COOH-terminal Region Research Articles

Na+/HCO3- cotransporter is expressed on β and α cells during rat pancreatic development.

AIMTo determine the expression and localization of the electrogenic Na+/HCO3- cotransporter (NBC1) in rat pancreas during development.METHODSThe rat pancreas from postnatal and embryos removed from the uterus of pregnant rats that had been sacrificed by CO2 asphyxiation were used. Rat pancreas from embryonic day (E) 15.5 and E18.5 rat embryos was isolated under a stereomicroscope. Rat pancreas from postnatal (P) days 0, 7, 14, 21 and adult was directly isolated by the unaided eye. The RT-PCR analysis of the NBC1 specific region on rat pancreas tissues from different developmental stages. The two antibodies which target the NBC1 common COOH-terminal region and NH2-terminal region detected a clear band of about 145 kDa in the Western blot analysis. The localization of NBC1 was examined by immuno-fluorescence detection.RESULTSThe results revealed the first peak of NBC1 expression at E18.5 and the second peak at P14. Meanwhile, the low NBC1 expression occurred at P7 and adult stages. Our results demonstrated, for the first time, the presence of NBC1 in the plasma membrane of β and α cells, as well as in the basolateral membrane of acinar cells of the rat pancreas at different stages of development.CONCLUSIONThe data strongly suggests that NBC1 is diversely expressed in the pancreas at different developmental stages, where it may exert its functions in pancreatic development especially islet cell growth through HCO3- transport and pH regulation.

Identification of regions within the Legionella pneumophila VipA effector protein involved in actin binding and polymerization and in interference with eukaryotic organelle trafficking.

The Legionella pneumophila effector protein VipA is an actin nucleator that co-localizes with actin filaments and early endosomes in infected macrophages and which interferes with organelle trafficking when expressed in yeast. To identify the regions of VipA involved in its subcellular localization and functions, we ectopically expressed specific VipA mutant proteins in eukaryotic cells. This indicated that the characteristic punctate distribution of VipA depends on its NH2 -terminal (amino acid residues 1-133) and central coiled-coil (amino acid residues 133-206) regions, and suggested a role for the COOH-terminal (amino acid residues 206-339) region in association with actin filaments and for the NH2 -terminal in co-localization with early endosomes. Co-immunoprecipitation and in vitro assays showed that the COOH-terminal region of VipA is necessary and sufficient to mediate actin binding, and is essential but insufficient to induce microfilament formation. Assays in yeast revealed that the NH2 and the COOH-terminal regions, and possibly an NPY motif within the NH2 region of VipA, are necessary for interference with organelle trafficking. Overall, this suggests that subversion of eukaryotic vesicular trafficking by VipA involves both its ability to associate with early endosomes via its NH2 -terminal region and its capacity to bind and polymerize actin through its COOH-terminal region.

Basic chemokine-derived glycosaminoglycan binding peptides exert antiviral properties against dengue virus serotype 2, herpes simplex virus-1 and respiratory syncytial virus

Chemokines attract leukocytes to sites of infection in a G protein-coupled receptor (GPCR) and glycosaminoglycan (GAG) dependent manner. Therefore, chemokines are crucial molecules for proper functioning of our antimicrobial defense mechanisms. In addition, some chemokines have GPCR-independent defensin-like antimicrobial activities against bacteria and fungi. Recently, high affinity for GAGs has been reported for the positively charged COOH-terminal region of the chemokine CXCL9. In addition to CXCL9, also CXCL12γ has such a positively charged COOH-terminal region with about 50% positively charged amino acids. In this report, we compared the affinity of COOH-terminal peptides of CXCL9 and CXCL12γ for GAGs and KD values in the low nM range were detected. Several enveloped viruses such as herpesviruses, hepatitis viruses, human immunodeficiency virus (HIV), dengue virus (DENV), etc. are known to bind to GAGs such as the negatively charged heparan sulfate (HS). In this way GAGs are important for the initial contacts between viruses and host cells and for the infection of the cell. Thus, inhibiting the virus-cell interactions, by blocking GAG-binding sites on the host cell, might be a way to target multiple virus families and resistant strains. This article reports that the COOH-terminal peptides of CXCL9 and CXCL12γ have antiviral activity against DENV serotype 2, clinical and laboratory strains of herpes simplex virus (HSV)-1 and respiratory syncytial virus (RSV). Moreover, we show that CXCL9(74-103) competes with DENV envelope protein domain III for binding to heparin. These short chemokine-derived peptides may be lead molecules for the development of novel antiviral agents.

Effect of carbohydrate feeding on the bone metabolic response to running.

Bone resorption is increased after running, with no change in bone formation. Feeding during exercise might attenuate this increase, preventing associated problems for bone. This study investigated the immediate and short-term bone metabolic responses to carbohydrate (CHO) feeding during treadmill running. Ten men completed two 7-day trials, once being fed CHO (8% glucose immediately before, every 20 min during, and immediately after exercise at a rate of 0.7 g CHO·kg body mass−1·h−1) and once being fed placebo (PBO). On day 4 of each trial, participants completed a 120-min treadmill run at 70% of maximal oxygen consumption (V̇o2 max). Blood was taken at baseline (BASE), immediately after exercise (EE), after 60 (R1) and 120 (R2) min of recovery, and on three follow-up days (FU1-FU3). Markers of bone resorption [COOH-terminal telopeptide region of collagen type 1 (β-CTX)] and formation [NH2-terminal propeptides of procollagen type 1 (P1NP)] were measured, along with osteocalcin (OC), parathyroid hormone (PTH), albumin-adjusted calcium (ACa), phosphate, glucagon-like peptide-2 (GLP-2), interleukin-6 (IL-6), insulin, cortisol, leptin, and osteoprotogerin (OPG). Area under the curve was calculated in terms of the immediate (BASE, EE, R1, and R2) and short-term (BASE, FU1, FU2, and FU3) responses to exercise. β-CTX, P1NP, and IL-6 responses to exercise were significantly lower in the immediate postexercise period with CHO feeding compared with PBO (β-CTX: P = 0.028; P1NP: P = 0.021; IL-6: P = 0.036), although there was no difference in the short-term response (β-CTX: P = 0.856; P1NP: P = 0.721; IL-6: P = 0.327). No other variable was significantly affected by CHO feeding during exercise. We conclude that CHO feeding during exercise attenuated the β-CTX and P1NP responses in the hours but not days following exercise, indicating an acute effect of CHO feeding on bone turnover.

The Positively Charged COOH-terminal Glycosaminoglycan-binding CXCL9(74–103) Peptide Inhibits CXCL8-induced Neutrophil Extravasation and Monosodium Urate Crystal-induced Gout in Mice

The ELR(-)CXC chemokine CXCL9 is characterized by a long, highly positively charged COOH-terminal region, absent in most other chemokines. Several natural leukocyte- and fibroblast-derived COOH-terminally truncated CXCL9 forms missing up to 30 amino acids were identified. To investigate the role of the COOH-terminal region of CXCL9, several COOH-terminal peptides were chemically synthesized. These peptides display high affinity for glycosaminoglycans (GAGs) and compete with functional intact chemokines for GAG binding, the longest peptide (CXCL9(74-103)) being the most potent. The COOH-terminal peptide CXCL9(74-103) does not signal through or act as an antagonist for CXCR3, the G protein-coupled CXCL9 receptor, and does not influence neutrophil chemotactic activity of CXCL8 in vitro. Based on the GAG binding data, an anti-inflammatory role for CXCL9(74-103) was further evidenced in vivo. Simultaneous intravenous injection of CXCL9(74-103) with CXCL8 injection in the joint diminished CXCL8-induced neutrophil extravasation. Analogously, monosodium urate crystal-induced neutrophil migration to the tibiofemural articulation, a murine model of gout, is highly reduced by intravenous injection of CXCL9(74-103). These data show that chemokine-derived peptides with high affinity for GAGs may be used as anti-inflammatory peptides; by competing with active chemokines for binding and immobilization on GAGs, these peptides may lower chemokine presentation on the endothelium and disrupt the generation of a chemokine gradient, thereby preventing a chemokine from properly performing its chemotactic function. The CXCL9 peptide may serve as a lead molecule for further development of inhibitors of inflammation based on interference with chemokine-GAG interactions.

FBXO21 mediates the ubiquitylation and proteasomal degradation of EID1.

Although identification of substrates for ubiquitin ligase (E3) is important for understanding its biological functions, detection of the interaction between an E3 and its substrates has remained challenging. We recently developed a new approach, termed differential proteomics-based identification of ubiquitylation substrates (DiPIUS), for the discovery of substrates of a given E3 ligase. We have now applied this approach to an uncharacterized human F-box protein, FBXO21, which serves as the substrate-recognition subunit of a SKP1-CUL1-F-box protein (SCF)-type E3, thereby identifying EID1 (EP300-interacting inhibitor of differentiation 1) as a candidate substrate. The central and COOH-terminal portion of FBXO21 was found to interact with the COOH-terminal region of EID1 in transfected cells. Over-expression of FBXO21 resulted in the down-regulation of EID1, whereas disruption of the FBXO21 gene with the CRISPR/Cas9 system stabilized EID1 and led to its accumulation in both the cytoplasm and nucleus. An in vitro ubiquitylation assay showed that EID1 is a direct substrate of SCF(FBXO)(21). Collectively, our results suggest that EID1 is a bona fide substrate of FBXO21 and that the control of EID1 abundance by SCF(FBXO)(21) might affect the transcriptional repression activity of EID1.

Amyloid fibril formation from a 9 amino acid peptide, 55th–63rd residues of human lysozyme

Hen egg white lysozyme (HEWL) readily forms amyloid fibrils in vitro. We have previously identified a core structure, termed HEWL K-peptide, involved in fibril formation. Two major peptides, peptide #3 (50th–102nd residues of human lysozyme (hLZ)) and peptide #5 (54th–102nd residues), were isolated from the hLZ amyloid fibrils that had been exposed to pH 2.0 and 58°C, and precipitated by ultra-centrifugation. These peptides cover most of the beta-domain and C-helix of hLZ including a 9 residues sequence corresponds to a human counterpart of HEWL K-peptide, GIFQINSRY (55th–63rd residues of hLZ, thus named “human K-peptide”). Chemically synthesized human K-peptide readily formed amyloid fibrils, as in HEWL K-peptide. It was demonstrated that both at least 9 residue length and Phe residue at 3rd position of the human K-peptide were crucial for amyloidogenesis in vitro. Short peptides covering COOH-terminal region of peptides #3 and #5 did not form amyloid fibrils. These data suggested that human K-peptide region with high propensity of amyloidogenesis plays a key role as a fibril-forming core sequence of hLZ. Interestingly, human K-peptide region is flanked by two predicted semi-disordered regions (39th–52nd and 67th–75th residues). We discuss the possible role of these regions.

Identification of the region of rice 13 kDa prolamin essential for the formation of ER-derived protein bodies using a heterologous expression system.

Cereal prolamins, which are alcohol-soluble seed storage proteins, can induce ER-derived protein bodies (PBs) in heterologous tissue. Like maize and wheat prolamins, rice prolamins can form ER-derived PBs, but the region of mature polypeptides that is essential for PB formation has not been identified. In this study, we examined the formation mechanisms of ER-derived PB-like structures by expressing rice 13 kDa prolamin-deletion mutants fused to green fluorescent protein (GFP) in heterologous tissues such as yeast. The 13 kDa prolamin-GFP fusion protein was stably accumulated in transgenic yeast and formed an ER-derived PB-like structure. In contrast, rice α-globulin-GFP fusion protein was transported to vacuoles. In addition, the middle and COOH-terminal regions of 13 kDa prolamin formed ER-derived PB-like structures, whereas the NH2-terminal region of 13 kDa prolamin did not form such structures. These results suggest that the middle and COOH-terminal regions of 13 kDa prolamin can be retained and thus can induce ER-derived PB in yeast.

Treponema pallidum subsp. pallidum TP0136 protein is heterogeneous among isolates and binds cellular and plasma fibronectin via its NH2-terminal end.

Adherence-mediated colonization plays an important role in pathogenesis of microbial infections, particularly those caused by extracellular pathogens responsible for systemic diseases, such as Treponema pallidum subsp. pallidum (T. pallidum), the agent of syphilis. Among T. pallidum adhesins, TP0136 is known to bind fibronectin (Fn), an important constituent of the host extracellular matrix. To deepen our understanding of the TP0136-Fn interaction dynamics, we used two naturally-occurring sequence variants of the TP0136 protein to investigate which region of the protein is responsible for Fn binding, and whether TP0136 would adhere to human cellular Fn in addition to plasma Fn and super Fn as previously reported. Fn binding assays were performed with recombinant proteins representing the two full-length TP0136 variants and their discrete regions. As a complementary approach, we tested inhibition of T. pallidum binding to Fn by recombinant full-length TP0136 proteins and fragments, as well as by anti-TP0136 immune sera. Our results show that TP0136 adheres more efficiently to cellular Fn than to plasma Fn, that the TP0136 NH2-terminal conserved region of the protein is primarily responsible for binding to plasma Fn but that binding sites for cellular Fn are also present in the protein’s central and COOH-terminal regions. Additionally, message quantification studies show that tp0136 is highly transcribed during experimental infection, and that its message level increases in parallel to the host immune pressure on the pathogen, which suggests a possible role for this protein in T. pallidum persistence. In a time where syphilis incidence is high, our data will help in the quest to identify suitable targets for development of a much needed vaccine against this important disease.

Binding of Tissue-type Plasminogen Activator to the Glucose-regulated Protein 78 (GRP78) Modulates Plasminogen Activation and Promotes Human Neuroblastoma Cell Proliferation in Vitro

The glucose-regulated protein 78 (GRP78) is a plasminogen (Pg) receptor on the cell surface. In this study, we demonstrate that GRP78 also binds the tissue-type plasminogen activator (t-PA), which results in a decrease in K(m) and an increase in the V(max) for both its amidolytic activity and activation of its substrate, Pg. This results in accelerated Pg activation when GRP78, t-PA, and Pg are bound together. The increase in t-PA activity is the result of a mechanism involving a t-PA lysine-dependent binding site in the GRP78 amino acid sequence (98)LIGRTWNDPSVQQDIKFL(115). We found that GRP78 is expressed on the surface of neuroblastoma SK-N-SH cells where it is co-localized with the voltage-dependent anion channel (VDAC), which is also a t-PA-binding protein in these cells. We demonstrate that both Pg and t-PA serve as a bridge between GRP78 and VDAC bringing them together to facilitate Pg activation. t-PA induces SK-N-SH cell proliferation via binding to GRP78 on the cell surface. Furthermore, Pg binding to the COOH-terminal region of GRP78 stimulates cell proliferation via its microplasminogen domain. This study confirms previous findings from our laboratory showing that GRP78 acts as a growth factor-like receptor and that its association with t-PA, Pg, and VDAC on the cell surface may be part of a system controlling cell growth.

Detection of HIV-1 matrix protein p17 quasispecies variants in plasma of chronic HIV-1-infected patients by ultra-deep pyrosequencing.

The HIV-1 matrix protein p17 (p17MA) is a pleiotropic protein that plays a key role in the HIV-1 life cycle. It has been long believed to have a highly conserved primary amino acid sequence and a well-preserved structural integrity to avoid severe fitness consequences. However, recent data revealed that the carboxy (COOH)-terminus of p17MA possesses high levels of predicted intrinsic disorder, which would subtend to at least partially unfolded status of this region. This finding pointed to the need of investigating p17MA heterogeneity. The degree of intrapatient variations in the p17MA primary sequence was assessed on plasma viral RNA by using ultra-deep pyrosequencing. Data obtained support a complex nature of p17MA quasispecies, with variants present at variable frequency virtually in all patients. Clusters of mutations were scattered along the entire sequence of the viral protein, but they were more frequently detected within the COOH-terminal region of p17MA. Moreover, deletions and insertions also occurred in a restricted area of the COOH-terminal region. On the whole, our data show that the intrapatient level of sequence diversity in the p17MA is much higher than predicted before. Our results pave the way for further studies aimed at unraveling possible correlations between the presence of distinct p17MA variants and peculiar clinical evolutions of HIV-1 disease. The presence of p17MA quasispecies diversity may offer new tools to improve our understanding of the viral adaptation during the natural history of HIV-1 infection.

Adenosine triphosphate regulates the activity of guinea pig Cav1.2 channel by direct binding to the channel in a dose-dependent manner

The present study is to investigate the mechanism by which ATP regulates Cav1.2 channel activity. Ventricular tissue was obtained from adult guinea pig hearts using collagenase. Ca(2+) channel activity was monitored using the patch-clamp technique. Proteins were purified using wheat germ agglutinin-Sepharose, and the concentration was determined using the Coomassie brilliant blue technique. ATP binding to the Cav1.2 channel was examined using the photoaffinity method. EDA-ATP-biotin maintains Ca(2+) channel activity in inside-out membrane patches. ATP directly bound to the Cav1.2 channel in a dose-dependent manner, and at least two molecules of ATP bound to one molecule of the Cav1.2 channel. Low levels of calmodulin (CaM) increased ATP binding to the Cav1.2 channel, but higher levels of CaM decreased ATP binding to the Cav1.2 channel. In addition, Ca(2+) was another regulator for ATP binding to the Cav1.2 channel. Furthermore, ATP bound to GST-fusion peptides of NH2-terminal region (amino acids 6-140) and proximal COOH-terminal region (amino acids 1,509-1,789) of the main subunit (α1C) of the Cav1.2 channel. Our data suggest that ATP might regulate Cav1.2 channel activity by directly binding to the Cav1.2 channel in a dose-dependent manner. In addition, the ATP-binding effect to the Cav1.2 channel was both CaM- and Ca(2+) dependent.

A3 Domain Region 1803–1818 Contributes to the Stability of Activated Factor VIII and Includes a Binding Site for Activated Factor IX

A recent chemical footprinting study in our laboratory suggested that region 1803-1818 might contribute to A2 domain retention in activated factor VIII (FVIIIa). This site has also been implicated to interact with activated factor IX (FIXa). Asn-1810 further comprises an N-linked glycan, which seems incompatible with a role of the amino acids 1803-1818 for FIXa or A2 domain binding. In the present study, FVIIIa stability and FIXa binding were evaluated in a FVIII-N1810C variant, and two FVIII variants in which residues 1803-1810 and 1811-1818 are replaced by the corresponding residues of factor V (FV). Enzyme kinetic studies showed that only FVIII/FV 1811-1818 has a decreased apparent binding affinity for FIXa. Flow cytometry analysis indicated that fluorescent FIXa exhibits impaired complex formation with only FVIII/FV 1811-1818 on lipospheres. Site-directed mutagenesis revealed that Phe-1816 contributes to the interaction with FIXa. To evaluate FVIIIa stability, the FVIII/FV chimeras were activated by thrombin, and the decline in cofactor function was followed over time. FVIII/FV 1803-1810 and FVIII/FV 1811-1818 but not FVIII-N1810C showed a decreased FVIIIa half-life. However, when the FVIII variants were activated in presence of FIXa, only FVIII/FV 1811-1818 demonstrated an enhanced decline in cofactor function. Surface plasmon resonance analysis revealed that the FVIII variants K1813A/K1818A, E1811A, and F1816A exhibit enhanced dissociation after activation. The results together demonstrate that the glycan at 1810 is not involved in FVIII cofactor function, and that Phe-1816 of region 1811-1818 contributes to FIXa binding. Both regions 1803-1810 and 1811-1818 contribute to FVIIIa stability.

Amino acid mutations in the caldesmon COOH-terminal functional domain increase force generation in bladder smooth muscle

Caldesmon (CaD), a component of smooth muscle thin filaments, binds actin, tropomyosin, calmodulin, and myosin and inhibits actin-activated ATP hydrolysis by smooth muscle myosin. Internal deletions of the chicken CaD functional domain that spans from amino acids (aa) 718 to 731, which corresponds to aa 512-530 including the adjacent aa sequence in mouse CaD, lead to diminished CaD-induced inhibition of actin-activated ATP hydrolysis by myosin. Transgenic mice with mutations of five aa residues (Lys(523) to Gln, Val(524) to Leu, Ser(526) to Thr, Pro(527) to Cys, and Lys(529) to Ser), which encompass the ATPase inhibitory determinants located in exon 12, were generated by homologous recombination. Homozygous (-/-) animals did not develop, but heterozygous (+/-) mice carrying the expected mutations in the CaD ATPase inhibitory domain (CaD mutant) matured and reproduced normally. The peak force produced in response to KCl and electrical field stimulation by the detrusor smooth muscle from the CaD mutant was high compared with that of the wild type. CaD mutant mice revealed nonvoiding contractions during bladder filling on awake cystometry, suggesting that the CaD ATPase inhibitory domain suppresses force generation during the filling phase and this suppression is partially released by mutations in 50% of CaD in heterozygous. Our data show for the first time a functional phenotype, at the intact smooth muscle tissue and in vivo organ levels, following mutation of a functional domain at the COOH-terminal region of CaD.

Two Salt Bridges Differentially Contribute to the Maintenance of Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Channel Function

Previous studies have identified two salt bridges in human CFTR chloride ion channels, Arg(352)-Asp(993) and Arg(347)-Asp(924), that are required for normal channel function. In the present study, we determined how the two salt bridges cooperate to maintain the open pore architecture of CFTR. Our data suggest that Arg(347) not only interacts with Asp(924) but also interacts with Asp(993). The tripartite interaction Arg(347)-Asp(924)-Asp(993) mainly contributes to maintaining a stable s2 open subconductance state. The Arg(352)-Asp(993) salt bridge, in contrast, is involved in stabilizing both the s2 and full (f) open conductance states, with the main contribution being to the f state. The s1 subconductance state does not require either salt bridge. In confirmation of the role of Arg(352) and Asp(993), channels bearing cysteines at these sites could be latched into a full open state using the bifunctional cross-linker 1,2-ethanediyl bismethanethiosulfonate, but only when applied in the open state. Channels remained latched open even after washout of ATP. The results suggest that these interacting residues contribute differently to stabilizing the open pore in different phases of the gating cycle.

Cancer‐associated missense mutations of caspase‐8 activate nuclear factor‐κB signaling

Head and neck squamous cell carcinoma (HNSCC) is an aggressive cancer with a 5-year survival rate of ~50%. With the use of a custom cDNA-capture system coupled with massively parallel sequencing, we have now investigated transforming mechanisms for this malignancy. The cDNAs of cancer-related genes (n=906) were purified from a human HNSCC cell line (T3M-1 Cl-10) and subjected to high-throughput resequencing, and the clinical relevance of non-synonymous mutations thus identified was evaluated with luciferase-based reporter assays. A CASP8 (procaspase-8) cDNA with a novel G-to-C point mutation that results in the substitution of alanine for glycine at codon 325 was identified, and the mutant protein, CASP8 (G325A), was found to activate nuclear factor-κB (NF-κB) signaling to an extent far greater than that achieved with the wild-type protein. Moreover, forced expression of wild-type CASP8 suppressed the growth of T3M-1 Cl-10 cells without notable effects on apoptosis. We further found that most CASP8 mutations previously detected in various epithelial tumors also increase the ability of the protein to activate NF-κB signaling. Such NF-κB activation was shown to be mediated through the COOH-terminal region of the second death effector domain of CASP8. Although CASP8 mutations associated with cancer have been thought to promote tumorigenesis as a result of attenuation of the proapoptotic function of the protein, our results now show that most such mutations, including the novel G325A identified here, separately confer a gain of function with regard to activation of NF-κB signaling, indicating another role of CASP8 in the transformation of human malignancies including HNSCC.

Feedback regulation of cone cyclic nucleotide channels by phosphoinositides. Focus on “CNGA3 achromatopsia-associated mutation potentiates the phosphoinositide sensitivity of cone photoreceptor CNG channels by altering intersubunit interactions”

<i>Feedback regulation of cone cyclic nucleotide channels by phosphoinositides</i>. Focus on “CNGA3 achromatopsia-associated mutation potentiates the phosphoinositide sensitivity of cone photoreceptor CNG channels by altering intersubunit interactions”

Protein cold adaptation strategy via a unique seven-amino acid domain in the icefish ( Chionodraco hamatus ) PEPT1 transporter

Adaptation of organisms to extreme environments requires proteins to work at thermodynamically unfavorable conditions. To adapt to subzero temperatures, proteins increase the flexibility of parts of, or even the whole, 3D structure to compensate for the lower thermal kinetic energy available at low temperatures. This may be achieved through single-site amino acid substitutions in regions of the protein that undergo large movements during the catalytic cycle, such as in enzymes or transporter proteins. Other strategies of cold adaptation involving changes in the primary amino acid sequence have not been documented yet. In Antarctic icefish (Chionodraco hamatus) peptide transporter 1 (PEPT1), the first transporter cloned from a vertebrate living at subzero temperatures, we came upon a unique principle of cold adaptation. A de novo domain composed of one to six repeats of seven amino acids (VDMSRKS), placed as an extra stretch in the cytosolic COOH-terminal region, contributed per se to cold adaptation. VDMSRKS was in a protein region uninvolved in transport activity and, notably, when transferred to the COOH terminus of a warm-adapted (rabbit) PEPT1, it conferred cold adaptation to the receiving protein. Overall, we provide a paradigm for protein cold adaptation that relies on insertion of a unique domain that confers greater affinity and maximal transport rates at low temperatures. Due to its ability to transfer a thermal trait, the VDMSRKS domain represents a useful tool for future cell biology or biotechnological applications.

CNGA3 achromatopsia-associated mutation potentiates the phosphoinositide sensitivity of cone photoreceptor CNG channels by altering intersubunit interactions

Cyclic nucleotide-gated (CNG) channels are critical for sensory transduction in retinal photoreceptors and olfactory receptor cells; their activity is modulated by phosphoinositides (PIPn) such as phosphatidylinositol 4,5-bisphosphate (PIP2) and phosphatidylinositol 3,4,5-trisphosphate (PIP3). An achromatopsia-associated mutation in cone photoreceptor CNGA3, L633P, is located in a carboxyl (COOH)-terminal leucine zipper domain shown previously to be important for channel assembly and PIPn regulation. We determined the functional consequences of this mutation using electrophysiological recordings of patches excised from cells expressing wild-type and mutant CNG channel subunits. CNGA3-L633P subunits formed functional channels with or without CNGB3, producing an increase in apparent cGMP affinity. Surprisingly, L633P dramatically potentiated PIPn inhibition of apparent cGMP affinity for these channels. The impact of L633P on PIPn sensitivity depended on an intact amino (NH2) terminal PIPn regulation module. These observations led us to hypothesize that L633P enhances PIPn inhibition by altering the coupling between NH2- and COOH-terminal regions of CNGA3. A recombinant COOH-terminal fragment partially restored normal PIPn sensitivity to channels with COOH-terminal truncation, but L633P prevented this effect. Furthermore, coimmunoprecipitation of channel fragments, and thermodynamic linkage analysis, also provided evidence for NH2-COOH interactions. Finally, tandem dimers of CNGA3 subunits that specify the arrangement of subunits containing L633P and other mutations indicated that the putative interdomain interaction occurs between channel subunits (intersubunit) rather than exclusively within the same subunit (intrasubunit). Collectively, these studies support a model in which intersubunit interactions control the sensitivity of cone CNG channels to regulation by phosphoinositides. Aberrant channel regulation may contribute to disease progression in patients with the L633P mutation.

The major allergen of the Parietaria pollen contains an LPS‐binding region with immuno‐modulatory activity

The major allergens in Parietaria pollen, Par j 1 and Par j 2, have been identified as lipid transfer proteins. The family of the Par j 1 allergens is composed of two isoforms, which differ by the presence of a 37 amino acid peptide (Par37) exclusive to the Par j 1.0101 isoform. The goal of this study was to elucidate the biological properties of the Par37 peptide. In silico analysis, spectrofluorimetric experiments and in vitro cell culture assays were used to identify the biological properties of Par37. In addition, a mouse model of sensitization was used to study the influence of Par37 in the murine immune response. In silico analysis predicted that Par37 displays characteristics of a host defence peptide. Spectrofluorimetric analysis, real-time PCR and ELISA assays demonstrated that Par37 possesses an LPS-binding activity influencing cell signalling in vitro. In RAW264.7 cells, LPS-induced IL-6 and TNF-α transcription and translation were inhibited after preincubation with Par37. Consistent with these data, inhibition of IFN-γ secretion was observed in murine spleen cells and in human PBMC. Finally, mice immunized with the two Par j 1 isoforms differing in the presence or absence of the Par37 peptide showed different immunological behaviours in vivo. This study demonstrates that the Par j 1.0101 allergen displays LPS-binding activity due to the presence of a 37 amino acid COOH-terminal region and that this region is capable of influencing cytokine and antibody responses in vitro and in vivo.