a group of 16 ALS patients with an age of onset ≤55 years and a group of 16 ALS patients with an age of onset ≥75 years, and matched healthy controls. We identified 43 differentially expressed proteins in the two groups of patients. Gene ontology analysis revealed that there was a significant enrichment in annotations associated with protein folding and response to stress. We next validated a selected number of proteins belonging to this functional group in 85 patients and 83 age- and sex-matched healthy controls using immunoassays. The results of the validation study confirmed that there was a decreased level of peptidyl-prolyl cis-trans isomerase A (also known as cyclophilin A), heat shock protein HSP 90-alpha, 78 kDa glucose-regulated protein (also known as BiP) and protein deglycase DJ-1 in PBMC of ALS patients with an early onset. Similar results were obtained in PBMC and spinal cord from two SOD1 G93A mouse models with an early and late disease onset. This study suggests that a different ability to upregulate proteins involved in proteostasis, such as foldase and chaperone proteins, may be at the basis of a different susceptibility to ALS, putting forward the development of therapeutic approaches aiming at boosting the protein quality control system.

The autodisplay technology has been applied for expression of a desired protein on the outer membrane (OM) of Escherichia coli. In this work, the OM fractions of E. coli with two autodisplayed proteins were separately prepared and mixed to demonstrate the feasibility of control over the ratio of two autodisplayed proteins. As the first model, Z-domain and streptavidin were autodisplayed, and their activities were tested by means of the combined OM layer in a 96-well microplate and a surface plasmon resonance (SPR) biosensor. As the second model, lipase and foldase were autodisplayed which required an interaction between two proteins to obtain the activity of lipase. The OM fractions of E. coli with an autodisplayed lipase and foldase were separately prepared and mixed to demonstrate the feasibility of control over the ratio of two autodisplayed proteins when the interaction of two proteins is required within the same OM layer for the activity of the lipase., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Pichia pastoris is generally considered as an expression host for heterologous proteins with the coding gene under control of the alcohol oxidase 1 (AOX1) promoter. The secretion of heterologous proteins in P. pastoris can be potentially affected by many factors. Based on our previous results, the secretion levels of human albumin (HSA) fusion protein IL2-HSA were only around 500 mg/L or less in fermentor cultures, which decreased more than 50% compared with that of HSA (>1 g/L). In this study, we selected five potential secretion helper factors, in which Ero1, Pdi1 and Kar2 were involved in protein folding and Sec1 and Sly1 were involved in vesicle trafficking. We evaluated the possible effects of individual overexpression of these secretion helper factors on the secretion of IL2-HSA in P. pastoris. Constitutive overexpression of the five selected secretion factors did not have an obvious negative effect on cell growth of the IL2-HSA secreting strain. Individual co-overexpression of Ero1, Kar2, Pdi1, Sec1 and Sly1 improved the secretion level of IL2-HSA to ~2.3-, 1.9-, 2.2-, 2.5- and 1.9-fold that in the control strain respectively in shake flasks. We evaluated the changes in mRNA and protein levels of the intracellular IL2-HSA, as well as the secretion helper factor genes in the co-overexpressing strains. Our results indicated that manipulating the expression level of ER resident protein Pdi1, Ero1, Kar2 and SM protein Sec1 and Sly1 could improve the secretion level of IL2-HSA fusion protein in P. pastoris, which provided new candidates for combinatorial engineering in future study. Copyright © 2016 John Wiley & Sons, Ltd., (Copyright © 2016 John Wiley & Sons, Ltd.)

Plasmepsin II is a malarial pepsin-like aspartic protease produced as a zymogen containing an N-terminal prosegment domain that is removed during activation. Despite structural similarities between active plasmepsin II and pepsin, their prosegments adopt different conformations in the respective zymogens. In contrast to pepsinogen, the proplasmepsin II prosegment is 80 residues longer, contains a transmembrane region and is non-essential for recombinant expression in an active form, thus calling into question the prosegment's precise function. The present study examines the role of the prosegment in the folding mechanism of plasmepsin II. Both a shorter (residues 77-124) and a longer (residues 65-124) prosegment catalyze plasmepsin II folding at rates more than four orders of magnitude faster compared to folding without prosegment. Native plasmepsin II is kinetically trapped and requires the prosegment both to catalyze folding and to shift the folding equilibrium towards the native conformation. Thus, despite low sequence identity and distinct zymogen conformations, the folding landscapes of plasmepsin II and pepsin, both with and without prosegment, are qualitatively identical. These results imply a conserved and unusual feature of the pepsin-like protease topology that necessitates prosegment-assisted folding., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Pepsin is initially produced as the zymogen pepsinogen, containing a 44 residue prosegment (PS) domain. When folded without the PS, pepsin forms a thermodynamically stable denatured state (refolded pepsin, Rp). To guide native folding, the PS binds to Rp, stabilizes the folding transition state, and binds tightly to native pepsin (Np), thereby driving the folding equilibrium to favor the native state. It is unknown whether these functionalities of the PS are encoded within the entire sequence or within discrete segments. PS residues 1p-29p correspond to a highly conserved region in pepsin-like aspartic proteases and we hypothesized that this segment is critical to PS-catalyzed folding. This notion was tested in the present study by characterizing the ability of various truncated PS peptides to bind Rp, catalyze folding from Rp to Np, and to inhibit Np. Four PS truncations were examined, corresponding to PS residues 1p-16p (PS1-16), 1p-29p (PS1-29), 17p-44p (PS17-44) and 30p-44p (PS30-44). The three PS functionalities could be ascribed primarily to discrete regions within the highly conserved motif: 1p-16p dictated Rp binding, 17p-29p dictated Np binding/inhibition, while the entire 1p-29p dictated transition state binding/catalyzing folding. Conversely, PS30-44 played no obvious role in PS-catalyzed folding; it is hypothesized that this more variable region may serve as a linker between PS1-29 and the mature domain. The high sequence conservation of PS1-29 and its role in catalyzing pepsin folding strongly suggest that there is a conserved PS-catalyzed folding mechanism shared by pepsin-like aspartic proteases with this motif., (Copyright © 2015 Elsevier B.V. All rights reserved.)

In Gram-positive bacteria, the secretion of proteins requires translocation of polypeptides across the bacterial membrane into the highly charged environment of the membrane-cell wall interface. Here, proteins must be folded and often further delivered across the matrix of the cell wall. While many aspects of protein secretion have been well studied in Gram-negative bacteria which possess both an inner and outer membrane, generally less attention has been given to the mechanics of protein secretion across the single cell membrane of Gram-positive bacteria. In this review, we focus on the role of a post-translocation secretion chaperone in Listeria monocytogenes known as PrsA2, and compare what is known regarding PrsA2 with PrsA homologs in other Gram-positive bacteria. PrsA2 is a member of a family of membrane-associated lipoproteins that contribute to the folding and stability of secreted proteins as they cross the bacterial membrane. PrsA2 contributes to the integrity of the L. monocytogenes cell wall as well as swimming motility and bacterial resistance to osmotic stress; however its most critical role may be its requirement for L. monocytogenes virulence and viability within host cells. A better understanding of the role of PrsA2 and PrsA-like homologs will provide insight into the dynamics of protein folding and stability in Gram-positive bacteria and may result in new strategies for optimizing protein secretion as well as inhibiting the production of virulence factors.