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To enhance the applied value of transglutaminase (TGase), various methods have been employed to improve its catalytic properties. However, most modifications have targeted the N-terminus, while the role of the C-terminus in determining TGase properties has been overlooked. In this study, we focused on enhancing the thermal stability of Streptomyces hygroscopicus TGase by engineering its C-terminal region. Modeling revealed that the C-terminal loop interacts with the N-terminal loop through hydrogen bonds between Trp331 and N-terminal residues (Asp19, Ala20, Tyr21). Removing the last C-terminal residue (Ser322) had no significant effect on TGase stability, but deleting additional residues (Trp331, Gly330, Gln299) led to inactivation. Substituting Trp331 with Ala reduced TGase's half-life at 50 °C and specific activity by 50% and 70%, respectively, highlighting the importance of C-terminal interactions in TGase stability. We also attempted to fuse three self-assembling amphipathic peptides (SAPs) (EAK16, KL15, ELK16) and a C-terminal sequence (IGCIILT) from Sulfolobus tokodaii RNase HI to TGase. The fusion of IGCIILT increased TGase's half-life by 1.5-fold without affecting specific activity, while the three SAPs had little effect on stability. Structural analysis showed that the fusion of IGCIILT raised TGase's melting temperature by 5.2 °C and altered its tertiary structure. Our results indicate that the C-terminus is important for modulating TGase properties, and fusing "stabilization tags" like IGCIILT at the C-terminus is a promising strategy to enhance thermal stability. [ABSTRACT FROM AUTHOR]

(1) Lipases are catalysts widely applied in industrial fields. To sustain the harsh treatments in industries, optimizing lipase activities and thermal stability is necessary to reduce production loss. (2) The thermostability of Thermomyces lanuginosus lipase (TLL) was evaluated via B-factor analysis and consensus-sequence substitutions. Five single-point variants (K24S, D27N, D27R, P29S, and A30P) with improved thermostability were constructed via site-directed mutagenesis. (3) The optimal reaction temperatures of all the five variants displayed 5 °C improvement compared with TLL. Four variants, except D27N, showed enhanced residual activities at 80 °C. The melting temperatures of three variants (D27R, P29S, and A30P) were significantly increased. The molecular dynamics simulations indicated that the 25-loop (residues 24–30) in the N-terminus of the five variants generated more hydrogen bonds with surrounding amino acids; hydrogen bond pair D254-I255 preserved in the C-terminus of the variants also contributes to the improved thermostability. Furthermore, the newly formed salt-bridge interaction (R27...E56) in D27R was identified as a crucial determinant for thermostability. (4) Our study discovered that substituting residues from the 25-loop will enhance the stability of the N-terminus and C-terminus simultaneously, restrict the most flexible regions of TLL, and result in improved thermostability. [ABSTRACT FROM AUTHOR]

In mitochondria, the major subunits of oxidative phosphorylation complexes are translated by the mitochondrial ribosome (mito-ribosome). The correct insertion and assembly of these subunits into the inner mitochondrial membrane (IMM) are facilitated by mitochondrial oxidase assembly protein 1 (Oxa1) during the translation process. This co-translational insertion process involves an association between the mito-ribosome and the C-terminus of Oxa1 (Oxa1-CTD) Nuclear magnetic resonance (NMR) methods were mainly used to investigate the structural characterization of yeast Oxa1-CTD and its mode of interaction with the E. coli 70S ribosome. Oxa1-CTD forms a transient α-helical structure within the residues P342–Q385, which were reported to form an α-helix when combining with the ribosome. Two conserved contact sites that could interact with the ribosome were further identified. The first site was located on the very end of the N-terminus (V321–I327), and the second one encompassed a stretch of amino acid residues I348–Q370. Based on our discoveries and previous reports, a model has been proposed in which Oxa1-CTD interacts with ribosomes, accompanied by transient-to-stable transitions at the second contact site. These observations may enhance our understanding of the potential role of Oxa1-CTD in facilitating the assembly of oxidative phosphorylation complexes and provide insight into the structural characteristics of Oxa1-CTD. [ABSTRACT FROM AUTHOR]

Unstructured regions in functional proteins have gained attention in recent years due to advancements in informatics tools and biophysical methods. G protein-coupled receptors (GPCRs), a large family of cell surface receptors, contain unstructured regions in the form of the i3 loop and C-terminus. This review provides an overview of the functional significance of these regions in GPCRs. GPCRs transmit signals from the extracellular environment to the cell interior, regulating various physiological processes. The i3 loop, located between the fifth and sixth transmembrane helices, and the C-terminus, connected to the seventh transmembrane helix, are determinant of interactions with G proteins and with other intracellular partners such as arrestins. Recent studies demonstrate that the i3 loop and C-terminus play critical roles in allosterically regulating GPCR activation. They can act as autoregulators, adopting conformations that, by restricting G protein access, modulate receptor coupling specificity. The length and unstructured nature of the i3 loop and C-terminus provide unique advantages in GPCR interactions with intracellular protein partners. They act as "fishing lines", expanding the radius of interaction and enabling GPCRs to tether scaffolding proteins, thus facilitating receptor stability during cell membrane movements. Additionally, the i3 loop may be involved in domain swapping between GPCRs, generating novel receptor dimers with distinct binding and coupling characteristics. Overall, the i3 loop and C-terminus are now widely recognized as crucial elements in GPCR function and regulation. Understanding their functional roles enhances our comprehension of GPCR structure and signaling complexity and holds promise for advancements in receptor pharmacology and drug development. [ABSTRACT FROM AUTHOR]

All proteins have a carboxyl terminus, and we previously summarized eight mutations in binding and trafficking sequence determinants in the C-terminus that, when disrupted, cause human diseases. These sequence elements for binding and trafficking sites, as well as post-translational modifications (PTMs), are called minimotifs or short linear motifs. We wanted to determine how frequently mutations in minimotifs in the C-terminus cause disease. We searched specifically for PTMs because mutation of a modified amino acid almost always changes the chemistry of the side chain and can be interpreted as loss-of-function. We analyzed data from ClinVar for disease variants, Minimotif Miner and the C-terminome for PTMs, and RefSeq for protein sequences, yielding 20 such potential disease-causing variants. After additional screening, they include six with a previously reported PTM disruption mechanism and nine with new hypotheses for mutated minimotifs in C-termini that may cause disease. These mutations were generally for different genes, with four different PTM types and several different diseases. Our study helps to identify new molecular mechanisms for nine separate variants that cause disease, and this type of analysis could be extended as databases grow and to binding and trafficking motifs. We conclude that mutated motifs in C-termini are an infrequent cause of disease. [ABSTRACT FROM AUTHOR]

NAD+-dependent formate dehydrogenase (EC 1.2.1.2, FDH) from pathogenic bacterium Staphylococcus aureus (SauFDH) differs significantly from other FDHs both in terms of primary structure and catalytic properties. A distinctive feature of SauFDH is the highest (about 2.5–3 times) specific activity compared to other formate dehydrogenases. At the same time, SauFDH has high Michaelis constants for both substrates. Based on the analysis of three-dimensional structures and the alignment of amino acid sequences, replacements promising in terms of changing catalytic parameters were selected. The replacement of I220H resulted in an increase in ; the value of kcat has not changed. When T250H is replaced, an increase in is observed, kcat decreases from 20 to 13 s–1. The replacement of K368H led to a slight increase in , kcat decreased from 20 to 6 s–1. The introduction of TGA and AGA additional inserts in α-helix at the C-terminus of the enzyme led to an increase in and . A bigger effect was observed for —the difference was more than 10 times. For mutant SauFDH with insertions kcat significantly reduced to 4 s–1. Similar results were observed for mutants with multipoint replacements. Thus, the C-terminal sequence has been shown to play an important role in the catalysis of SauFDH. [ABSTRACT FROM AUTHOR]

P53 immunohistochemical staining with antibodies targeted to epitopes at or near the N-terminus are commonly used in diagnostic pathology practice as a surrogate for TP53 mutations. The abnormal staining patterns indicating TP53 mutations include nuclear overexpression, null, and the recently described cytoplasmic staining. The latter staining pattern occurs with the less common TP53 mutations affecting its nuclear localization and/or tetramerization domains that are located toward the C-terminus. Here we describe the first two cases of pediatric sarcomas with cytoplasmic staining with P53 antibody against N-terminus epitope and the absence of staining with P53 antibody against C-terminus epitope. We propose that a more precise description of P53 immunohistochemical staining patterns should include the nature of the antibody used. [ABSTRACT FROM AUTHOR]

Main conclusion: A synthetic peptide from the C-terminal end of C4-phosphoenolpyruvate carboxylase is implicated in the proteolysis of the enzyme, and Glc-6P or phosphorylation of the enzyme modulate this effect. Phosphoenolpyruvate carboxylase (PEPC) is a cytosolic, homotetrameric enzyme that performs a variety of functions in plants. Among them, it is primarily responsible for CO2 fixation in the C4 photosynthesis pathway (C4-PEPC). Here we show that proteolysis of C4-PEPC by cathepsin proteases present in a semi-purified PEPC fraction was enhanced by the presence of a synthetic peptide containing the last 19 amino acids from the C-terminal end of the PEPC subunit (pC19). Threonine (Thr)944 and Thr948 in the peptide are important requirements for the pC19 effect. C4-PEPC proteolysis in the presence of pC19 was prevented by the PEPC allosteric effector glucose 6-phosphate (Glc-6P) and by phosphorylation of the enzyme. The role of these elements in the regulation of PEPC proteolysis is discussed in relation to the physiological context. [ABSTRACT FROM AUTHOR]

Background: 4-vinylphenols produced by phenolic acid degradation catalyzed by phenolic acid decarboxylase can be used in food additives as well as flavor and fragrance industry. Improving the catalytic characters of phenolic acid decarboxylase is of great significance to enhance its practical application. Results: A phenolic acid decarboxylase (P-WT) was created from Bacillus amyloliquefaciens ZJH-01. Mutants such as P-C, P-N, P-m1, P-m2, P-Nm1, and P-Nm2 were constructed by site-directed mutagenesis of P-WT. P-C showed better substrate affinities and higher turnover rates than P-WT for p-coumaric acid, ferulic acid, and sinapic acid; however, P-N had reduced affinity toward p-coumaric acid. The extension of the C-terminus increased its acid resistance, whereas the extension of the N-terminus contributed to the alkali resistance and heat resistance. The affinity of P-m1 to four substrates and that of P-m2 to p-coumaric acid and ferulic acid were greatly improved. However, the affinity of P-Nm2 to four phenolic acids was greatly reduced. The residual enzyme activities of P-Nm1 and P-Nm2 considerably improved compared with those of P-m1 and P-m2 after incubation at 50 °C for 60 min. Conclusions: The extension of the N-terminus may be more conducive to the combination of the binding cavity with the substrate in an alkaline environment and may make its structure more stable. [ABSTRACT FROM AUTHOR]

Several water-soluble variants of the human mu opioid receptor (wsMORs) have been designed and expressed, which enables the detection of opioids in the nM to pM range using biosensing platforms. The tools previously developed allowed us to investigate MOR and G-protein interactions in a lipid free system to demonstrate that the lipid bilayer might not be essential for the G-protein recognition and binding. In this study, we are able to investigate G-protein interactions with MOR by using graphene enabled technology, in a lipid free system, with a high sensitivity in a real time manner. A new wsMOR with the native C-terminus was designed, expressed and then immobilized on the surfaces of scalable graphene field effect transistor (GFET)-based biosensors, enabling the recording of wsMOR/G-protein interaction with an electronic readout. G-protein only interacts with the wsMOR in the presence of the native MOR C-terminus with a $\text{K}_{A}$ of 32.3± 11.1 pM. The electronic readout of such interaction is highly reproducible with little variance across 50 devices in one biosensor array. For devices with receptors that do not have the native C-terminus, no significant electronic response was observed in the presence of G-protein, indicating an absence of interaction. These findings reveal that lipid environment is not essential for the G-protein interaction with MOR, however, the C-terminus of MOR is essential for G-protein recognition and high affinity binding. A system to detect MOR-G protein interaction is developed. wsMOR-G2_Cter provides a novel tool to investigate the role of C terminus in the signaling pathway. [ABSTRACT FROM AUTHOR]

Background: Multiple C2 domains and transmembrane region proteins (MCTPs) may act as transport mediators of other regulators. Although increased number of MCTPs in higher plants implies their diverse and specific functions in plant growth and development, only a few plant MCTPs have been studied and no study on the MCTPs in cotton has been reported. Results: In this study, we identified 31 MCTPs in G. hirsutum, which were classified into five subfamilies according to the phylogenetic analysis. GhMCTPs from subfamily V exhibited isoelectric points (pIs) less than 7, whereas GhMCTPs from subfamily I, II, III and IV exhibited pIs more than 7.5, implying their distinct biological functions. In addition, GhMCTPs within subfamily III, IV and V exhibited more diverse physicochemical properties, domain architectures and expression patterns than GhMCTPs within subfamily I and II, suggesting that GhMCTPs within subfamily III, IV and V diverged to perform more diverse and specific functions. Analyses of conserved motifs and pIs indicated that the N-terminus was more divergent than the C-terminus and GhMCTPs' functional divergence might be mainly contributed by the N-terminus. Furthermore, yeast two-hybrid assay indicated that the N-terminus was responsible to interact with target proteins. Phylogenetic analysis classified multiple N-terminal C2 domains into four subclades, suggesting that these C2 domains performed different molecular functions in mediating the transport of target proteins. Conclusions: Our systematic characterization of MCTPs in G. hirsutum will provide helpful information to further research GhMCTPs' molecular roles in mediating other regulators' transport to coordinate growth and development of various cotton tissues. [ABSTRACT FROM AUTHOR]

The misfolding of transactive response DNA-binding protein (TDP-43) is a major contributor to the pathogenesis of TDP-43 proteinopathies, including amyotrophic lateral sclerosis and frontotemporal lobar degeneration with TDP-43 inclusions, but also plays a role in other neurodegenerative diseases including Alzheimer disease. It is thought that different truncations at the N- and C-termini of TDP-43 contribute to its misfolding and aggregation in the brain, and that these aberrant TDP-43 fragments contribute to disease. Despite this, little is known about whether different truncation events influence the protein's transmissibility between cells and how this cell-to-cell transfer occurs. In this study, we use a well-established cellular model to study the efficiency by which full-length and truncated TDP-43 fragments are transferred between neuron-like cells. We demonstrate that preservation of the N-terminus of TDP-43 enhances its transmissibility between cells and that this protein transmission occurs in a manner exclusive of extracellular vesicles, instead requiring cellular proximity for efficient propagation. These data indicate that the N-terminus of TDP-43 might be a useful target in the generation of therapeutics to limit the spread of TDP-43 pathology. [ABSTRACT FROM AUTHOR]

Extracellular adenine nucleotides play important roles in cell–cell communication and tissue homeostasis. High concentrations of extracellular ATP released by dying cells are sensed as a danger signal by the P2X7 receptor, a non-specific cation channel. Studies in P2X7 knockout mice and numerous disease models have demonstrated an important role of this receptor in inflammatory processes. P2X7 activation has been shown to induce a variety of cellular responses that are not usually associated with ion channel function, for example changes in the plasma membrane composition and morphology, ectodomain shedding, activation of lipases, kinases, and transcription factors, as well as cytokine release and apoptosis. In contrast to all other P2X family members, the P2X7 receptor contains a long intracellular C-terminus that constitutes 40% of the whole protein and is considered essential for most of these effects. So far, over 50 different proteins have been identified to physically interact with the P2X7 receptor. However, few of these interactions have been confirmed in independent studies and for the majority of these proteins, the interaction domains and the physiological consequences of the interactions are only poorly described. Also, while the structure of the P2X7 extracellular domain has recently been resolved, information about the organization and structure of its C-terminal tail remains elusive. After shortly describing the structure and assembly of the P2X7 receptor, this review gives an update of the identified or proposed interaction domains within the P2X7 C-terminus, describes signaling pathways in which this receptor has been involved, and provides an overlook of the identified interaction partners. [ABSTRACT FROM AUTHOR]

All proteins end with a carboxyl terminus that has unique biophysical properties and is often disordered. Although there are examples of important C-termini functions, a more global role for the C-terminus is not yet established. In this review, we summarize research on C-termini, a unique region in proteins that cells exploit. Alternative splicing and proteolysis increase the diversity of proteins and peptides in cells with unique C-termini. The C-termini of proteins contain minimotifs, short peptides with an encoded function generally characterized as binding, posttranslational modifications, and trafficking. Many of these activities are specific to minimotifs on the C-terminus. Approximately 13% of C-termini in the human proteome have a known minimotif, and the majority, if not all of the remaining termini have conserved motifs inferring a function that remains to be discovered. C-termini, their predictions, and their functions are collated in the C-terminome, Proteus, and Terminus Oriented Protein Function INferred Database (TopFIND) database/web systems. Many C-termini are well conserved, and some have a known role in health and disease. We envision that this summary of C-termini will guide future investigation of their biochemical and physiological significance. [ABSTRACT FROM AUTHOR]

Anterograde cell surface transport of nascent G protein‐coupled receptors (GPCRs) en route from the endoplasmic reticulum (ER) through the Golgi apparatus represents a crucial checkpoint to control the amount of the receptors at the functional destination and the strength of receptor activation‐elicited cellular responses. However, as compared with extensively studied internalization and recycling processes, the molecular mechanisms of cell surface trafficking of GPCRs are relatively less defined. Here, we will review the current advances in understanding the ER‐Golgi‐cell surface transport of GPCRs and use angiotensin II type 1 receptor as a representative GPCR to discuss emerging roles of receptor‐interacting proteins and specific motifs embedded within the receptors in controlling the forward traffic of GPCRs along the biosynthetic pathway. As compared with internalization and recycling, anterograde cell surface transport of nascent G protein‐coupled receptors (GPCRs) en route from the endoplasmic reticulum through the Golgi apparatus remains poorly understood. Here, we will review the current understanding of the cell surface trafficking of the GPCR superfamily and use AT1R as an example to discuss emerging roles of receptor‐interacting proteins and specific motifs. [ABSTRACT FROM AUTHOR]

Background: Adenocarcinoma of the pancreas is one of the most aggressive tumor diseases affecting the human body. The oncogenic potential of pancreatic cancer is mainly characterized by extremely rapid growth triggered by the activation of oncogenic signaling cascades, which suggests a change in the regulation of important transcription factors. Amongst others, NFAT transcription factors are assumed to play a central role in the carcinogenesis of pancreatic cancer. Recent research has shown the importance of the transcription factor Sp1 in the transcriptional activity of NFATc2 in pancreatic cancer. However, the role of the interaction between these two binding partners remains unclear. The current study investigated the role of Sp1 proteins in the expression of NFATc2 target genes and identified new target genes and their function in cells. A further objective was the domain of the Sp1 protein that mediates interaction with NFATc2. The involvement of Sp1 proteins in NFATc2 target genes was shown by means of a gene expression profile analysis, and the results were confirmed by quantitative RT-PCR. The functional impact of this interaction was shown in a thymidine incorporation assay. A second objective was the physical interaction between NFATc2 and different Sp1 deletion mutants that was investigated by means of immunoprecipitation. Results: In pancreatic cancer, the proto-oncogene c-Fos, the tumor necrosis factor TNF-alpha, and the adhesion molecule integrin beta-3 are target genes of the interaction between Sp1 and NFATc2. Loss of just one transcription factor inhibits oncogenic complex formation and expression of cell cycle-regulating genes, thus verifiably decreasing the carcinogenic effect. The current study also showed the interaction between the transcription factor NFATc2 and the N-terminal domain of Sp1 in pancreatic cancer cells. Sp1 increases the activity of NFATc2 in the NFAT-responsive promoter. Conclusions: The regulation of gene promotors during transcription is a rather complex process because of the involvement of many proteins that – as transcription factors or co-factors – regulate promotor activity as required and control cell function. NFATc2 and Sp1 seem to play a key role in the progression of pancreatic cancer. [ABSTRACT FROM AUTHOR]

Adenovirus Early 1A proteins (E1A) are crucial for initiation of the viral life cycle after infection. The E1A gene is encoded at the left end of the viral genome and consists of two exons, the first encoding 185 amino acids in the 289 residues adenovirus 5 E1A, while the second exon encodes 104 residues. The second exon-encoded region of E1A is conserved across all E1A isoforms except for the 55 residues protein, which has a unique C-terminus due to a frame shift following splicing into the second exon. This region of E1A contributes to a variety of processes including the regulation of viral and cellular gene expression, immortalization and transformation. Here we evaluated the contributions that different regions of the second exon of E1A make to the viral life cycle using deletion mutants. The region of E1A encoded by the second exon was found to be important for overall virus growth, induction of viral and cellular gene expression, viral genome replication and deregulation of the cell cycle. Efficient viral replication was found to require exon 2 and the nuclear localization signal, as loss of either resulted in severe growth deficiency. Induction of cellular DNA synthesis was also deficient with any deletion of E1A within the C-terminus even if these deletions were outside of conserved region 4. Overall, our study provides the first comprehensive insight into the contributions of the C-terminus of E1A to the replicative fitness of human adenovirus 5 in arrested lung fibroblasts. [ABSTRACT FROM AUTHOR]

The human orexin 2 receptor (OX2R) is a G-protein-coupled receptor (GPCR) that has been implicated in a number of diverse physiological functions. Recent studies have identified a number of functions of the C-termini of GPCRs. However, the importance of the OX2R C-terminus in regulating signaling and surface expression remains unclear. In the present study, the function of the OX2R C-terminus was investigated using three C-terminal mutants, which were truncated at residues 368, 384 and 414, respectively, and the wild-type control, which expressed the full-length OX2R. HEK-293 cells were transfected with the mutated and control OX2R constructs. ELISA, western blot analysis and calcium assays were used to investigate the effects of the mutations on OX2R function. The present results demonstrated that residues 385-414 and 415-444 exhibited a cumulative effect on the surface expression of OX2R. Residues 369-384 exhibited a significant influence on inositol phosphate production and extracellular signal-regulated kinase 1/2 phosphorylation. Residues 385-414 significantly influenced agonist-induced internalization, whereas residues 369-384 and 385-414 significantly influenced Ca2+ release. The results of the present study suggest that the C-terminus of OX2R is important for its role in various physiological and pathological processes, and may therefore be associated with such disorders as depression and anorexia. [ABSTRACT FROM AUTHOR]

With increasing interests of phytoestrogens for their potential applications, a rapid and simple tool for screening these phytochemicals is still required. In this study, a simple assay to detect phytoestrogens was developed based on the competition binding between the tested samples and the fluorescently labelled oestrogen (E2) to the human ligand binding domain of oestrogen receptor (LBD‐ER) that was immobilised on the magnetite nanoparticles (MNPs). The 40‐kDa LBD‐ER peptide was produced in an Escherichia coli system. The synthesised 68.7‐nm MNPs were silanised and subsequently covalently linked to the C‐terminus of LBD‐ER peptide. The LBD‐ER immobilised MNPs demonstrated the specific binding for the standard E2 with the equilibrium dissociation constant of 9.56 nM and the binding capacity of 0.08 pmol/1 mg of the MNPs. The LBD‐ER immobilised MNPs could evaluate oestrogenic activity of the extracts of Asparagus racemosus and Curcuma comosa, the reported phytoestrogenic plants, but not progesterone (P4) and Raphanus sativus extract, the negative controls. The results of this work clearly demonstrated a potential assay for detecting phytoestrogens of crude plant extracts, which is simple and easily adapted to a high throughput format. [ABSTRACT FROM AUTHOR]

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The doublesex gene regulates the somatic sexual development of Bombyx mori by alternatively splicing into sex-specific splice forms. In our previous study, the splice form Bmdsx, which encodes the BmDSX protein, was found to be expressed in a female-specific manner and to contain a novel C-terminus. In this study, we aimed to investigate the role of this C-terminus. Two transgenic lines, L1 and L2, were constructed to ectopically express Bmdsx in males. Phenotype and W chromosome-specific polymerase chain reaction (PCR) analysis showed that developmental abnormalities and sex reversal did not occur. Moreover, the sex ratio was also normal. Quantitative PCR revealed that the expression levels of SP1 and Vg were upregulated in the fat body of transgenic males. Additionally, the expression level of PBP was downregulated in the antenna of transgenic males. The results suggested that the C-terminus of BmDSX functioned as a regulatory domain during regulation of downstream target gene expression and that BmDSX participated in the sexual development of somatic cells together with other DSX proteins in B. mori. [ABSTRACT FROM AUTHOR]

Chikungunya virus (CHIKV) is an arthropod-borne agent that causes severe arthritic disease in humans and is considered a serious health threat in areas where competent mosquito vectors are prevalent. CHIKV has recently been responsible for several millions of cases of disease, involving over 40 countries. The recent re-emergence of CHIKV and its potential threat to human health has stimulated interest in better understanding of the biology and pathogenesis of the virus, and requirement for improved treatment, prevention and control measures. In this study, we mapped the binding sites of a panel of eleven monoclonal antibodies (mAbs) previously generated towards the capsid protein (CP) of CHIKV. Using N- and C-terminally truncated recombinant forms of the CHIKV CP, two putative binding regions, between residues 1-35 and 140-210, were identified. Competitive binding also revealed that five of the CP-specific mAbs recognized a series of overlapping epitopes in the latter domain. We also identified a smaller, N-terminally truncated product of native CP that may represent an alternative translation product of the CHIKV 26S RNA and have potential functional significance during CHIKV replication. Our data also provides evidence that the C-terminus of CP is required for authentic antigenic structure of CP. This study shows that these anti-CP mAbs will be valuable research tools for further investigating the structure and function of the CHIKV CP. [ABSTRACT FROM AUTHOR]

The N-terminus of parathyroid hormone--related protein regulates bone marrow stromal cell differentiation. We hypothesized that the nuclear localization sequence and C-terminus are involved. MicroRNA and gene expression analyses were performed on bone marrow stromal cells from mice lacking the nuclear localization sequence and C-terminus (PthrpΔ/Δ) and age-matched controls. Differentiation assays with microRNA, cytochemical/histologic/morphologic, protein, and gene expression analyses were performed. PthrpΔ/Δ bone marrow stromal cells are anti-osteochondrogenic, pro-adipogenic, and pro-myogenic, expressing more Klf4, Gsk-3β, Lif, Ct-1, and microRNA-434 but less β-catenin, Igf-1, Taz, Osm, and microRNA-22 (p ≤ 0.024). PthrpΔ/Δ osteoblasts had less mineralization, osteocalcin, Runx2, Osx, Igf-1, and leptin (p ≤ 0.029). PthrpΔ/Δ produced more adipocytes, Pparγ, and aP2, but less Lpl (p ≤ 0.042). PthrpΔ/Δ cartilage pellets were smaller with less Sox9 and Pth1r, but greater Col2a1 (p ≤ 0.024). PthrpΔ/Δ produced more myocytes, Des, and Myog (p ≤ 0.021). MicroRNA changes supported these findings. In conclusion, the nuclear localization sequence and C-terminus are pro-osteochondrogenic, anti-adipogenic, and anti-myogenic. [ABSTRACT FROM AUTHOR]

Background: Human immunodeficiency virus type 1 (HIV-1) Vif hijacks an E3 ligase to suppress natural APOBEC3 restriction factors, and core binding factor â (CBF-â) is required for this process. Although an extensive region of Vif spanning most of its N-terminus is known to be critical for binding with CBF-â, involvement of the Vif C-terminus in the interaction with CBF-â has not been fully investigated. Results: Here, through immunoprecipitation analysis of Vif C-terminal truncated mutants of various lengths, we identified that CBF-â binding requires not only certain amino acids (G126A, E134A, Y135A and G138A) in the HCCH region but also the HCCH motif itself, which also affects the Vif-mediated suppression of APOBEC3G/APOBEC3F (A3G/A3F). These mutants still maintained interactions with substrate A3G or A3F as well as other cellular factors ElonginB/C (ELOB/C), indicating that their structures were not functionally affected. Moreover, by determining that the BC box also is necessary for CBF-â interaction in vivo, we speculate that binding to ELOB/C induces conformational changes in Vif, facilitating its interaction with CBF-â and consequent interaction with CUL5. Conclusions: These results provide important information on the assembly of the Vif-CUL5-E3 ubiquitin ligase. Identification of the new binding interface with CBF-â at the C-terminus of HIV-1 Vif also provides novel targets for the development of HIV-1 inhibitors. [ABSTRACT FROM AUTHOR]

ABSTRACT Dibenzothiophene (DBT) is a typical sulfur-containing compound found in fossil fuels. This compound and its derivatives are resistant to the hydrodesulfurization method often used in industry, but they are susceptible to enzymatic desulfurization via the 4S pathway, which is a well-studied biochemical pathway consisting of four enzymes. DBT monooxygenase (DszC) from Rhodococcus erythropolis is involved in the first step of the 4S pathway. We determined the crystal structure of DszC, which reveals that, in contrast to several homologous proteins, the C-terminus (410-417) of DszC participates in the stabilization of the substrate-binding pocket. Analytical ultracentrifugation analysis and enzymatic assays confirmed that the C-terminus is important for the stabilization of the active conformation of the substrate-binding pocket and the tetrameric state. Therefore, the C-terminus of DszC plays a significant role in the catalytic activity of this enzyme. Proteins 2014; 82:2733-2743. © 2014 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

Cardiac Ca1.2 channels play a critical role in cardiac function. It has been proposed that the carboxyl-terminal intracellular tail of the Ca1.2 channel is the target of Ca-dependent and Ca-independent regulation of the channel. Recent studies on C-terminal truncated forms of the Ca1.2 channel reported neonatal death, reduced Ca1.2 current, and failure of β-adrenergic stimulation of these channels in ventricular cardiomyocytes (CMs). Here, we used atrial CMs at embryonic day 18.5 that expressed a C-terminal truncated form of the Ca1.2 channel (Stop/Stop). Surprisingly, the atrial CMs showed robust L-type Ca currents which could be stimulated by forskolin, an activator of adenylyl cyclase. These currents exhibited a left-ward shift in the voltage-dependent activation curve and a reduced sensitivity to the Ca channel blocker isradipine as compared to currents in wild-type atrial CMs. RT-PCR analysis revealed normal levels of mRNA for the Ca1.2 channel but a twofold increase in the level of mRNA for the Ca1.3 channel in the Stop/Stop atrium as compared to wild-type atrium. A Western blot analysis indicated an increase of Ca1.3 protein in the Stop/Stop atrium. We suggest that, in contrast to Stop/Stop ventricular CMs, Stop/Stop atrial CMs can compensate the functional loss of the truncated Ca1.2 channel with an upregulation of the Ca1.3 channel. [ABSTRACT FROM AUTHOR]

The Na,K-ATPase is essential to all animals, since it maintains the electrochemical gradients that energize the plasma membrane. Naturally occurring inhibitors of the pump from plants have been used pharmaceutically in cardiac treatment for centuries. The inhibitors block the pump by binding on its extracellular side and thereby locking it. To explore the possibilities for designing an alternative way of targeting the pump function, we have examined the structural requirements for binding to a pocket that accommodates the two C-terminal residues, YY, in the crystal structures of the pump. To cover the sample space of two residues, we first performed docking studies with the 400 possible dipeptides. For validation of the in silico predictions, pumps with 13 dipeptide sequences replacing the C-terminal YY were expressed in Xenopus laevis oocytes and examined with electrophysiology. Our data show a significant correlation between the docking scores from two different methods and the experimentally determined sodium affinities, which strengthens the previous hypothesis that sodium binding is coupled to docking of the C-terminus. From the dipeptides that dock the best and better than wild-type YY, it may therefore be possible to develop specific drugs targeting a previously unexplored binding pocket in the sodium pump. [ABSTRACT FROM AUTHOR]

The C-terminus (CT) of rCx46 consists of 186 residues (H230-I416). Recent studies showed that rCx46, truncated after H243, altered the formation of functional hemichannels when expressed in Xenopus oocytes, while rCx46, truncated after A333 formed gap junction hemichannels similarly to rCx46. To analyze the role of the CT up to A333 in functional expression with cell imaging and dye-transfer techniques, different mutants were generated by C-terminal truncation between H243-A333, labeled with EGFP and expressed in HeLa cells. These rCx46 variants were characterized according to their compartmentalization in organelles, their presence in microscopic detectable vesicles and their ability to form gap junction plaques. rCx46 truncated after A311 (rCx46) was compartmentalized, was found in vesicles and formed functional gap junction plaques similarly to rCx46. With a truncation after P284 (rCx46), the protein was not compartmentalized and the amount of vesicles containing the protein were reduced; however, functional gap junction plaque formation was not affected as compared to rCx46. rCx46 did not form functional gap junction plaques; it was not found in vesicles or in cellular compartments. Live-cell imaging and detection of annular junctions for rCx46 and rCx46 revealed that the truncation after P284 reduced the frequency of vesicle budding from gap junction plaques and the formation of annular junctions. These results suggest that the C-terminal region of rCx46 up to A311 (rCx46) is necessary for its correct compartmentalization and internalization in the form of annular junctions, while the H230-P284 C-terminal region (rCx46) is sufficient for the formation of dye coupled gap junction channels. [ABSTRACT FROM AUTHOR]

Abstract: Ncd, a member of kinesin-14 family motors, uses the power stroke, a lever-like pivoting action of a long and stiff element, to exert force and generate movement. To better understand the role of the Ncd C-terminus in this process we produced four Ncd mutants in which this segment was altered or deleted. For these proteins we measured their affinity to the microtubule, steady-state ATPase and gliding velocity in multiple motor assays. The mutations had a dramatic effect on all three parameters measured, suggesting that the C-terminal residues of Ncd play an important role in modulating the interaction of the motor with the microtubule. [Copyright &y& Elsevier]

C-terminal regions of the protein phosphatases PP1 and PP2B were seldom studied. C-terminal 24 amino acids of PP1 was deleted, its enzymatic activity increased 3-fold while its stability declined. When the truncated PP1 was fused with the terminal (residues 483–511) of PP2B, both its enzymatic activity and its stability remained low. This indicates that the termini of PP2B and PP1 have inhibitory effect on the catalytic domain of PP1. PP1-(1–306) and PP1wt differ in their activation by metal ions, showing that the sites interacting with metal ions are not located in its C-terminus; while metal ions activated notably to PP1/PP2B chimera. In addition, the sensitivity results of PP1-(1–306) to the inhibitors, TM and NCTD, proved that these two inhibitors also did not bind to the C-terminus. However, the IC50s of PP1/PP2B chimera were higher than for PP1-(1–306), indicating that the C-terminal region interferes interactions with these inhibitors to some extent. Although 483–511 segment of PP2B was not the functional domain, it played important role in interaction with metal ions and inhibitors. It further indicates although PP1 and PP2B have high sequence identity, their non-conserved termini have different roles. [ABSTRACT FROM PUBLISHER]

Abstract: In this study, we identified a variable region in the C-terminus of hemocyanin from the shrimp Litopenaeus vannamei (2288–2503bp, HcSC) by sequence alignments. A total of 13 SNPs were identified by PCR-SSCP and HcSC clone sequencing. The SSCP patterns of HcSC could be modulated in Vibro parahaemolyticus-treated shrimps. A novel SSCP band with four SNP sites was identified in V. parahaemolyticus-resistant shrimps. More importantly, three of these four SNPs introduced variations in amino acid sequence and possibly secondary structure of the HcSC polypeptide and resulted in a higher agglutinative activity against seven pathogenic bacteria. These results suggest that the C-terminus of shrimp L. vannamei hemocyanin possesses SNPs, which may be related to shrimp resistance to different pathogens. [Copyright &y& Elsevier]

Deacetoxy/deacetylcephalosporin C synthase (acDAOC/DACS) from Acremonium chrysogenum is a bifunctional enzyme that catalyzes both the ring-expansion of penicillin N to deacetoxycephalosporin C and the hydroxylation of the latter to deacetylcephalosporin C. The R308 residue located in close proximity to the C-terminus of acDAOC/DACS was mutated to the other 19 amino acids. In the resulting mutant pool, R308L, R308I, R308T and R308V showed significant improvement in their ability to convert penicillin analogs, thus confirming the role of R308 in controlling substrate selectivity, the four amino acids all possess short aliphatic sidechains that may improve hydrophobic interactions with the substrates. The mutant R308I showed the highest reactivity for penicillin G, with 3-fold increase in k/K ratio and 7-fold increase in relative activity. [ABSTRACT FROM AUTHOR]

Abstract: Substantial evidence implicates that the aggregation of α-synuclein (αSyn) is a critical factor in the pathogenesis of Parkinson’s disease. This study focuses on the role of αSyn C-terminus. We introduced two additional cysteine residues at positions 107 and 124 (A107C and A124C) to our previous construct. Five X-isomers of oxidative-folded mutation of α-synuclein with three disulfides were isolated and their secondary structures and aggregating features were analyzed. All isomers showed similar random coil structures as wild-type α-synuclein. However, these isomers did not form aggregates or fibrils, even with prolonged incubation, suggesting that the interactions between the C-terminal and N-terminal or central NAC region are important in maintaining the natively unfolded structure of αSyn and thus prevent αSyn from changing conformation, which is a critical step for fibrillation. [Copyright &y& Elsevier]

Amyloid precursor protein (APP) and its paralogs, amyloid precursor-like protein-1 and amyloid precursor-like protein-2, appear to have redundant but essential role(s) during development. To gain insights into the physiological and possibly pathophysiological functions of APP, we used a functional proteomic approach to identify proteins that interact with the highly conserved C-terminal region of APP family proteins. Previously, we characterized an interaction between APP and ubiquitous mitochondrial creatine kinase. Here, we describe an interaction between APP and a novel protein, 4-nitrophenylphosphatase domain and non-neuronal SNAP25-like protein homolog 1 (NIPSNAP1). The interaction between APP and NIPSNAP1 was confirmed both in transiently transfected COS7 cells and in the mouse brain, where NIPSNAP1 is expressed at a high level. We demonstrate that NIPSNAP1 is targeted to the mitochondria via its N-terminal targeting sequence, and interacts with mitochondrial chaperone translocase of the outer membrane 22. Mitochondrial localization of NIPSNAP1 appears to be critical for its interaction with APP, and overexpression of APP appeared to disrupt NIPSNAP1 mitochondrial localization. Moreover, APP overexpression resulted in downregulation of NIPSNAP1 levels in cultured cells. Our data suggest that APP may affect mitochondrial function through a direct interaction with NIPSNAP1 as well as with other mitochondrial proteins. [ABSTRACT FROM AUTHOR]

Acute oxygen sensing in the heart is thought to occur through redox regulation and phosphorylation of membrane channels. Here we report a novel O2-sensing mechanism involving the C-terminus of the L-type Ca2+ channel and regulated by PKA phosphorylation. In patch-clamped myocytes, oxygen deprivation decreased ICa within 40 s. The suppressive effect of anoxia was relieved by PKA-mediated phosphorylation only when Ca2+ was the charge carrier, whereas phosphorylated IBa remained sensitive to O2 withdrawal. Suppression of Ca2+ release by thapsigargin did not alter the response of ICa to anoxia, suggesting a mandatory role for Ca2+ influx and not Ca2+-induced Ca2+ release (CICR) in O2 regulation of the channel. Consistent with this idea, mutation of 80 amino acids in the Ca2+/CaM-binding domain of the recombinant α1C subunit that removes Ca2+ dependent inactivation (CDI) abolished O2 sensitivity of the channel. Our findings suggest that the Ca2+/CaM binding domain of the L-type Ca2+ may represent a molecular site for O2 sensing of the heart. [ABSTRACT FROM AUTHOR]

The cytoplasmic C-terminus plays regulatory roles in the gating of many ion channels. However, lack of structural information on the C-terminus prevents the elucidation of how the C-terminal domain interacts with the gating machinery to exert its effects on the channel gating. In this report, we investigated the regulatory role of the C-terminus with functional study and structural modeling of a succession of C-terminal truncations of the Kv1.2 and Kv1.2427-KcsA112-160 chimeric channels. Functional study demonstrated a length-dependent shift of the activation curves for the C-terminal truncations of the Kv1.2 channel. Structural modeling indicated that the C-terminus of one subunit could dynamically interact with the S4-S5 linker of a neighboring subunit and the probability of interaction was dependent on the length of the C-terminal truncated Kv1.2 channels. In contrast, no length-dependent shift of the activation curve and probability of interaction between C-terminus and the neighboring S4-S5 linker were observed for the truncations of the Kv1.2-KcsA chimeric channel, suggesting that the native C-terminus of the Kv1.2 channel is essential for the interaction. Furthermore, surface plasmon resonance measurements indicated that there is direct interaction between the C-terminal domain and the S4-S5 linker of the Kv1.2 channel. These results imply that the dynamic interaction of the C-terminus with the S4-S5 linker from a neighboring subunit of the Kv1.2 channel provides a mechanism for its C-terminus to regulate the channel activation. [ABSTRACT FROM AUTHOR]

Cargo transport within cells is essential to healthy cells, which requires microtubules-based motors, including kinesin. The C-terminal tails (E-hooks) of alpha and beta tubulins of microtubules have been proven to play important roles in interactions between the kinesins and tubulins. Here, we implemented multi-scale computational methods in E-hook-related analyses, including flexibility investigations of E-hooks, binding force calculations at binding interfaces between kinesin and tubulins, electrostatic potential calculations on the surface of kinesin and tubulins. Our results show that E-hooks have several functions during the binding process: E-hooks utilize their own high flexibilities to increase the chances of reaching a kinesin; E-hooks help tubulins to be more attractive to kinesin. Besides, we also observed the differences between alpha and beta tubulins: beta tubulin shows a higher flexibility than alpha tubulin; beta tubulin generates stronger attractive forces (about twice the strengths) to kinesin at different distances, no matter with E-hooks in the structure or not. Those facts may indicate that compared to alpha tubulin, beta tubulin contributes more to attracting and catching a kinesin to microtubule. Overall, this work sheds the light on microtubule studies, which will also benefit the treatments of neurodegenerative diseases, cancer treatments, and preventions in the future. [ABSTRACT FROM AUTHOR]

Many members of the TRP superfamily oligomerize in the ER before trafficking to the plasma membrane. For membrane localization of the non-selective cation channel TRPV4 specific domains in the N-terminus are required, but the role of the C-terminus in the oligomerization and trafficking process has been not determined until now. Therefore, the localization of recombinant TRPV4 in two cell models was analyzed: HaCaT keratinocytes that express TRPV4 endogenously were compared to CHO cells that are devoid of endogenous TRPV4. When deletions were introduced in the C-terminal domain three states of TRPV4 localization were defined: a truncated TRPV4 protein of 855 amino acids was exported to the plasma membrane like the full-length channel (871 aa) and was also functional. Mutants with a length of 828 to 844 amino acids remained in the ER of CHO cells, but in HaCaT cells plasma membrane localization was partially rescued by oligomerization with endogenous TRPV4. This was confirmed by coexpression of recombinant full-length TRPV4 together with these deletion mutants, which resulted in an almost complete plasma membrane localization of both proteins and significant FRET in the plasma membrane and the ER. All deletions upstream of amino acid 828 resulted in total ER retention that could not rescued by coexpression with the full-length protein. However, these deletion mutants did not impair export of full-length TRPV4, implying that no oligomerization took place. These data indicate that the C-terminus of TRPV4 is required for oligomerization, which takes place in the ER and precedes plasma membrane trafficking. [ABSTRACT FROM AUTHOR]

The Group C G protein-coupled receptors include the metabotropic glutamate receptors (mGluRs), the GABAB receptor, the calcium sensor and several taste receptors, most of which are obligate dimers, indeed recent work has shown that dimerization is necessary for the activation of these receptors. Consequently factors that regulate their ability to homo- or heterodimerize are important. The Group 1 mGluRs include mGluR1 and mGluR5 both of which have splice variants with altered C-termini. In this study, we show that mGluR1b is a dimer and that it does not efficiently heterodimerize with mGluR1a, unlike the two splice variants of mGluR5 that can heterodimerize. Mutation of a positively charged motif (RRKK) at the C-terminus of the mGluR1b tail permits mGluR1b to heterodimerize with mGluR1a. Co-expression of mGluR1a and mGluR1b in COS-7 cells results in the accumulation of mGluR1b in intracellular inclusions that do not contain mGluR1a. This behaviour is mimicked by a chimera of the lymphocyte antigen CD2 with the C-terminus of mGluR1b (pCD1b) and depends on the presence of the RRKK motif. These accumulations are immunoreactive for endoplasmic reticulum (ER) markers, but not Golgi and ERGIC markers. This segregation of mGluR1b from other ER proteins may contribute to its failure to dimerize with mGluR1a. [ABSTRACT FROM AUTHOR]

Abstract: Desensitization of the cannabinoid CB1 receptor is mediated by the interaction with arrestin. In this study, we report the structural changes of a synthetic diphosphorylated peptide corresponding to residues 419–439 of the CB1 C-terminus upon binding to arrestin-2. This segment is pivotal to the desensitization of CB1. Using high-resolution proton NMR, we observe two helical segments in the bound peptide that are separated by the presence a glycine residue. The binding we observe is with a diphoshorylated peptide, whereas a previous study reported binding of a highly phosphorylated rhodopsin fragment to visual arrestin. The arrestin bound conformations of the peptides are compared. [Copyright &y& Elsevier]

The human anion exchanger 1 (AE1) is the most abundant integral membrane protein in red cells and is responsible for the exchange of Cl- for HCO3 -. However, the detailed role played by the AE1 C-terminal region in the anion translocation and membrane trafficking process remains unclear. In this paper, we created four mutants in the human AE1 C-terminus by deletion of the residues Ala891-Phe895, Asp896-Glu899, Asp902-Glu906 and Val907-Val911, to investigate the role of these sequences in functional expression of AE1. WT AE1 and its deletion mutant constructs were expressed in HEK 293 cells. Western blotting showed that deletions of Ala891-Phe895, Asp896-Glu899, and Val907-Val911 induced high expression of AE1, whereas loss of Asp902-Glu906 results in stable low expression. Pulse chase assays of WT AE1 and its mutants showed that the stability of protein is unaffected by the levels of expression of the AE1 and its mutants. Ala891-Phe895, Asp902-Glu906 and Val907-Val911 mutants exhibited lower levels of trafficking to the plasma membrane compared with WT AE1, while the Asp896-Glu899 mutant was more highly expressed at the plasma membrane. The decreased ability of the mutants to mediate Cl-/HCO3 - exchange in transfected cells revealed that the deletion sequences have an important role in transport activity. These results demonstrate that the studied residues in the AE1 C-terminus differently affect the expression, membrane trafficking and functional folding of AE1. [ABSTRACT FROM AUTHOR]

Analysis of DNA sequences coding for the C-terminus of spider silk proteins from a range of spiders suggests that many silk C-termini share a common origin, and that their physical properties have been highly conserved over several hundred million years. These physical properties are compatible with roles in protein synthesis, silk function and in recruiting accessory proteins. Phylogenetic relationships among different silk genes suggest that any recombination has been insufficient to homogenize the different types of silk gene, which appear to have evolved independently of one another. The types of nucleotide substitutions that have occurred suggest that selection may have operated differently in the various silk lineages. Amino acid sequences of flagelliform silk C-termini differ substantially from the other types of spider silk studied, but they are expected to have very similar physical properties and may perform a similar function. [ABSTRACT FROM AUTHOR]

Voltage gated potassium channels are tetrameric membrane proteins, which have a central role in cellular excitability. Human Kv1.4 channels open on membrane depolarization and inactivate rapidly by a ‘ball and chain’ mechanism whose molecular determinants have been mapped to the cytoplasmic N terminus of the channel. Here we show that the other terminal end of the channel also plays a role in channel inactivation. Swapping the C-terminal residues of hKv1.4 with those from two non-inactivating channels (hKv1.1 and hKv1.2) affects the rates of inactivation, as well as the recovery of the channel from the inactivated state. Secondary structure predictions of the hKv1.4 sequence reveal a helical structure at its distal C-terminal. Complete removal or partial disruption of this helical region results in channels with remarkably slowed inactivation kinetics. The ionic selectivity and voltage-dependence of channel opening were similar to hKv1.4, indicative of an unperturbed channel pore. These results demonstrate that fast inactivation is modulated by structural elements in the C-terminus, suggesting that the process involves the concerted action of the N- and C-termini. [ABSTRACT FROM AUTHOR]

Abstract: Streptomyces RecA proteins are characterized by a conserved, positively charged extension of unknown function appended at their C-termini. To investigate the function of this element, we introduced the Streptomyces rimosus recA gene and its mutant form encoding the protein with a C-terminal deletion into S. rimosus. Both transcript and protein levels were dramatically increased in the strain expressing the truncated gene compared to the strain bearing the wild-type recA, indicating involvement of the characteristic C-terminal extension in regulating the recA expression in Streptomyces. Considering that RecA acts as a major regulator of DNA damage response in bacteria, this mode of regulation is expected to have broader implications and significance that outreaches our current understanding of RecA autoregulation. [Copyright &y& Elsevier]

Prestin is a unique molecular-motor protein expressed in the lateral plasma membrane of outer hair cells (OHC) in the organ of Corti of the mammalian cochlea. It is thought that prestin undergoes conformational changes driven by the cell's membrane potential. The resulting alterations in OHC-length are assumed to constitute the cochlear amplifier. Prestin is a member of the anion solute carrier family 26 (SCL26A), but it is different from other family members in its unique function of voltage-driven motility. Because the C-terminus is the least conserved region in the family, we investigated its influence with a series of deletion, point and chimeric mutants. The function and cellular expression of mutants were examined in a heterologous expression system by measurement of nonlinear capacitance (NLC) and immunofluorescence. Each mutant produced a unique mixture of patterns of cell morphologies, which were classified as to the location of prestin within the cell. The data from deletion mutants (Del516, Del525, Del630, Del590, Del709, Del719) revealed that nearly the full length (>708 amino acids) of the protein was required for normal prestin expression and function. Since most deletion mutations eliminated plasma membrane targeting, chimeric proteins were constructed by fusing prestin, at amino acid 515 or 644, with the homologous portion of the C-terminus from the two most closely related SLC26A members, pendrin and putative anion exchanger 1. These chimeric proteins were again improperly (but differently) targeted than simple truncation mutants, and all lacked functional phenotype. When two of the potential basolateral membrane-targeting motifs were mutated (Y520A/Y526A), incomplete plasma membrane expression was seen. We also show that some double point mutations (V499G1Y501H) fully express in the plasma membrane but lack NLC. These non-charged amino acids may have unrevealed important roles in prestin's function. Together, these data suggest that certain specific sequences and individual amino acids in the C-terminus are necessary for correct cellular distribution and function. [ABSTRACT FROM AUTHOR]

Congenital long QT syndrome (LQTS) is a genetically heterogeneous disease in which six ion-channel genes have been identified. The phenotype-genotype relationships of the HERG (human ether-a-go-go-related gene) mutations are not fully understood. The objective of this study is to identify the underlying genetic basis of a Chinese family with LQTS and to characterize the clinical manifestations properties of the mutation. Single strand conformation polymorphism (SSCP) analyses were conducted on DNA fragments amplified by polymerase chain reaction from five LQT-related genes. Aberrant conformers were analyzed by DNA sequencing. A novel splice mutation in C-terminus of HERG was identified in this Chinese LQTS family, leading to the deletion of 11-bp at the acceptor splice site of Exon9 [Exon9 IVS del (−12→−2)]. The, mutation might affect, through deficient splicing, the putative cyclic nucleotide binding domain (CNBD) of the HERG K+ channel. This mutation resulted in a mildly affected phenotype. Only the proband had a history of syncopes, while the other three individuals with long QT interval had no symptoms. Two other mutation carriers displayed normal phenotype. No sudden death occurred in the family. The 4 affected individuals and the two silent mutation carriers were all heterozygous for the mutation. It is the first splice mutation of HERG reported in Chinese LQTS families. Clinical data suggest that the CNBD mutation may be less malignant than mutations occurring in the pore region and be partially dominant over wild-type function. [ABSTRACT FROM AUTHOR]

Abstract: Deacetoxy/deacetylcephalosporin C synthase (acDAOC/DACS) from Acremonium chrysogenum is a bifunctional enzyme that catalyzes both the ring-expansion of penicillin N to deacetoxycephalosporin C (DAOC) and the hydroxylation of the latter to deacetylcephalosporin C (DAC). Three residues N305, R307 and R308 located in close proximity to the C-terminus of acDAOC/DACS were each mutated to leucine. The N305L and R308L mutant acDAOC/DACSs showed significant improvement in their ability to convert penicillin analogs. R308 was identified for the first time as a critical residue for DAOC/DACS activity. Kinetic analyses of purified R308L enzyme indicated its improved catalytic efficiency is due to combined improvements of Km and kcat. Comparative modeling of acDAOC/DACS supports the involvement of R308 in the formation of substrate-binding pocket. [Copyright &y& Elsevier]

1. Block of the human two-pore domain potassium (2-PK) channel TREK-1 by fluoxetine (Prozac) and its active metabolite, norfluoxetine, was investigated using whole-cell patch-clamp recording of currents through recombinant channels in tsA 201 cells. 2. Fluoxetine produced a concentration-dependent inhibition of TREK-1 current that was reversible on wash. The IC50 for block was 19 microM. Block by fluoxetine was voltage-independent. Fluoxetine (100 microM) produced an 84% inhibition of TREK-1 currents, but only a 31% block of currents through a related 2-PK channel, TASK-3. 3. Norfluoxetine was a more potent inhibitor of TREK-1 currents with an IC50 of 9 microM. Block by norfluoxetine was also voltage-independent. 4. Truncation of the C-terminus of TREK-1 (delta89) resulted in a loss of channel function, which could be restored by intracellular acidification or the mutation E306A. The mutation E306A alone increased basal TREK-1 current and resulted in a loss of the slow phase of TREK-1 activation. 5. Progressive deletion of the C-terminus of TREK-1 had no effect on the inhibition of the channel by fluoxetine. The E306A mutation, on the other hand, reduced the magnitude of fluoxetine inhibition, with 100 microM producing only a 40% inhibition. 6. It is concluded that fluoxetine and norfluoxetine are potent inhibitors of TREK-1. Block of TREK-1 by fluoxetine may have important consequences when the drug is used clinically in the treatment of depression. [ABSTRACT FROM AUTHOR]

Three C-terminal variants of the human norepinephrine transporter (hNET) are known: the wild-type hNET in which exon 14 encodes the last seven amino acids and two variants with either three or 18 amino acids encoded by an alternatively spliced exon 15. In transfected HEK293 cells we compared by means of[3H]norepinephrine ([3H]NE) uptake and[3H]nisoxetine ([3H]NIS) binding the functional properties of the wild-type hNET with those of the more abundant long splice variant containing exon 15 (hNET-Ex15L) and of two artificial hNET mutants lacking either the last three (hNET-Ex14–4) or all seven (hNET-Ex14–0) C-terminal amino acids of exon 14. No differences among the NET isoforms were observed concerning theKm for uptake of NE and theKD for binding of NIS. However, compared with the wild-type hNET, the three isoforms (hNET-Ex15L, hNET-Ex14–4 and hNET-Ex14–0) showed a pronounced decrease inVmax of[3H]NE uptake andBmax of[3H]NIS binding which correlated with strongly reduced surface expression of the transporter isoforms. The decrease in surface expression of the hNET isoforms is probably a consequence of the lack of the three amino acids leucine, alanine and isoleucine at the C-terminal end which may represent a motif facilitating cell surface expression of the hNET. Expression of hNET-Ex15L exerted a dominant negative effect on plasma membrane expression of the wild-type hNET and thus may represent a novel mechanism for regulation of noradrenergic neurotransmission. [ABSTRACT FROM AUTHOR]

Mammalian cells poorly express rNav1.8 channels. In contrast, rNav1.7 dorsal root ganglion channels have 90-fold higher peak Na+ current densities. We investigated the role of rNav1.7 and rNav1.8 carboxy-termini in modulating the expression of rNav1.7 and rNav1.8 channels in tsA201 cells. Mutations in the ubiquitination site of the C-terminus did not improve rNav1.8 current levels. However, rNav1.8 chimeras containing the entire or the proximal portion of the rNav1.7 C-terminus expressed 3.2-fold and 4.8-fold higher peak current densities, respectively, than parent rNav1.8 channels. We conclude that the two Na+ channels may have different endoplasmic reticulum processing signals. [Copyright &y& Elsevier]