membrane-associated folate-bindingprotein and cannot grow under similar low folate conditions. In these studies, 3T3 cells were transfected with a vector containing the cDNA that codes for the KB cell folate-bindingprotein. In contrast to the wild-type 3T3 cells, the transfected 3T3 cells express a level of folate-bindingprotein similar to KB cells, 1 and 1.4 ng/micrograms protein, respectively. The capacity for binding [3H] folate to the surface of transfected 3T3 cells cultured in folate-deficient medium is 7.7 pmol/10(6) cells, and this is approximately 50% of the surface binding capacity of KG cells under similar culture
concentrations of folate. A function for the soluble form has not yet been identified. We constructed a cDNA library from a human carcinoma cell line, Caco-2, which expresses the membrane form abundantly. The library was screened and a near full-length cDNA for the folate binder was isolated. Transfection of COS cells with the cDNA inserted in an
Folate, also known as vitamin B9, is essential in cell metabolism and very important especially for pregnant women and lactating mothers. Natural folate is available in food but it is very unstable. Synthetic folate is generally used as an alternative to meet daily needs due to its stability, even though it has a negative effect causing a variety of metabolic disorders. Some lactic acid bacteria have been reported as being able to synthesize natural folate during the fermentation process. Lactic acid bacteria are the main microorganisms for lactic fermentation such as fermented milk, fruits, and vegetables. Milk is the most nutritious food and contains folate-bindingprotein, hence it is considered the ideal fermentation medium to increase folate stability during storage. Fermentation of milk with folate-producing lactic acid bacteria can be used as a technique to produce natural folate-rich fermented foods as an attempt to prevent folate deficiency without side effects to the consumers.
The influence of extracellular folate concentration on cellular levels of the folate transport protein and its soluble product was studied directly in cultured human nasopharyngeal carcinoma (KB) cells. As determined by radioimmunoassay, levels of the folate transport protein and the soluble folate-bindingprotein were 58 +/- 17 (mean +/- SD) and 5 +/- 2 pmol/mg cell protein, respectively, in KB cells maintained in standard medium (containing 2,300 nM folic acid). These levels significantly increased to 182 +/- 34 and 26 +/- 6 pmol/mg cell protein, respectively, in KB cells serially passaged in low folate medium (containing 2- 10 nM 5-methyltetrahydrofolate). Increases in folate-bindingprotein levels occurred more rapidly in KB cells serially passaged in very low folate medium containing less than 2 nM folate and were prevented by the addition of 100 nM 5-methyltetrahydrofolate or 0.1-1 microM 5-formyltetrahydrofolate to this medium. When KB cells which had been passaged in low folate medium were passaged back into either standard medium or low folate
saturable, and was much greater for pteroylmonoglutamate and 5-methyltetrahydrofolate than 5-formyltetrahydrofolate and amethopterin. On detergent solubilization of membranes, two peaks of specific folatebinding with Mr greater than or equal to 200,000 and 160,000 were identified on Sephacryl S-200 gel filtration chromatography in Triton X-100, and this corresponded to two similar peaks of immunoprecipitated material when solubilized iodinated membranes were probed with anti-human placental folate receptor antiserum. Age-dependent separation of erythrocytes by Stractan density gradients revealed a sevenfold greater folatebinding capacity in membranes purified from younger compared with aged erythrocytes. Since this difference was not reflected in proportionately higher immunoreactive folatebindingprotein, (as determined by a specific radioimmunoassay for these proteins), or differences in affinity in younger than aged cells, these findings indicate that erythrocyte folatebinding proteins become progressively nonfunctional at the onset of red cell aging.
Surface modification of drug carriers with bioactive molecules that can be adsorbed, coated, conjugated or linked to them which interact with cell receptors demon- strate a selective affinity for a specific cell or tissue type and can subsequently enhance drug uptake (Figure 1). The modified-coating (e.g. combined albumin and chitosan) can also be used to prevent enzymatic degradation both on the GI tract and plasma . Monoclonal antibodies (or fragments) or non-antibody ligands like carbohy- drates specific for cell surface such as lectins have been investigated . Also, most recently small molecules or peptides agonists/substracts or antagonists/inhibitors for receptors that are overexpressed on cell surface of spe- cific tissue (e.g. folate, transferrin as well as galactosa- mine) have shown promising results [6,54,79,80]. Sev- eral considerations have to be taken as the use of target- ing ligands which can enhance distribution to secondary target sites of non-intended tissues . In fact, the dis- advantage of using non-antibody ligands is their non- selective expression . On the other hand, immuno- conjugates poses problems related to immunogenicity and retention in the reticuloendothelial system (RES) . The carrier surface modification can also incorporate coatings to change the lipophilicity/hydrophilicity profile, prevent the uptake by immune cells and improve cell recognition (e.g. the synergy between the distribution and signaling of antibodies). As a result, per example, once IV injection occurs, nanoparticles are cleared from the plasma within a few minutes due to opsonization and subsequent phagocytosis by the cells of the RES . Opsonization can be reduced by applying some surface ligands. An example is PEG, a hydrophilic polymer, which promotes the resistance to the binding of plasma proteins and prevents aggregation induced by salts and proteins in the serum . This fact prevents opsoniza- tion and recognition from phagocytes and thus avoiding immune responses. Moreover, PEG can also reduce the
Recently, the question concerning integration of the PG layer and septal junction complex formation has been addressed. If sep- tal junction proteins from adjacent cells interact with each other, they should traverse the septal PG layer(s). The presence of perfo- rations, also termed nanopores, in septal PG discs has been dem- onstrated for Nostoc punctiforme recently (23). The periplasmic amidase AmiC2 has been shown to be involved in the formation of such structures (23). Based on cell-cell communication analysis, an ortholog of AmiC2 in Anabaena, encoded by alr0092 and an- notated AmiC1, has also been found to influence septal junction formation (24). Thus, those perforations can be the channels through which septal junction complexes traverse the septal PG. However, a possible regulation of the dimension of the septal junction channel is unknown. Here we describe a further protein factor encoded by all1861 that influences septal junction channel formation in Anabaena. This factor restricts the size of the chan- nels in the PG layer, leading to its annotation as peptidoglycan septal junction channel formation protein no. 1 (SjcF1).
In recent years, atomic force microscopy (AFM) has been in- creasingly used to study the molecular basis of staphylococcal ad- hesion (15–22), providing novel insight into the binding mecha- nisms of MSCRAMMs. So far, however, the technique has never been applied to investigate the binding strength and molecular elasticity of Cna. Here, we used single-cell and single-molecule AFM to measure the mechanical strength of Cna in living bacteria and to determine the contribution of the B region to the protein mechanics. We show that single Cna-Cn bonds are much stronger than those generally measured for bacterial adhesins, consistent with the high-affinity collagen hug mechanism. The B region is required for strong binding and displays nanospring properties that, we believe, fulfill an important function: by acting as a rigid spring, the B repeats aid Cna to project away from the cell surface and to maintain bacterial adhesion even under conditions of high mechanical stress. We also quantify the antiadhesion activity of a FIG 1 Studying Cna-mediated adhesion. (A) Schematic structure of Cna showing the ligand-binding A region made of the N1N2N3 subdomains (beginning at residues 31, 140, and 344, respectively) and the repeated B region consisting of two B-repeat units (beginning at residues 535 and 722) and whose functional role is unclear. The number of B repeats is strain dependent: the Phillips strain used here has 2 B repeats, whereas the one used in structural studies has 3 repeats (5). (B) Microscale adherence assay using optical microscopy. Images of S. aureus Phillips bacteria expressing or not expressing full-length Cna [Cna ( ⫹ ) and
systems using deuterium or carbon relaxation methods  . These studies have shown that many proteins display three classes of motion that involve various degrees of rotameric interconversion on the picosecond-nanosecond timescale . Of particular interest is that these motions appear to report on the changes in conformational entropy associated with a change in protein functional state [10, 28-‐29]. Indeed, recent advances indicate that appropriate use of NMR relaxation methodologies can quantitatively access this important thermodynamic feature of protein molecules [30, 53]. These types of investigations have been carried out almost exclusively in the solution state. However, protein powders hydrated to various degrees are often employed in biophysical investigations using techniques such as neutron scattering [54-55]. Thus it is of interest to compare the internal dynamics of fully solvated and partially hydrated protein molecules. Here we use a small subdomain of the villin headpiece (HP36) and employ deuterium relaxation to illuminate the dynamics of the protein in free aqueous solution and compare them to those observed in a partially hydrated powder.
SpA mediates bacterial adhesion to immobilized vWF through speciﬁc bonds that are much stronger ( ⬃ 2 nN) than most receptor-ligand interactions studied to date. Compared to its soluble form, immobilized vWF is biologically important as it promotes platelet adhesion at sites of vascular injury (8). Weaker bonds are detected on endo- thelial cells, reﬂecting rupture of vWF weakly anchored in the ECM. Notably, vWF binding to SpA is tightly mechanoregulated: the bond is weak at low tensile force, but is dramatically increased at high force. To understand the molecular origin of the force-dependent SpA-vWF adhesion, it is interesting to compare our results with the forces measured so far for other staphylococcal adhesins, especially the structurally and functionally related Staphylococcus epidermidis SdrG and S. aureus ClfA and ClfB. The structural features and molecular biology of these three proteins have been widely investigated (26). They bind to their ligands (e.g., ﬁbrinogen) via the well-known dock, lock, and latch (DLL) mechanism. The binding site of these adhesins is a cleft of 30 Å in length between the N2 and N3 subdomains located in the A region of the protein. Once the ligand peptide is docked and stabilized by hydrophobic interactions and hydrogen bonds, a C-terminus extension of the N3 subdomain folds over the ligand to insert and complement a ␤ -sheet in the N2 subdomain. This DLL mechanism thus greatly stabi- lizes the conformation of the complex (27). Single-molecule AFM experiments have shown that SdrG, ClfA, and ClfB display an extreme mechanical stability (20, 21, 28), with a binding strength much larger than those of all biomolecular bonds studied so far. Recently, the Gaub group used simulations to unravel the mechanism behind this extreme mechanostability (29, 30). They found that the target peptide is conﬁned in a screwlike manner in the binding pocket of SdrG and that the binding strength of the complex results from numerous hydrogen bonds between the peptide backbone and SdrG, independent of peptide side chains. Rupture of the complex requires all hydro- gen bonds to be broken simultaneously. Interestingly, ClfA and ClfB may bind their ligands through a catch bond as their binding strength increases as mechanical force is applied (20, 21).
virus resistance, the effect of knocking down ORP1L protein levels with RNA interference (RNAi) on WNV replication was first an- alyzed. It was hypothesized that if ORP1L was required for the genetic resistance phenotype, the extracellular WNV yield as well as intracellular viral RNA and protein levels would increase when ORP1L levels were decreased. ORP1L protein expression was re- duced by stable expression of an ORP1L-specific shRNA in resis- tant KI MEFs, and then clonal cell lines were selected as described in Materials and Methods. Among the 19 lines tested, the KI shOrp1L-6 clone had the lowest ORP1L protein levels compared to that in the nonspecific control shRNA clone, KI shCont-7 (Fig. 4A). KI shCont-7 and KI shOrp1L-6 cultures were infected with WNV Eg101 at an MOI of 1, and clarified culture fluid and either total cell RNA or cell lysates were harvested at various times after infection from duplicate wells. Virus yields were measured by plaque assay on BHK cells. Counter to what was expected, lower WNV yields were produced by KI shOrp1L-6 cells at 24 to 48 h after infection than from KI shCont-7 cells (Fig. 4B). Intracellular viral RNA levels were assessed by real-time qRT-PCR, and intra- cellular viral protein levels were detected by Western blotting. Both WNV RNA (Fig. 4C) and NS3 protein levels (Fig. 4D) were also reduced in KI shOrp1L-6 cells compared to KI shCont-7 at 24 through 48 h after infection. A loss in cell viability of approxi- mately 15% was detected by an MTT assay at 48 h after WNV infection in both types of shRNA-expressing cell lines compared to mock-infected shRNA-expressing cells (Fig. 4E), suggesting that the decreased viral yields observed were not due to off-target cytotoxic effects of the shRNA. It was noted that the efficiency of ORP1L knockdown by shRNA decreased with time after WNV infection, suggesting that the RNAi pathway may be antagonized in infected cells (Fig. 4D).
deficient cells. Movement of Daxx between the condensed chromatin and PML-NB reflects the variable equilibrium between the chromatin and NB and how dynamic is this process which is in the same time needs the accumulation of SUMO-1 modified PML to make the basic structure and framework of NB (Ishov et al., 1999). The Daxx-PML interaction therefore determines the correct of this global repressor, keeping it mainly in the nucleus, despite its locations potential to interact with c-fos although a very tiny proportion of Daxx was reported in the cytoplasmic fraction. In the absence of any inducers of the dissociation of co-repressors, such as all-trans- RA (atRA), Daxx can interact with the PML-RARα complex (Li et al., 2000). This interaction was shown in vivo but could not be confirmed in vitro since it needs highly sumoylated PML, or maybe other cellular factors to associate and facilitate this interaction. Immunofluorescent antibody staining has shown a specific co- localization between Daxx and PML in different tumour cell lines such as HeLa and A549 in addition to normal human fibroblasts. Using reporter gene assays, the ability of PML to inhibit the Daxx repression function in HEK293 cells was measured. Compared to samples which were transfected with GAL4- DNA Binding Domain (DBD) alone, reporter gene expression was greatly reduced when cells were transfected with GAL4-Daxx, and this reduction was rescued in a dose-dependent manner by co-expression of increasing doses of full length PML VI plasmid (Li et al., 2000). This isoform of PML at least is therefore a negative regulator of Daxx repression and its interaction with Daxx may be important for mounting a potent anti-viral response against the invading viruses as it will permit the up-regulation of antiviral response genes.
completely to the envelope. the binding rates reduced as a function of lowering of the pH values to 8.0. only slight binding occurred at pHs 8.5 to 10 (fig. 3). When the established hemoglobin-envelope complex formed in the pH 4.5 solution was immersed in the pH 9.0 solution, hemoglobin released entirely from the envelope. However, dissociation was not vigorous in neutral and acidic solutions. these findings demon- strate that effects of pH on binding and dissociation among hemoglobin and the envelope is rightly recipro- cal; HbbP in the envelope prefers acidic conditions to alkaline conditions in the hemoglobin binding reac- tion. Additionally, hemoglobin bound to the envelope easily dissociate in the alkaline solutions (fig. 2).
M alaria caused by Plasmodium vivax is the most predominant form of malaria outside Africa, with over 130 million clinical cases annually (1, 2). Unlike Plasmodium falciparum malaria, P. vivax blood-stage infection is limited to reticulocytes and indi- viduals who are positive for the Duffy blood group antigen (Fy), also known as the Duffy antigen receptor for chemokines (DARC) (3, 4). Preference for this blood cell type is believed to be mediated by specific ligand-receptor interactions between the parasite merozoites and the host reticulocytes during the invasion process (5, 6). It is believed that the P. vivax Duffy bindingprotein (DBP) on the merozoite interacts with DARC on the reticulocyte surface, precipitating the junction formation step necessary for invasion. Historically, the vital need for the DBP-DARC interaction was evident from the virtual absence of P. vivax malaria in populations with a high prevalence of DARC negativity (3, 7). However, recent studies have reported evidence of Duffy (Fy)-independent inva- sion of human reticulocytes (8, 9). In Madagascar, with a mixture of Duffy-positive (Fy ⫹ ) and -negative (Fy ⫺ ) populations of di- verse ethnic backgrounds, there was a significant reduction in the
morphology and other effects such as the increase of metastatic potential in tumor cells (Shestakova et al., 1999). In order to establish protrusive force, transcripts of all seven subunits of the actin-related protein 2/3 (ARP2/3) complex are localized to the leading edge of fibroblasts as well (Mingle et al., 2005). This complex provides the nucleation of actin filaments and consequently, is required in a spatial context to establish cell motility (Machesky and Gould, 1999). The synaptic plasticity of developing neurons is established in a similar fashion. For instance, transcript of Calcium/Calmodulin dependent Kinase II (CaMKII α) is localized to dendrites (Mayford et al., 1996). There, directed protein synthesis can lead to rearrangement of synapses, which is important for higher brain functions such as learning and memory. mRNA localization may also contribute to the sorting of proteins into various organelles. Usually, protein sorting is provided by peptide signals, which directs the translated protein to the target organelle. However, there is increasing evidence that some mRNAs are already localized to the vicinity of organelles to facilitate and maximize the import of the corresponding proteins. There are several reports on mRNAs, which display perinuclear localization (Fig. 6E). For instance, transcripts encoding for Metallothionein-1 (MT-1) and transcription factors c-FOS and c-MYC accumulate at the nuclear periphery. These transcripts have also been reported to associate with the perinuclear cytoskeleton in order to become effectively imported into the nucleus (Levadoux et al., 1999; Mahon et al., 1997; Veyrune et al., 1996). Sorting of a subset of nuclear-encoded proteins to mitochondria involves mRNA localization. In yeast, for instance, the 3’-UTR of ATM1 and ATP2 mRNAs direct these transcripts to the vicinity of mitochondria. The latter transcript encodes a subunit of the mitochondrial ATP synthase. Interestingly, impaired ATP2 mRNA sorting correlates with a severe respiratory deficiency indicating a link between mRNA localization and protein function (Corral-Debrinski, 2007; Corral-Debrinski et al., 2000; Margeot et al., 2002; Sylvestre et al., 2003). Local enrichment of transcripts at mitochondrial-bound polysomes may ensure an effective cotranslational import into these organelles.
of ER stress markers C/EBP homologous protein (CHOP; DDIT3) and BiP (HSPA5) in RA FLS. mRNA levels of these genes were significantly increased in a dose- and time-dependent manner following thapsigargin treatment (Fig. 1a). Induction was readily observed 2 h after treat- ment and continued to increase for as long as 24 h. Not- ably, although thapsigargin is typically used at micromolar concentrations [17, 19], 10 nM thapsigargin was sufficient for maximum induction of DDIT3 and HSPA5 in FLS. To quantitatively assess signaling emanating directly from the ER, we developed an assay in which expression of XBP1 splice variants is followed by real-time PCR using primers recognizing only its IRE1α-processed (spliced) or unpro- cessed (unspliced) versions or detecting both variants indiscriminately (total). At baseline, most of XBP1 was in its unspliced form, confirming a low level of IRE1α ac- tivity (Fig. 1b). The amount of spliced XBP1 increased
tifying lysozyme resistance suppressor mutations via whole- genome sequencing. These suppressor strains were derived in the pgdA mutant background in order to circumvent the identifica- tion of mutations that upregulated pgdA. Upon genome sequenc- ing, we observed that four of the five individually derived strains contained an identical mutation in the promoter of an operon encoding two copies of the gene spoVG, resulting in its significant downregulation. spoVG is broadly conserved, especially among Gram-positive bacteria (12), and spoVG mutants display remark- able phenotypes in many species, including reduced methicillin resistance, decreased capsule production, and decreased enzyme secretion in Staphylococcus aureus (13–15) and altered asymmetric cell division, decreased hemolysin production, and sporulation phenotypes in Bacillus subtilis (16–18). Additionally, our lab iden- tified spoVG in a separate suppressor screen for mutants that res- cued virulence defects of (p)ppGpp-deficient L. monocytogenes (19). Despite these phenotypes and despite being initially charac- terized nearly 40 years ago (20), the function of the spoVG- encoded protein has remained unclear. In this study, we deter- mined that SpoVG is an RNA-bindingprotein that interacts with noncoding RNAs, regulates genes in cooperation with RNases, and controls pleiotropic aspects of bacterial physiology, including motility, carbon metabolism, and virulence. Together, these char- acteristics are similar to those of posttranscriptional gene regula- tors such as CsrA, a class of RNA-binding molecules that are fun- damental for synchronizing environmental cues with gene regulation in order to adapt bacteria to their diverse ecological domains (11). Considering that these proteins have been primar- ily characterized in Gram-negative bacteria, we hypothesize that SpoVG may act as a functionally conserved counterpart to these molecules in Gram-positive organisms.
Although ABCF3 is normally expressed in cells, its anti-flaviviral function was only observed when Oas1b was also present. ABCF3 was also shown to interact with Oas1bt and Oas1a in both yeast two-hybrid and in vitro interaction assays. The main difference between these Oas1 proteins is their localization within the cell. Oas1b was shown in the present study to localize to the ER membrane and is the only member of the OAS family of proteins in any species tested so far found to localize to this compartment of the cell. These data imply that the interaction between Oas1b and ABCF3 specifically localizes ABCF3 in close proximity to WNV RNA translation/replication sites. Future studies could investigate whether ER membrane localization of ABCF3 is required for its antiviral affect. ABCF3 cDNA could be cloned into an expression vector that fuses a transmembrane ER localization signal to the ABCF3 protein (pER- ABCF3). The overexpression of this construct in C3H/He (He; Oas1bt-expressing) cells would be expected to directly localize ABCF3 to the ER membrane. Control He, He-ABCF3 and He- ER-ABCF3 cell lines could be created and then infected with WNV and the effect on WNV yield, viral RNA and viral protein levels determined. It would be expected that He-ABCF3 cells will produce WNV yields similar to the control cells since Oas1b is not expressed in these cells and therefore cannot localize ABCF3 to the sites of WNV replication. In contrast, He-ER- ABCF3 cells might produce reduced viral yields since the ABCF3 protein is localized to the ER membrane.