N-terminal Cytosolic Domain Research Articles

Methylation of protein aspartates and deamidated asparagines as a function of blood bank storage and oxidative stress in human red blood cells

Being devoid of de novo protein synthesis capacity, red blood cells (RBCs) have evolved to recycle oxidatively damaged proteins via mechanisms that involve methylation of dehydrated and deamidated aspartate and asparagine residues. Here we hypothesize that such mechanisms are relevant to routine storage in the blood bank. Within the framework of the REDS-III RBC-Omics (Recipient Epidemiology Donor Evaluation Study III Red Blood Cell-Omics) study, packed RBC units (n = 599) were stored under blood bank conditions for 10, 23, and 42 days and profiled for oxidative hemolysis and time-dependent metabolic dysregulation of the trans-sulfuration pathway. In these units, methionine consumption positively correlated with storage age and oxidative hemolysis. Mechanistic studies show that this phenomenon is favored by oxidative stress or hyperoxic storage (sulfur dioxide >95%), and prevented by hypoxia or methyltransferase inhibition. Through a combination of proteomics approaches and 13 C-methionine tracing, we observed oxidation-induced increases in both Asn deamidation to Asp and formation of methyl-Asp on key structural proteins and enzymes, including Band 3, hemoglobin, ankyrin, 4.1, spectrin beta, aldolase, glyceraldehyde 3-phosphate dehydrogenase, biphosphoglycerate mutase, lactate dehydrogenase and catalase. Methylated regions tended to map proximal to the active site (e.g., N316 of glyceraldehyde 3-phosphate dehydrogenase) and/or residues interacting with the N-terminal cytosolic domain of Band 3. While methylation of basic amino acid residues serves as an epigenetic modification in nucleated cells, protein methylation at carboxylate side chains and deamidated asparagines is a nonepigenetic posttranslational sensor of oxidative stress and refrigerated storage in anucleated human RBCs.

SHOU4 Proteins Regulate Trafficking of Cellulose Synthase Complexes to the Plasma Membrane

Cell walls play critical roles in plants, regulating tissue mechanics, defining the extent and orientation of cell expansion, and providing a physical barrier against pathogen attack [1]. Cellulose microfibrils, which are synthesized by plasma membrane-localized cellulose synthase (CESA) complexes, are the primary load-bearing elements of plant cell walls [2]. Cell walls are dynamic structures that are regulated in part by cell wall integrity (CWI)-monitoring systems that feed back to modulate wall properties and the synthesis of new wall components [3]. Several receptor-like kinases have been implicated as sensors of CWI [3-5], including the FEI1/FEI2 receptor-like kinases [4]. Here, we characterize two genes encoding novel plant-specific plasma membrane proteins (SHOU4 and SHOU4L) that were identified in a suppressor screen of the cellulose-deficient fei1 fei2 mutant. shou4 shou4l double mutants display phenotypes consistent with elevated levels of cellulose, and elevated levels of non-crystalline cellulose are present in this mutant. Disruption of SHOU4 and SHOU4L increases the abundance of CESA proteins at the plasma membrane as a result of enhanced exocytosis. The SHOU4/4L N-terminal cytosolic domains directly interact with CESAs. Our results suggest that the SHOU4 proteins regulate cellulose synthesis in plants by influencing the trafficking of CESA complexes to the cell surface.

Structure of the mouse TRPC4 ion channel

Members of the transient receptor potential (TRP) ion channels conduct cations into cells. They mediate functions ranging from neuronally mediated hot and cold sensation to intracellular organellar and primary ciliary signaling. Here we report a cryo-electron microscopy (cryo-EM) structure of TRPC4 in its unliganded (apo) state to an overall resolution of 3.3 Å. The structure reveals a unique architecture with a long pore loop stabilized by a disulfide bond. Beyond the shared tetrameric six-transmembrane fold, the TRPC4 structure deviates from other TRP channels with a unique cytosolic domain. This unique cytosolic N-terminal domain forms extensive aromatic contacts with the TRP and the C-terminal domains. The comparison of our structure with other known TRP structures provides molecular insights into TRPC4 ion selectivity and extends our knowledge of the diversity and evolution of the TRP channels.

Distinct domains of the β1-subunit cytosolic N terminus control surface expression and functional properties of large-conductance calcium-activated potassium (BK) channels

The properties and function of large-conductance calcium- and voltage-activated potassium (BK) channels are modified by the tissue-specific expression of regulatory β1-subunits. Although the short cytosolic N-terminal domain of the β1-subunit is important for controlling both BK channel trafficking and function, whether the same, or different, regions of the N terminus control these distinct processes remains unknown. Here we demonstrate that the first six N-terminal residues including Lys-3, Lys-4, and Leu-5 are critical for controlling functional regulation, but not trafficking, of BK channels. This membrane-distal region has features of an amphipathic helix that is predicted to control the orientation of the first transmembrane-spanning domain (TM1) of the β1-subunit. In contrast, a membrane-proximal leucine residue (Leu-17) controls trafficking without affecting functional coupling, an effect that is in part dependent on controlling efficient endoplasmic reticulum exit of the pore-forming α-subunit. Thus cell surface trafficking and functional coupling with BK channels are controlled by distinct domains of the β1-subunit N terminus.

Maintaining protein homeostasis: early and late endosomal dual recycling for the maintenance of intracellular pools of the plasma membrane protein Chs3.

The major chitin synthase activity in yeast cells, Chs3, has become a paradigm in the study of the intracellular traffic of transmembrane proteins due to its tightly regulated trafficking. This includes an efficient mechanism for the maintenance of an extensive reservoir of Chs3 at the trans-Golgi network/EE, which allows for the timely delivery of the protein to the plasma membrane. Here we show that this intracellular reservoir of Chs3 is maintained not only by its efficient AP-1-mediated recycling, but also by recycling through the retromer complex, which interacts with Chs3 at a defined region in its N-terminal cytosolic domain. Moreover, the N-terminal ubiquitination of Chs3 at the plasma membrane by Rsp5/Art4 distinctly labels the protein and regulates its retromer-mediated recycling by enabling Chs3 to be recognized by the ESCRT machinery and degraded in the vacuole. Therefore the combined action of two independent but redundant endocytic recycling mechanisms, together with distinct labels for vacuolar degradation, determines the final fate of the intracellular traffic of the Chs3 protein, allowing yeast cells to regulate morphogenesis, depending on environmental constraints.

AltitudeOmics: Red Blood Cell Metabolic Adaptation to High Altitude Hypoxia.

Red blood cells (RBCs) are key players in systemic oxygen transport. RBCs respond to in vitro hypoxia through the so-called oxygen-dependent metabolic regulation, which involves the competitive binding of deoxyhemoglobin and glycolytic enzymes to the N-terminal cytosolic domain of band 3. This mechanism promotes the accumulation of 2,3-DPG, stabilizing the deoxygenated state of hemoglobin, and cytosol acidification, triggering oxygen off-loading through the Bohr effect. Despite in vitro studies, in vivo adaptations to hypoxia have not yet been completely elucidated. Within the framework of the AltitudeOmics study, erythrocytes were collected from 21 healthy volunteers at sea level, after exposure to high altitude (5260 m) for 1, 7, and 16 days, and following reascent after 7 days at 1525 m. UHPLC-MS metabolomics results were correlated to physiological and athletic performance parameters. Immediate metabolic adaptations were noted as early as a few hours from ascending to >5000 m, and maintained for 16 days at high altitude. Consistent with the mechanisms elucidated in vitro, hypoxia promoted glycolysis and deregulated the pentose phosphate pathway, as well purine catabolism, glutathione homeostasis, arginine/nitric oxide, and sulfur/H2S metabolism. Metabolic adaptations were preserved 1 week after descent, consistently with improved physical performances in comparison to the first ascendance, suggesting a mechanism of metabolic memory.

Role of the Kinase Domain in TRPM7 Channel Activity and Function

Transient Receptor Potential Melastatin 7 is an integral membrane protein comprising a non-specific cation channel, a cytosolic C-terminal kinase and a functionally undefined cytosolic N-terminal domain. TRPM7 is ‘ubiquitously’ expressed, playing vital roles in cell survival, proliferation and differentiation. Proliferating cells generally exhibit larger TRPM7 currents than non-dividing, terminally differentiated cells. Channel properties are well described and are thought to be responsible for the Mg2+/Ca2+ influx supporting cell biological functions. The channel exhibits strong rectification in the presence of extracellular or cytosolic Mg2+. Kinase functionality is less well understood other than that channel properties are unaltered between wild type (WT) and kinase-inactive mutants. Recent work shows that the kinase domain can be cleaved from the channel in vivo [Krapivinsky et al. Cell. 157 (2014) 1061]. The ectopically expressed kinase domain localizes to the nucleoplasm and the kinase-deleted channel (ΔK) exhibits a possible length-dependent range of activities relative to WT. Here we addressed the role of the kinase domain in regulating channel activation. We confirmed the nuclear expression of ectopically expressed murine TRPM7 kinase (residues 1511-1863) and a similar localization of endogenous TRPM7 in proliferating liver cells (WIF-B) suggesting that these cells express the cleaved kinase. Conversely non-dividing hepatocytes are labelled in the nuclear envelop indicating that TRPM7 is mainly intact. Fractional currents (I/Imax) in HEK293 cells at break-in are significantly lower in ΔK (0.18±0.08) compared with WT (0.47±0.12) expressors. Mean activation time constants are 2.5 times lower in ΔK versus WT cells. Removal of the kinase domain increases sensitivity to both extra- and intracellular Mg2+. Endogenous currents in WIF-B cells reflect a hybrid of these properties. We conclude that a complex dynamic exists between subcellular compartmentalization, proteolysis and the cellular role(s) of TRPM7.

Membrane-associated collagens with interrupted triple-helices (MACITs): evolution from a bilaterian common ancestor and functional conservation in C. elegans.

BackgroundCollagens provide structural support and guidance cues within the extracellular matrix of metazoans. Mammalian collagens XIII, XXIII and XXV form a unique subgroup of type II transmembrane proteins, each comprising a short N-terminal cytosolic domain, a transmembrane domain and a largely collagenous ectodomain. We name these collagens as MACITs (Membrane-Associated Collagens with Interrupted Triple-helices), and here investigate their evolution and conserved properties. To date, these collagens have been studied only in mammals. Knowledge of the representation of MACITs in other extant metazoans is lacking. This question is of interest for understanding structural/functional relationships in the MACIT family and also for insight into the evolution of MACITs in relation to the secreted, fibrillar collagens that are present throughout the metazoa.ResultsMACITs are restricted to bilaterians and are represented in the Ecdysozoa, Hemichordata, Urochordata and Vertebrata (Gnathostomata). They were not identified in available early-diverging metazoans, Lophotrochozoa, Echinodermata, Cephalochordata or Vertebrata (Cyclostomata). Whereas invertebrates encode a single MACIT, collagens XIII/XXIII/XXV of jawed vertebrates are paralogues that originated from the two rounds of en-bloc genome duplication occurring early in vertebrate evolution. MACITs have conserved domain architecture in which a juxta-membrane furin-cleavage site and the C-terminal 34 residues are especially highly conserved, whereas the cytoplasmic domains are weakly conserved. To study protein expression and function in a metazoan with a single MACIT gene, we focused on Caenorhabditis elegans and its col-99 gene. A col-99 cDNA was cloned and expressed as protein in mammalian CHO cells, two antibodies against COL-99 protein were generated, and a col-99-bearing fosmid gene construct col-99::egfp::flag was used to generate transgenic C. elegans lines. The encoded COL-99 polypeptide is 85 kDa in size and forms a trimeric protein. COL-99 is plasma membrane-associated and undergoes furin-dependent ectodomain cleavage and shedding. COL-99 is detected in mouth, pharynx, body wall and the tail, mostly in motor neurons and muscle systems and is enriched at neuromuscular junctions.ConclusionsThrough identification of MACITs in multiple metazoan phyla we developed a model for the evolution of MACITs. The experimental data demonstrate conservation of MACIT molecular and cellular properties and tissue localisations in the invertebrate, C. elegans.Electronic supplementary materialThe online version of this article (doi:10.1186/s12862-015-0554-3) contains supplementary material, which is available to authorized users.

EPR Spectroscopic Study of the Voltage-Sensor Domain (VSD) of the Human KCNQ1 Potassium Ion Channel

KCNQ1 is a voltage-gated potassium channel modulated by members of the KCNE protein family. KCNQ1 is essential to both the cardiac action potential that mediates heartbeat and K+ homeostasis in the inner ear. KCNQ1 dysfunction has been linked to multiple diseases, including various cardiac arrhythmias, congenital deafness, and type II diabetes mellitus. Human KCNQ1 is a 676-residue protein consisting of 100-residue N-terminal cytosolic domain, followed by an ∼260-residue channel domain containing six transmembrane (TMD) helices, and a 300-residue cytosolic C-terminus. The first four TMD helices (S1-S4) form the voltage-sensor domain (VSD) that is linked to the pore domain (helices S5 and S6). Roughly 40% of the >200 reported disease-related mutations in the KCNQ1 gene result in amino acid substitutions in the VSD, making structural and dynamic studies of this domain important in unraveling molecular mechanisms in human pathophysiology. Site-directed spin labeling EPR is a very powerful structural biology technique to study the structural and conformational dynamics of membrane proteins in membrane environment. In this study, we have successfully expressed the Cysteine substituted Q1-VSD in E. Coli, purified into detergent micelles, labeled with MTSL spin labels, and collected CW-EPR spectra. We are developing several advanced EPR spectroscopic techniques (such as double electron-electron resonance (DEER) and electron spin echo envelope modulation (ESEEM)) for probing the structural and dynamic properties of Q1-VSD in a lipid bilayer environment. This study will provide important mechanistic information on the Q1-VSD channel upon binding with KCNE1.

Expression, purification and functional reconstitution of FeoB, the ferrous iron transporter from Pseudomonas aeruginosa

The FeoB Fe(II) transporter from the drug resistant pathogen, Pseudomonas aeruginosa is essential for ferrous iron transport and is implicated in virulence and biofilm development. Hence it is an attractive target for the development of new anti-infective drugs. FeoB is an intriguing protein that consists of a cytosolic N-terminal GTPase domain and an integral membrane domain which most likely acts as ferrous iron permease. Characterisation of FeoB is critical for developing therapeutics aimed at inhibiting this protein. However, structural and functional analysis of FeoB is hampered by the lack of high yield homogenously pure protein which is monodisperse, stable and active in solution. Here we describe the optimised procedure for the recombinant expression of FeoB from P. aeruginosa and provide an evaluation of the most favourable purification, pH and detergent conditions. The functional reconstitution of FeoB in liposomes is also described. This represents the first detailed procedure for obtaining a pure, active and stable FeoB solution in milligram quantities which would be amenable to biochemical, biophysical and structural studies.

Structural basis for catalysis in a CDP-alcohol phosphotransferase.

The CDP-alcohol phosphotransferase (CDP-AP) family of integral membrane enzymes catalyzes the transfer of a substituted phosphate group from a CDP-linked donor to an alcohol-acceptor. This is an essential reaction for phospholipid biosynthesis across all kingdoms of life, and it is catalyzed solely by CDP-APs. Here we report the 2.0 Å resolution crystal structure of a representative CDP-AP from Archaeoglobus fulgidus. The enzyme (AF2299) is a homodimer, with each protomer consisting of six transmembrane helices and an N-terminal cytosolic domain. A polar cavity within the membrane accommodates the active site, lined with the residues from an absolutely conserved CDP-AP signature motif (D1xxD2G1xxAR…G2xxxD3xxxD4). Structures in the apo, CMP-bound, CDP-bound and CDP-glycerol-bound states define functional roles for each of these eight conserved residues and allow us to propose a sequential, base-catalyzed mechanism universal for CDP-APs, in which the fourth aspartate (D4) acts as the catalytic base.

Functional role of evolutionarily highly conserved residues, N-glycosylation level and domains of the Leishmania miltefosine transporter-Cdc50 subunit

Cdc50 (cell-cycle control protein 50) is a family of conserved eukaryotic proteins that interact with P4-ATPases (phospholipid translocases). Cdc50 association is essential for the endoplasmic reticulum export of P4-ATPases and proper translocase activity. In the present study, we analysed the role of Leishmania infantum LiRos3, the Cdc50 subunit of the P4-ATPase MLF (miltefosine) transporter [LiMT (L. infantum MLF transporter)], on trafficking and complex functionality using site-directed mutagenesis and domain substitution. We identified 22 invariant residues in the Cdc50 proteins from L. infantum, human and yeast. Seven of these residues are found in the extracellular domain of LiRos3, the conservation of which is critical for ensuring that LiMT arrives at the plasma membrane. The substitution of other invariant residues affects complex trafficking to a lesser extent. Furthermore, invariant residues located in the N-terminal cytosolic domain play a role in the transport activity. Partial N-glycosylation of LiRos3 reduces MLF transport and total N-deglycosylation completely inhibits LiMT trafficking to the plasma membrane. One of the N-glycosylation residues is invariant along the Cdc50 family. The transmembrane and exoplasmic domains are not interchangeable with the other two L. infantum Cdc50 proteins to maintain LiMT interaction. Taken together, these findings indicate that both invariant and N-glycosylated residues of LiRos3 are implicated in LiMT trafficking and transport activity.

The Sec62-Sec63 translocon facilitates translocation of the C-terminus of membrane proteins.

The Sec62-Sec63 complex mediates post-translational translocation of a subset of primarily secretory proteins into the endoplasmic reticulum (ER) in yeast. Therefore, it has been thought that membrane proteins, which are mainly co-translationally targeted into the ER, are not handled by the Sec62-Sec63 translocon. By systematic analysis of single and multi-spanning membrane proteins with broad sequence context [with differing hydrophobicity, flanking charged residues and orientation of transmembrane (TM) segments], we show that mutations in the N-terminal cytosolic domain of yeast Sec62 impair its interaction with Sec63 and lead to defects in membrane insertion and translocation of the C-terminus of membrane proteins. These results suggest that there is an unappreciated function of the Sec62-Sec63 translocon in regulating topogenesis of membrane proteins in the eukaryotic cell.

Cellular vimentin regulates construction of dengue virus replication complexes through interaction with NS4A protein.

Dengue virus (DENV) interacts with host cellular factors to construct a more favorable environment for replication, and the interplay between DENV and the host cellular cytoskeleton may represent one of the potential antiviral targeting sites. However, the involvement of cellular vimentin intermediate filaments in DENV replication has been explored less. Here, we revealed the direct interaction between host cellular vimentin and DENV nonstructural protein 4A (NS4A), a known component of the viral replication complex (RC), during DENV infection using tandem affinity purification, coimmunoprecipitation, and scanning electron microscopy. Furthermore, the dynamics of vimentin-NS4A interaction were demonstrated by using confocal three-dimensional (3D) reconstruction and proximity ligation assay. Most importantly, we report for the first time the discovery of the specific region of NS4A that interacts with vimentin lies within the first 50 amino acid residues at the cytosolic N-terminal domain of NS4A (N50 region). Besides identifying vimentin-NS4A interaction, vimentin reorganization and phosphorylation by calcium calmodulin-dependent protein kinase II occurs during DENV infection, signifying that vimentin reorganization is important in maintaining and supporting the DENV RCs. Interestingly, we found that gene silencing of vimentin by small interfering RNA induced a significant alteration in the distribution of RCs in DENV-infected cells. This finding further supports the crucial role of intact vimentin scaffold in localizing and concentrating DENV RCs at the perinuclear site, thus facilitating efficient viral RNA replication. Collectively, our findings implicate the biological and functional significance of vimentin during DENV replication, as we propose that the association of DENV RCs with vimentin is mediated by DENV NS4A.

A novel intermediate mucolipidosis II/IIIαβ caused by GNPTAB mutation in the cytosolic N-terminal domain

Mucolipidosis (ML) II and ML IIIα/β are allelic autosomal recessive metabolic disorders due to mutations in GNPTAB. The gene encodes the enzyme UDP-GlcNAc-1-phosphotransferase (GNPT), which is critical to proper trafficking of lysosomal acid hydrolases. The ML phenotypic spectrum is dichotomous. Criteria set for defining ML II and ML IIIα/β are inclusive for all but the few patients with phenotypes that span the archetypes. Clinical and biochemical findings of the 'intermediate' ML in eight patients with the c.10A>C missense mutation in GNPTAB are presented to define this intermediate ML and provide a broader insight into ML pathogenesis. Extensive clinical information, including radiographic examinations at various ages, was obtained from a detailed study of all patients. GNPTAB was sequenced in probands and parents. GNPT activity was measured and cathepsin D sorting assays were performed in fibroblasts. Intermediate ML patients who share the c.10A>C/p.K4Q mutation in GNPTAB demonstrate a distinct, consistent phenotype similar to ML II in physical and radiographic features and to ML IIIα/β in psychomotor development and life expectancy. GNPT activity is reduced to 7-12% but the majority of newly synthesized cathepsin D remains intracellular. The GNPTAB c.10A>C/p.K4Q missense allele results in an intermediate ML II/III with distinct clinical and biochemical characteristics. This delineation strengthens the utility of the discontinuous genotype-phenotype correlation in ML II and ML IIIα/β and prompts additional studies on the tissue-specific pathogenesis in GNPT-deficient ML.

Effects of optional structural elements, including two alternative amino termini and a new splicing cassette IV, on the function of the sodium–bicarbonate cotransporter NBCn1 (SLC4A7)

The SLC4A7 gene encodes the electroneutral sodium/HCO3 cotransporter NBCn1, which plays important physiological and pathophysiological roles in many cell types. Previous work identified six NBCn1 variants differing in the sequence of the extreme N terminus--MEAD in rat only, MERF in human only--as well as in the optional inclusion of cassettes I, II, and III. Earlier work also left open the question of whether optional structural elements (OSEs) affect surface abundance or intrinsic (per-molecule) transport activity. Here, we demonstrate for the first time that SLC4A7 from one species can express both MEAD- and MERF-NBCn1. We also identify a novel cassette IV of 20 aa, and extend by 10 the number of full-length NBCn1 variants. The alternative N termini and four cassettes could theoretically produce 32 major variants. Moreover, we identify a group of cDNAs predicted to encode just the cytosolic N-terminal domain (Nt) of NBCn1. A combination of electrophysiology and biotinylation shows that the OSEs can affect surface abundance and intrinsic HCO3(-) transport activity of NBCn1, as expressed in Xenopus oocytes. Specifically, MEAD tends to increase whereas novel cassette IV reduces surface abundance. Cassettes II, III and novel cassette IV all appear to increase the intrinsic activity of NBCn1.

Presenilins regulate the cellular activity of ryanodine receptors differentially through isotype-specific N-terminal cysteines

Presenilins (PS), endoplasmic reticulum (ER) transmembrane proteins, form the catalytic core of γ-secretase, an amyloid precursor protein processing enzyme. Mutations in PS lead to Alzheimer's disease (AD) by altering γ-secretase activity to generate pathologic amyloid beta and amyloid plaques in the brain. Here, we identified a novel mechanism where binding of a soluble, cytosolic N-terminal domain fragment (NTF) of PS to intracellular Ca2+ release channels, ryanodine receptors (RyR), controls Ca2+ release from the ER. While PS1NTF decreased total RyR-mediated Ca2+ release, PS2NTF had no effect at physiological Ca2+ concentrations. This differential function and isotype-specificity is due to four cysteines absent in PS1NTF, present, however, in PS2NTF. Site-directed mutagenesis targeting these cysteines converted PS1NTF to PS2NTF function and vice versa, indicating differential RyR binding. This novel mechanism of intracellular Ca2+ regulation through the PS–RyR interaction represents a novel target for AD drug development and the treatment of other neurodegenerative disorders that critically depend on RyR and PS signaling.

Deoxygenation of leucofiltered erythrocyte concentrates preserves proteome stability during storage in the blood bank.

While prospective clinical trials are currently underway in several countries throughout the world1, the safety and effectiveness of long-stored erythrocyte concentrates have been questioned by controversial retrospective clinical evidence2,3 and accumulating biochemical investigations4–6. Laboratory investigations have recently highlighted that a significant number of biochemical and mechanical lesions accumulate in erythrocyte concentrates during their storage in the blood bank. These alterations, often referred to as “storage lesions”, include: (i) the progressive impairment of cell metabolism, leading to the consumption of high energy phosphate compounds (ATP and 2,3-diphosphoglycerate)7; (ii) the accumulation of oxidative stress, resulting in the oxidation8, non-enzymatic glycosylation9, fragmentation or aggregation of the red blood cell (RBC) membrane10–13 and cytosolic proteins14; (iii) the progressive loss of metabolic modulation15, as a result of oxidative stress (especially in non-leucoreduced units)16 and protease activity-dependent fragmentation of the N-terminal cytosolic domain of band 317, which results in impairment of the regulatory function of the so-called “respiratory metabolon”18,19; (iv) the progressive oxidation of lipids and deregulation of lipid homeostasis20,21; (v) the exacerbation of membrane blebbing and shedding of microvesicles, a self-protective mechanism22, closely mimicking apoptosis23, which allows cells to get rid of no-longer functional or irreversibly altered proteins and lipids24, while retaining rheological properties of the donor25, which might result in pro-immunogenic potential in the recipient, together with newly appearing storage-dependent biomarkers promoting RBC phagocytosis, such as phosphatidylserine exposure; and (vi) microvesiculation events that compromise RBC morphology (changes from a discocytic to a spheroechnicocytic or spherocytic phenotype), which ends up impairing the surface-to-volume ratio, thereby increasing osmotic fragility26,27, and increasing membrane rigidity, as a result of progressive leaching and intercalation of the plasticizers (such as DEHP) into the membrane lipid bilayer28. In the light of such changes, alternative storage strategies have been proposed over the years, such as deoxygenation of packed erythrocyte concentrates through other methods29,30. The rationale underpinning such a strategy is that deoxygenation of erythrocyte concentrates would remove the main substrate for the production of reactive oxygen species, while promoting energy metabolism through the Embden-Meyerhoff pathway in the light of the oxygen-dependent metabolic modulation mediated by the competitive binding of deoxyhemoglobin and glycolytic enzymes to the N-terminal cytosolic domain of band 318,19. Early studies by independent groups (and through independent processing strategies29,30) found deoxygenation-dependent improvement of hemolysis and morphological parameters, as well as prolonged preservation of high energy phosphate compounds29–31, though at the expenses of the NADPH-generating potential via the pentose phosphate pathway, as gleaned through metabolomics approaches31. In the light of these encouraging results, we decided to apply a proteomics workflow to understand whether the RBC membrane proteome of erythrocytes stored under deoxygenation in leucofiltered-units is better preserved than in untreated controls. In our previous investigation on deoxygenated units of non-leucofiltered erythrocyte concentrates12 we concluded that the overall spot numbers in two-dimensional electrophoresis (2DE) gels could represent a diagnostic marker of proteome homeostasis during storage in the blood bank. Indeed, RBC are devoid of nuclei and organelles and are, therefore, incapable of synthesising proteins de novo. Any alteration to the overall number of spots in 2DE gels can, therefore, be attributed to storage-dependent effects on the membrane proteome, which we previously identified as fragmentation and aggregation phenomena during the first two-three weeks of storage (increased number of detected spots), followed by vesiculation of damaged proteins (decreased number of spots)11,12. Furthermore, in control units, we previously correlated the storage-dependent alteration of the erythrocyte membrane proteome stability to an impairment in osmotic fragility26. Concordantly, in the present study we performed 2DE assays of the membrane proteome of leucofiltered erythrocytes stored under control conditions or in a deoxygenated environment (following oxygen removal, as previously described)30. As a result, we could confirm that deoxygenation had beneficial effects in terms of membrane proteome homeostasis and preserved osmotic resistance.

Substitution of transmembrane domain Cys residues alters pHo-sensitive anion transport by AE2/SLC4A2 anion exchanger

AE2/SLC4A2 is the most widely expressed of the Na(+)-independent SLC4 Cl(-)/HCO3 (-) exchangers and is essential for postnatal survival, but its structure remains unknown. We have generated and expressed a mouse AE2 construct devoid of transmembrane domain cysteine (Cys) residues, mAE2Cys-less, to enhance the utility of Cys-substitution mutagenesis for structural and structure-function studies of mAE2. mAE2Cys-less expressed in Xenopus oocytes exhibited partial reduction of stilbene disulfonate-sensitive anion exchange activity. This activity was independent of the mAE2 N-terminal cytosolic domain and was accompanied by near-normal surface expression, without change in K 1/2 for extracellular Cl(-). mAE2Cys-less exhibited wildtype activation of anion exchange by hypertonicity and by NH4Cl, and wildtype inhibition of anion exchange by acidic intracellular pH (pHi) in the absence of NH4 (+). However, inhibition of anion exchange by extracellular pH (pHo) exhibited an alkaline shifted pHo(50) value of at least 0.6-0.7 pH units. Although SO4 (2-) transport by mAE2Cys-less resembled wildtype mAE2 in its stimulation by acidic pHo, the absence of transmembrane domain Cys residues abrogated activation of oxalate transport by acidic pHo. The contrasting enhancement of SO4 (2-) transport by alkaline pHo in the mAE1 anion translocation pathway mutant E699Q (Am J Physiol Cell Physiol 295: C302) was phenocopied by the corresponding mutant E1007Q in both AE2 and AE2Cys-less. However, the absence of transmembrane domain Cys residues exacerbated the reduced basal anion transport function exhibited by this and other missense substitutions at AE2 residue E1007. AE2Cys-less will be a valuable experimental tool for structure-function studies of the SLC4 gene family, but its utility for studies of AE2 regulation by extracellular pH must be evaluated in the context of its alkaline-shifted pHo sensitivity, resembling that of AE2 gastric parietal cell variant AE2c1.

Crystal Structure of the Klebsiella pneumoniae NFeoB/FeoC Complex and Roles of FeoC in Regulation of Fe 2+ Transport by the Bacterial Feo System

Feo is a transport system commonly used by bacteria to acquire environmental Fe(2+). It consists of three proteins: FeoA, FeoB, and FeoC. FeoB is a large protein with a cytosolic N-terminal domain (NFeoB) that contains a regulatory G protein domain and a helical S domain. The C-terminal region of FeoB is a transmembrane domain that likely acts as the Fe(2+) permease. NFeoB has been shown to form a trimer pore that may function as an Fe(2+) gate. FeoC is a small winged-helix protein that possesses four conserved cysteine residues with a consensus sequence that likely provides binding sites for the [Fe-S] cluster. Therefore, FeoC is presumed to be an [Fe-S] cluster-dependent regulator that directly controls transcription of the feo operon. Despite the apparent significance of the Feo system, however, the function of FeoC has not been experimentally demonstrated. Here, we show that Klebsiella pneumoniae FeoC (KpFeoC) forms a tight complex with the intracellular N-terminal domain of FeoB (KpNFeoB). The crystal structure of the complex reveals that KpFeoC binds to KpNFeoB between the switch II region of the G protein domain and the effector S domain and that the long KpFeoC W1 loop lies above the KpNFeoB nucleotide-binding site. These interactions suggest that KpFeoC modulates the guanine nucleotide-mediated signal transduction process. Moreover, we showed that binding of KpFeoC disrupts pore formation by interfering with KpNFeoB trimerization. These results provide strong evidence suggesting that KpFeoC plays a crucial role in regulating Fe(2+) transport in Klebsiella pneumonia in addition to the presumed gene regulator role.