PBP-A, a cyanobacterial DD-peptidase with high specificity for amidated muropeptides, exhibits pH-dependent promiscuous activity harmful to Escherichia coli.

Penicillin binding proteins (PBPs) are involved in biosynthesis, remodeling and recycling of peptidoglycan (PG) in bacteria. PBP-A from Thermosynechococcus elongatus belongs to a cyanobacterial family of enzymes sharing close structural and phylogenetic proximity to class A ß-lactamases. With the long-term aim of converting PBP-A into a ß-lactamase by directed evolution, we simulated what may happen when an organism like Escherichia coli acquires such a new PBP and observed growth defect associated with the enzyme activity. To further explore the molecular origins of this harmful effect, we decided to characterize deeper the activity of PBP-A both in vitro and in vivo. We found that PBP-A is an enzyme endowed with DD-carboxypeptidase and DD-endopeptidase activities, featuring high specificity towards muropeptides amidated on the D-iso-glutamyl residue. We also show that a low promiscuous activity on non-amidated peptidoglycan deteriorates E. coli's envelope, which is much higher under acidic conditions where substrate discrimination is mitigated. Besides expanding our knowledge of the biochemical activity of PBP-A, this work also highlights that promiscuity may depend on environmental conditions and how it may hinder rather than promote enzyme evolution in nature or in the laboratory.

Plasma membrane nanodeformations promote actin polymerization through CIP4/CDC42 recruitment and regulate type II IFN signaling.

In their environment, cells must cope with mechanical stresses constantly. Among these, nanoscale deformations of plasma membrane induced by substrate nanotopography are now largely accepted as a biophysical stimulus influencing cell behavior and function. However, the mechanotransduction cascades involved and their precise molecular effects on cellular physiology are still poorly understood. Here, using homemade fluorescent nanostructured cell culture surfaces, we explored the role of Bin/Amphiphysin/Rvs (BAR) domain proteins as mechanosensors of plasma membrane geometry. Our data reveal that distinct subsets of BAR proteins bind to plasma membrane deformations in a membrane curvature radius-dependent manner. Furthermore, we show that membrane curvature promotes the formation of dynamic actin structures mediated by the Rho GTPase CDC42, the F-BAR protein CIP4, and the presence of PI(4,5)P2. In addition, these actin-enriched nanodomains can serve as platforms to regulate receptor signaling as they appear to contain interferon-γ receptor (IFNγ-R) and to lead to the partial inhibition of IFNγ-induced JAK/STAT signaling.

The yeast Gdt1 protein mediates the exchange of H+ for Ca2+ and Mn2+ influencing the Golgi pH.

The GDT1 family is broadly spread and highly conserved among living organisms. GDT1 members have functions in key processes like glycosylation in humans and yeasts and photosynthesis in plants. These functions are mediated by their ability to transport ions. While transport of Ca2+ or Mn2+ is well established for several GDT1 members, their transport mechanism is poorly understood. Here, we demonstrate that H+ ions are transported in exchange for Ca2+ and Mn2+ cations by the Golgi-localized yeast Gdt1 protein. We performed direct transport measurement across a biological membrane by expressing Gdt1p in Lactococcus lactis bacterial cells and by recording either the extracellular pH or the intracellular pH during the application of Ca2+, Mn2+ or H+ gradients. Besides, in vivo cytosolic and Golgi pH measurements were performed in Saccharomyces cerevisiae with genetically encoded pH probes targeted to those subcellular compartments. These data point out that the flow of H+ ions carried by Gdt1p could be reversed according to the physiological conditions. Together, our experiments unravel the influence of the relative concentration gradients for Gdt1p-mediated H+ transport and pave the way to decipher the regulatory mechanisms driving the activity of GDT1 orthologs in various biological contexts.

N-BAR and F-BAR proteins-endophilin-A3 and PSTPIP1-control clathrin-independent endocytosis of L1CAM.

Recent advances in the field demonstrate the high diversity and complexity of endocytic pathways. In the current study, we focus on the endocytosis of L1CAM. This glycoprotein plays a major role in the development of the nervous system, and is involved in cancer development and is associated with metastases and poor prognosis. Two L1CAM isoforms are subject to endocytosis: isoform 1, described as a clathrin-mediated cargo; isoform 2, whose endocytosis has never been studied. Deciphering the molecular machinery of isoform 2 internalisation should contribute to a better understanding of its pathophysiological role. First, we demonstrated in our cellular context that both isoforms of L1CAM are mainly a clathrin-independent cargo, which was not expected for isoform 1. Second, the mechanism of L1CAM endocytosis is specifically mediated by the N-BAR domain protein endophilin-A3. Third, we discovered PSTPIP1, an F-BAR domain protein, as a novel actor in this endocytic process. Finally, we identified galectins as endocytic partners and negative regulators of L1CAM endocytosis. In summary, the interplay of the BAR proteins endophilin-A3 and PSTPIP1, and galectins fine tune the clathrin-independent endocytosis of L1CAM.

Rac1, the actin cytoskeleton and microtubules are key players in clathrin-independent endophilin-A3-mediated endocytosis.

Endocytic mechanisms actively regulate plasma membrane composition and sustain fundamental cellular functions. Recently, we identified a clathrin-independent endocytic (CIE) modality mediated by the BAR domain protein endophilin-A3 (endoA3, encoded by SH3GL3), which controls the cell surface homeostasis of the tumor marker CD166 (also known as ALCAM). Deciphering the molecular machinery of endoA3-dependent CIE should therefore contribute to a better understanding of its pathophysiological role, which remains so far unknown. Here, we investigate the role of actin, Rho GTPases and microtubules, which are major players in CIE processes, in this mechanism. We show that the actin cytoskeleton is dynamically associated with endoA3- and CD166-positive endocytic carriers, and that its perturbation strongly inhibits the process of CD166 uptake. We also reveal that the Rho GTPase Rac1, but not Cdc42, is a master regulator of this endocytic route. Finally, we provide evidence that microtubules and kinesin molecular motors are required to potentiate endoA3-dependent endocytosis. Of note, our study also highlights potential compensation phenomena between endoA3-dependent CIE and macropinocytosis. Altogether, our data deepen our understanding of this CIE modality and further differentiate it from other unconventional endocytic mechanisms. This article has an associated First Person interview with the first author of the paper.

Competence shut-off by intracellular pheromone degradation in salivarius streptococci.

Competence for DNA transformation is a major strategy for bacterial adaptation and survival. Yet, this successful tactic is energy-consuming, shifts dramatically the metabolism, and transitory impairs the regular cell-cycle. In streptococci, complex regulatory pathways control competence deactivation to narrow its development to a sharp window of time, a process known as competence shut-off. Although characterized in streptococci whose competence is activated by the ComCDE signaling pathway, it remains unclear for those controlled by the ComRS system. In this work, we investigate competence shut-off in the major human gut commensal Streptococcus salivarius. Using a deterministic mathematical model of the ComRS system, we predicted a negative player under the control of the central regulator ComX as involved in ComS/XIP pheromone degradation through a negative feedback loop. The individual inactivation of peptidase genes belonging to the ComX regulon allowed the identification of PepF as an essential oligoendopeptidase in S. salivarius. By combining conditional mutants, transcriptional analyses, and biochemical characterization of pheromone degradation, we validated the reciprocal role of PepF and XIP in ComRS shut-off. Notably, engineering cleavage site residues generated ultra-resistant peptides producing high and long-lasting competence activation. Altogether, this study reveals a proteolytic shut-off mechanism of competence in the salivarius group and suggests that this mechanism could be shared by other ComRS-containing streptococci.

Role of the Redox State of Human Peroxiredoxin-5 on Its TLR4-Activating DAMP Function.

Human peroxiredoxin-5 (PRDX5) is a unique redox-sensitive protein that plays a dual role in brain ischemia-reperfusion injury. While intracellular PRDX5 has been reported to act as a neuroprotective antioxidative enzyme by scavenging peroxides, once released extracellularly from necrotic brain cells, the protein aggravates neural cell death by inducing expression of proinflammatory cytokines in macrophages through activation of Toll-like receptor (TLR) 2 (TLR2) and 4 (TLR4). Although recent evidence showed that PRDX5 was able to interact directly with TLR4, little is known regarding the role of the cysteine redox state of PRDX5 on its DAMP function. To gain insights into the role of PRDX5 redox-active cysteine residues in the TLR4-dependent proinflammatory activity of the protein, we used a recombinant human PRDX5 in the disulfide (oxidized) form and a mutant version lacking the peroxidatic cysteine, as well as chemically reduced and hyperoxidized PRDX5 proteins. We first analyzed the oxidation state and oligomerization profile by Western blot, mass spectrometry, and SEC-MALS. Using ELISA, we demonstrate that the disulfide bridge between the enzymatic cysteines is required to allow improved TLR4-dependent IL-8 secretion. Moreover, single-molecule force spectroscopy experiments revealed that TLR4 alone is not sufficient to discriminate the different PRDX5 redox forms. Finally, flow cytometry binding assays show that disulfide PRDX5 has a higher propensity to bind to the surface of living TLR4-expressing cells than the mutant protein. Taken together, these results demonstrate the importance of the redox state of PRDX5 cysteine residues on TLR4-induced inflammation.

Trophic state alters the mechanism whereby energetic coupling between photosynthesis and respiration occurs in Euglena gracilis.

The coupling between mitochondrial respiration and photosynthesis plays an important role in the energetic physiology of green plants and some secondary-red photosynthetic eukaryotes (diatoms), allowing an efficient CO2 assimilation and optimal growth. Using the flagellate Euglena gracilis, we first tested if photosynthesis-respiration coupling occurs in this species harbouring secondary green plastids (i.e. originated from an endosymbiosis between a green alga and a phagotrophic euglenozoan). Second, we tested how the trophic state (mixotrophy and photoautotrophy) of the cell alters the mechanisms involved in the photosynthesis-respiration coupling. Energetic coupling between photosynthesis and respiration was determined by testing the effect of respiratory inhibitors on photosynthesis, and measuring the simultaneous variation of photosynthesis and respiration rates as a function of temperature (i.e. thermal response curves). The mechanism involved in the photosynthesis-respiration coupling was assessed by combining proteomics, biophysical and cytological analyses. Our work shows that there is photosynthesis-respiration coupling and membrane contacts between mitochondria and chloroplasts in E. gracilis. However, whereas in mixotrophy adjustment of the chloroplast ATP/NADPH ratio drives the interaction, in photoautotrophy the coupling is conditioned by CO2 limitation and photorespiration. This indicates that maintenance of photosynthesis-respiration coupling, through plastic metabolic responses, is key to E. gracilis functioning under changing environmental conditions.

K+-specific importers Trk1 and Trk2 play different roles in Ca2+ homeostasis and signalling in Saccharomyces cerevisiae cells.

The maintenance of K+ and Ca2+ homeostasis is crucial for many cellular functions. Potassium is accumulated in cells at high concentrations, while the cytosolic level of calcium, to ensure its signalling function, is kept at low levels and transiently increases in response to stresses. We examined Ca2+ homeostasis and Ca2+ signalling in Saccharomyces cerevisiae strains lacking plasma-membrane K+ influx (Trk1 and Trk2) or efflux (Tok1, Nha1 and Ena1-5) systems. The lack of K+ exporters slightly increased the cytosolic Ca2+, but did not alter the Ca2+ tolerance or Ca2+-stress response. In contrast, the K+-importers Trk1 and Trk2 play important and distinct roles in the maintenance of Ca2+ homeostasis. The presence of Trk1 was vital mainly for the growth of cells in the presence of high extracellular Ca2+, whilst the lack of Trk2 doubled steady-state intracellular Ca2+ levels. The absence of both K+ importers highly increased the Ca2+ response to osmotic or CaCl2 stresses and altered the balance between Ca2+ flux from external media and intracellular compartments. In addition, we found Trk2 to be important for the tolerance to high KCl and hygromycin B in cells growing on minimal media. All the data describe new interconnections between potassium and calcium homeostasis in S. cerevisiae.

From the Uncharacterized Protein Family 0016 to the GDT1 family: Molecular insights into a newly-characterized family of cation secondary transporters.

The Uncharacterized Protein Family 0016 (UPF0016) gathers poorly studied membrane proteins well conserved through evolution that possess one or two copies of the consensus motif Glu-x-Gly-Asp-(Arg/Lys)-(Ser/Thr). Members are found in many eukaryotes, bacteria and archaea. The interest for this protein family arose in 2012 when its human member TMEM165 was linked to the occurrence of Congenital Disorders of Glycosylation (CDGs) when harbouring specific mutations. Study of the UPF0016 family is undergone through the characterization of the bacterium Vibrio cholerae (MneA), cyanobacterium Synechocystis (SynPAM71), yeast Saccharomyces cerevisiae (Gdt1p), plant Arabidopsis thaliana (PAM71 and CMT1), and human (TMEM165) members. These proteins have all been identified as transporters of cations, more precisely of Mn2+, with an extra reported function in Ca2+ and/or H+ transport for some of them. Apart from glycosylation in humans, the UPF0016 members are required for lactation in humans, photosynthesis in plants and cyanobacteria, Ca2+ signaling in yeast, and Mn2+ homeostasis in the five aforementioned species. The requirement of the UPF0016 members for key physiological processes most likely derives from their transport activity at the Golgi membrane in human and yeast, the chloroplasts membranes in plants, the thylakoid and plasma membranes in cyanobacteria, and the cell membrane in bacteria. In the light of these studies on various UPF0016 members, this family is not considered as uncharacterized anymore and has been renamed the Gdt1 family according to the name of its S. cerevisiae member. This review aims at assembling and confronting the current knowledge in order to identify shared and distinct features in terms of transported molecules, mode of action, structure, etc., as well as to better understand their corresponding physiological roles.

Endophilin-A3 and Galectin-8 control the clathrin-independent endocytosis of CD166.

While several clathrin-independent endocytic processes have been described so far, their biological relevance often remains elusive, especially in pathophysiological contexts such as cancer. In this study, we find that the tumor marker CD166/ALCAM (Activated Leukocyte Cell Adhesion Molecule) is a clathrin-independent cargo. We show that endophilin-A3-but neither A1 nor A2 isoforms-functionally associates with CD166-containing early endocytic carriers and physically interacts with the cargo. Our data further demonstrates that the three endophilin-A isoforms control the uptake of distinct subsets of cargoes. In addition, we provide strong evidence that the construction of endocytic sites from which CD166 is taken up in an endophilin-A3-dependent manner is driven by extracellular galectin-8. Taken together, our data reveal the existence of a previously uncharacterized clathrin-independent endocytic modality, that modulates the abundance of CD166 at the cell surface, and regulates adhesive and migratory properties of cancer cells.

A new pH sensor localized in the Golgi apparatus of Saccharomyces cerevisiae reveals unexpected roles of Vph1p and Stv1p isoforms.

The gradual acidification of the secretory pathway is conserved and extremely important for eukaryotic cells, but until now there was no pH sensor available to monitor the pH of the early Golgi apparatus in Saccharomyces cerevisiae. Therefore, we developed a pHluorin-based sensor for in vivo measurements in the lumen of the Golgi. By using this new tool we show that the cis- and medial-Golgi pH is equal to 6.6-6.7 in wild type cells during exponential phase. As expected, V-ATPase inactivation results in a near neutral Golgi pH. We also uncover that surprisingly Vph1p isoform of the V-ATPase is prevalent to Stv1p for Golgi acidification. Additionally, we observe that during changes of the cytosolic pH, the Golgi pH is kept relatively stable, mainly thanks to the V-ATPase. Eventually, this new probe will allow to better understand the mechanisms involved in the acidification and the pH control within the secretory pathway.

The human Golgi protein TMEM165 transports calcium and manganese in yeast and bacterial cells.

Cases of congenital disorders of glycosylation (CDG) have been associated with specific mutations within the gene encoding the human Golgi TMEM165 (transmembrane protein 165), belonging to UPF0016 (uncharacterized protein family 0016), a family of secondary ion transporters. To date, members of this family have been reported to be involved in calcium, manganese, and pH homeostases. Although it has been suggested that TMEM165 has cation transport activity, direct evidence for its Ca2+- and Mn2+-transporting activities is still lacking. Here, we functionally characterized human TMEM165 by heterologously expressing it in budding yeast (Saccharomyces cerevisiae) and in the bacterium Lactococcus lactis Protein production in these two microbial hosts was enhanced by codon optimization and truncation of the putatively autoregulatory N terminus of TMEM165. We show that TMEM165 expression in a yeast strain devoid of Golgi Ca2+ and Mn2+ transporters abrogates Ca2+- and Mn2+-induced growth defects, excessive Mn2+ accumulation in the cell, and glycosylation defects. Using bacterial cells loaded with the fluorescent Fura-2 probe, we further obtained direct biochemical evidence that TMEM165 mediates Ca2+ and Mn2+ influxes. We also used the yeast and bacterial systems to evaluate the impact of four disease-causing missense mutations identified in individuals with TMEM165-associated CDG. We found that a mutation leading to a E108G substitution within the conserved UPF0016 family motif significantly reduces TMEM165 activity. These results indicate that TMEM165 can transport Ca2+ and Mn2+, which are both required for proper protein glycosylation in cells. Our work also provides tools to better understand the pathogenicity of CDG-associated TMEM165 mutations.

Determination of the Cellular Ion Concentration in Saccharomyces cerevisiae Using ICP-AES.

The yeast Saccharomyces cerevisiae has been perceived over decades as a highly valuable model organism for the investigation of ion homeostasis. Indeed, many of the genes and biological systems that function in yeast ion homeostasis are conserved throughout unicellular eukaryotes to humans. In this context, measurement of the yeast cellular ionic content provides information regarding yeast response to gene deletion or exposure to chemicals for instance. We propose here a protocol that we tested for the analysis of 12 elements (Ba2+, Ca2+, Cd2+, Co2+, Cu2+, Fe2+, K+, Mg2+, Mn2+, Na+, Ni2+, Zn2+) in yeast using Inductively Coupled Plasma-Atomic Emission Spectrometry (ICP-AES). This technique enables determination of the cellular content of numerous ions from one biological sample.

Manganese Superoxide Dismutase Activity Assay in the Yeast Saccharomyces cerevisiae.

Superoxide dismutases (SODs) act as a primary defence against reactive oxygen species (ROS) by converting superoxide anion radicals (O2 -) into molecular oxygen (O2) and hydrogen peroxide (H2O2). Members of this enzyme family include CuZnSODs, MnSODs, FeSODs, and NiSODs, depending on the nature of the cofactor that is required for proper activity. Most eukaryotes, including yeast, possess CuZnSOD and MnSOD. This protocol aims at assessing the activity of the yeast Saccharomyces cerevisiae MnSOD Sod2p from cellular extracts using nitroblue tetrazolium staining. This method can be used to estimate the cellular bioavailability of Mn2+ as well as to evaluate the redox state of the cell.

Plasma membrane aquaporins interact with the endoplasmic reticulum resident VAP27 proteins at ER-PM contact sites and endocytic structures.

Plasma membrane (PM) intrinsic proteins (PIPs) are aquaporins facilitating the diffusion of water and small solutes. The functional importance of the PM organisation of PIPs in the interaction with other cellular structures is not completely understood. We performed a pull-down assay using maize (Zea mays) suspension cells expressing YFP-ZmPIP2;5 and validated the protein interactions by yeast split-ubiquitin and bimolecular fluorescence complementation assays. We expressed interacting proteins tagged with fluorescent proteins in Nicotiana benthamiana leaves and performed water transport assays in oocytes. Finally, a phylogenetic analysis was conducted. The PM-located ZmPIP2;5 physically interacts with the endoplasmic reticulum (ER) resident ZmVAP27-1. This interaction requires the ZmVAP27-1 cytoplasmic major sperm domain. ZmPIP2;5 and ZmVAP27-1 localise in close vicinity in ER-PM contact sites (EPCSs) and endocytic structures upon exposure to salt stress conditions. This interaction enhances PM water permeability in oocytes. Similarly, the Arabidopsis ZmVAP27-1 paralogue, AtVAP27-1, interacts with the AtPIP2;7 aquaporin. Together, these data indicate that the PIP2-VAP27 interaction in EPCSs is evolutionarily conserved, and suggest that VAP27 might stabilise the aquaporins and guide their endocytosis in response to salt stress.

Pyridoxamine-phosphate oxidases and pyridoxamine-phosphate oxidase-related proteins catalyze the oxidation of 6-NAD(P)H to NAD(P).

6-NADH and 6-NADPH are strong inhibitors of several dehydrogenases that may form spontaneously from NAD(P)H. They are known to be oxidized to NAD(P)+ by mammalian renalase, an FAD-linked enzyme mainly present in heart and kidney, and by related bacterial enzymes. We partially purified an enzyme oxidizing 6-NADPH from rat liver, and, surprisingly, identified it as pyridoxamine-phosphate oxidase (PNPO). This was confirmed by the finding that recombinant mouse PNPO oxidized 6-NADH and 6-NADPH with catalytic efficiencies comparable to those observed with pyridoxine- and pyridoxamine-5'-phosphate. PNPOs from Escherichia coli, Saccharomyces cerevisiae and Arabidopsis thaliana also displayed 6-NAD(P)H oxidase activity, indicating that this 'side-activity' is conserved. Remarkably, 'pyridoxamine-phosphate oxidase-related proteins' (PNPO-RP) from Nostoc punctiforme, A. thaliana and the yeast S. cerevisiae (Ygr017w) were not detectably active on pyridox(am)ine-5'-P, but oxidized 6-NADH, 6-NADPH and 2-NADH suggesting that this may be their main catalytic function. Their specificity profiles were therefore similar to that of renalase. Inactivation of renalase and of PNPO in mammalian cells and of Ygr017w in yeasts led to the accumulation of a reduced form of 6-NADH, tentatively identified as 4,5,6-NADH3, which can also be produced in vitro by reduction of 6-NADH by glyceraldehyde-3-phosphate dehydrogenase or glucose-6-phosphate dehydrogenase. As 4,5,6-NADH3 is not a substrate for renalase, PNPO or PNPO-RP, its accumulation presumably reflects the block in the oxidation of 6-NADH. These findings indicate that two different classes of enzymes using either FAD (renalase) or FMN (PNPOs and PNPO-RPs) as a cofactor play an as yet unsuspected role in removing damaged forms of NAD(P).

Yeast α-arrestin Art2 is the key regulator of ubiquitylation-dependent endocytosis of plasma membrane vitamin B1 transporters.

Endocytosis of membrane proteins in yeast requires α-arrestin-mediated ubiquitylation by the ubiquitin ligase Rsp5. Yet, the diversity of α-arrestin targets studied is restricted to a small subset of plasma membrane (PM) proteins. Here, we performed quantitative proteomics to identify new targets of 12 α-arrestins and gained insight into the diversity of pathways affected by α-arrestins, including the cell wall integrity pathway and PM-endoplasmic reticulum contact sites. We found that Art2 is the main regulator of substrate- and stress-induced ubiquitylation and endocytosis of the thiamine (vitamin B1) transporters: Thi7, nicotinamide riboside transporter 1 (Nrt1), and Thi72. Genetic screening allowed for the isolation of transport-defective Thi7 mutants, which impaired thiamine-induced endocytosis. Coexpression of inactive mutants with wild-type Thi7 revealed that both transporter conformation and transport activity are important to induce endocytosis. Finally, we provide evidence that Art2 mediated Thi7 endocytosis is regulated by the target of rapamycin complex 1 (TORC1) and requires the Sit4 phosphatase but is not inhibited by the Npr1 kinase.

Yeast as a Tool for Deeper Understanding of Human Manganese-Related Diseases.

The biological importance of manganese lies in its function as a key cofactor for numerous metalloenzymes and as non-enzymatic antioxidant. Due to these two essential roles, it appears evident that disturbed manganese homeostasis may trigger the development of pathologies in humans. In this context, yeast has been extensively used over the last decades to gain insight into how cells regulate intra-organellar manganese concentrations and how human pathologies may be related to disturbed cellular manganese homeostasis. This review first summarizes how manganese homeostasis is controlled in yeast cells and how this knowledge can be extrapolated to human cells. Several manganese-related pathologies whose molecular mechanisms have been studied in yeast are then presented in the light of the function of this cation as a non-enzymatic antioxidant or as a key cofactor of metalloenzymes. In this line, we first describe the Transmembrane protein 165-Congenital Disorder of Glycosylation (TMEM165-CDG) and Friedreich ataxia pathologies. Then, due to the established connection between manganese cations and neurodegeneration, the Kufor-Rakeb syndrome and prion-related diseases are finally presented.

Mixed Polymer Brushes for the Selective Capture and Release of Proteins.

Selective protein adsorption is a key challenge for the development of biosensors, separation technologies, and smart materials for medicine and biotechnologies. In this work, a strategy was developed for selective protein adsorption, based on the use of mixed polymer brushes composed of poly(ethylene oxide) (PEO), a protein-repellent polymer, and poly(acrylic acid) (PAA), a weak polyacid whose conformation changes according to the pH and ionic strength of the surrounding medium. A mixture of lysozyme (Lyz), human serum albumin (HSA), and human fibrinogen (Fb) was used to demonstrate the success of this strategy. Polymer brush formation and protein adsorption were monitored by quartz crystal microbalance, whereas protein identification after adsorption from the mixture was performed by time-of-flight secondary ion mass spectrometry (ToF-SIMS) with principal component analysis and gel electrophoresis with silver staining. For the ToF-SIMS measurements, adsorption was first performed from single-protein solutions in order to identify characteristic peaks of each protein. Next, adsorption was performed from the mixture of the three proteins. Proteins were also desorbed from the brushes and analyzed by gel electrophoresis with silver staining for further identification. Selective adsorption of Lyz from a mixture of Lyz/HSA/Fb was successfully achieved at pH 9.0 and ionic strength of 10-3 M, while Lyz and HSA, but not Fb, were adsorbed at ionic strength 10-2 M and pH 9.0. The results demonstrate that by controlling the ionic strength, selective adsorption can be achieved from protein mixtures on PEO/PAA mixed brushes, predominantly because of the resulting control on electrostatic interactions. In well-chosen conditions, the selectively adsorbed proteins can also be fully recovered from the brushes by a simple ionic strength stimulus. The developed systems will find applications as responsive biointerfaces in the fields of separation technologies, biosensing, drug delivery, and nanomedicine.