Amino-terminal Sequence Research Articles

Structural and Functional Analysis of Hemoglobin Binding to the Peritrophic Matrix During Blood Digestion in Aedes aegypti

The Aedes aegypti mosquito is responsible for transmitting pathogens such as the Dengue, Zika, and Chikungunya viruses. The peritrophic matrix (PM) is an extracellular chitin-rich structure that lines the midgut of arthropods, providing a crucial protective barrier for the gut epithelium against mechanical damage, ingested pathogens, and toxic substances. During blood digestion, hemoglobin is lysed, releasing free heme into the midgut lumen. Part of this heme binds strongly to the PM, mitigating its harmful effects on the mosquito epithelial cells. Our study focused on investigating the interaction dynamic between hemoglobin and the PM during blood digestion in A. aegypti. Optical microscopy was employed to observe the temporal progression of blood digestion in the A. aegypti midgut, highlighting significant morphological changes in the blood bolus. An electrophoresis analysis revealed distinct protein bands in the PM extract, some of which were associated with hemoglobin and its subunits. The presence of PM-associated hemoglobin was confirmed by amino-terminal sequencing and an immunoblot analysis using anti-hemoglobin antibodies. Furthermore, fluorescence microscopy revealed overlapping labeling between hemoglobin and chitin, suggesting an interaction between hemoglobin and PM chitin. Corroborating these results, hemoglobin showed an affinity with chitin in the chromatography and molecular docking assays, in which the hemoglobin subunits interacted with the oligosaccharide (NAG)4. Thus, hemoglobin may perform a function similar to that of peritrophins. Further experiments demonstrated the protective role of the PM against hemoglobin proteolysis during blood digestion. Overall, this study provides valuable insights into the intricate interactions between hemoglobin and the PM, enhancing our understanding of mosquito digestive physiology and potentially contributing to the development of vector control strategies.

Evaluating AlphaFold for Clinical Pharmacology and Pharmacogenetics: A Case-Study of Huntingtin Variants Linked to Huntington's Disease.

To evaluate the artificial intelligence (AI)-guided AlphaFold algorithm for studying the binding interactions of human huntingtin and the aggregation of huntingtin peptides. Variants of huntingtin protein implicated in Huntington's disease were used as a model system to evaluate AlphaFold. Variants of huntingtin and huntingtin peptides with polyglutamine tracts (PQT) containing 21, 31, 51, or 78 glutamines were studied. The 3-dimensional structures of huntingtin variants and their interactions with huntingtin-associated protein-40 (HAP40) were obtained. Aggregation experiments were conducted with peptide sequences corresponding to variants of PQT, amino terminal sequence (NTS) plus PQT, NTS plus PQT plus proline rich region (PRR), and the 300 amino acid sequence from the NTS through HEAT3 of huntingtin. Oligomerization experiments with 1, 3, 6, or 12 peptide sequences were used to assess the quaternary structures of aggregates. The PQT and PQT plus NTS peptides formed a helical secondary structure that formed a central core in the quaternary structure of the aggregates The PRR formed an extended type II polyproline helix that did not participate in central core the aggregates. The distance between the amino and carboxyl termini of disease-linked 31Q, 51Q, and 78Q variants of full-length huntingtin was prominently decreased compared to the 21Q huntingtin. The interaction of HAP40 with the 78Q variant increased the distance between the amino and carboxyl termini. AlphaFold identified key tertiary structure changes in human huntingtin that have been independently corroborated in experimental models. The results highlight the utility of AlphaFold for hypothesis generation in pharmaceutical research.

A Novel Kunitz Trypsin Inhibitor from Enterolobium gummiferum Seeds Exhibits Antibiofilm Properties against Pathogenic Yeasts.

Plant peptidase inhibitors play crucial roles in plant defence mechanisms and physiological processes. In this study, we isolated and characterised a Kunitz trypsin inhibitor from Enterolobium gummiferum seeds named EgPI (E. gummiferum peptidase inhibitor). The purification process involved two chromatography steps using size exclusion and hydrophobic resins, resulting in high purity and yield. EgPI appeared as a single band of ~20 kDa in SDS-PAGE. Under reducing conditions, the inhibitor exhibited two polypeptide chains, with 15 and 5 kDa. Functional characterisation revealed that EgPI displayed an inhibition stoichiometry of 1:1 against trypsin, with a dissociation constant of 8.4 × 10-9 mol·L-1. The amino-terminal sequencing of EgPI revealed the homology with Kunitz inhibitors. Circular dichroism analysis provided insights into the secondary structure of EgPI, which displayed the signature typical of Kunitz inhibitors. Stability studies demonstrated that EgPI maintained the secondary structure necessary to exhibit its inhibitory activity up to 70 °C and over a pH range from 2 to 8. Microbiological screening revealed that EgPI has antibiofilm properties against pathogenic yeasts at 1.125 μmol·L-1, and EgPI reduced C. albicans biofilm formation by 82.7%. The high affinity of EgPI for trypsin suggests potential applications in various fields. Furthermore, its antibiofilm properties recommended its usefulness in agriculture and antimicrobial therapy research, highlighting the practical implications of our research.

Molecular and Biochemical Characterization of Xylanase Produced by Streptomyces viridodiastaticus MS9, a Newly Isolated Soil Bacterium.

A xylan-degrading bacterial strain, MS9, was recently isolated from soil samples collected in Namhae, Gyeongsangnam-do, Republic of Korea. This strain was identified as a variant of Streptomyces viridodiastaticus NBRC13106T based on 16S rRNA gene sequencing, DNA-DNA hybridization analysis, and other chemotaxonomic characteristics, and was named S. viridodiastaticus MS9 (=KCTC29014= DSM42055). In this study, we aimed to investigate the molecular and biochemical characteristics of a xylanase (XynCvir) identified from S. viridodiastaticus MS9. XynCvir (molecular weight ≍ 21 kDa) was purified from a modified Luria-Bertani medium, in which cell growth and xylanase production considerably increased after addition of xylan. Thin layer chromatography of xylan-hydrolysate showed that XynCvir is an endo-(1,4)-β-xylanase that degrades xylan into a series of xylooligosaccharides, ultimately converting it to xylobiose. The Km and Vmax values of XynCvir for beechwood xylan were 1.13 mg/ml and 270.3 U/mg, respectively. Only one protein (GHF93985.1, 242 amino acids) containing an amino acid sequence identical to the amino-terminal sequence of XynCvir was identified in the genome of S. viridodiastaticus. GHF93985.1 with the twin-arginine translocation signal peptide is cleaved between Ala-50 and Ala-51 to form the mature protein (21.1 kDa; 192 amino acids), which has the same amino-terminal sequence (ATTITTNQT) and molecular weight as XynCvir, indicating GHF93985.1 corresponds to XynCvir. Since none of the 100 open reading frames most homologous to GHF93985.1 listed in GenBank have been identified for their biochemical functions, our findings greatly contribute to the understanding of their biochemical characteristics.

HGF-Based CAR-T Cells Target Hepatocellular Carcinoma Cells That Express High Levels of c-Met

ABSTRACT Background CAR-T is emerging as an effective treatment strategy for hematologic malignancies, however its effectiveness for treating solid tumors, such as Hepatocellular Carcinoma (HCC) is limited. Here, we screened a variety of CAR-T cells that target c-Met to investigate their potential to induce HCC cell death in vitro. Methods Human T cells were transduced to express CARs by lentiviral vector transfection. c-Met expression in human HCC cell lines and CARs expression were monitored by flow cytometry. Tumor cell killing was evaluated by Luciferase Assay System Kit. The concentrations of cytokine were tested by Enzyme-linked immunosorbent assays. Knock down and overexpression studies targeting c-Met were conducted to assess the targeting specificity of CARs. Results We found that CAR T cells expressing a minimal amino-terminal polypeptide sequence comprising the first kringle (kringle 1) domain (denoted as NK1 CAR-T cells), efficiently killed HCC cell lines that expressed high levels of the HGF receptor c-Met. Furthermore, we report that while NK1 CAR-T cells were efficient at targeting SMMC7221 cells for destruction, and its potency was significantly attenuated in parallel experiments with cells stably expressing short hairpin RNAs (shRNAs) that suppressed c-Met expression. Correspondingly, overexpression of c-Met in the embryonic kidney cell line HEK293T led to their enhanced killing by NK1 CAR-T cells. Conclusion Our studies demonstrate that a minimal amino-terminal polypeptide sequence comprising the kirngle1 domain of HGF is highly relevant to the design of effective CAR-T cell therapies that kill HCC cells expressing high levels of c-Met.

Purification and characterization of a serine protease from organic dust and elucidation of its inductive effects on lung inflammatory mediators.

Organic dust inhalation is associated with the development of respiratory diseases. Serine protease activities in organic dusts were previously reported to contribute to the induction of lung inflammatory mediators however, the identities of the proteases and the mechanisms by which they induce inflammatory mediators are unknown. The goal of this study was to purify and characterize serine protease(s) from organic dust and elucidate mechanisms by which they induce lung inflammatory mediators. A serine protease was purified from poultry organic dust by benzamidine-agarose affinity chromatography. Mass spectrometry and amino-terminal sequence analysis identified the purified protease as chicken trypsin II-P29. Purified protease induced proinflammatory cytokine levels in Beas2B and NHBE epithelial and THP-1 macrophage cells. Treatment with the purified protease increased cellular and mitochondrial reactive oxygen species (ROS) generation. Induction of inflammatory mediators and ROS were suppressed by serine protease inhibitors and antioxidants. Purified protease activated protein kinase C (PKC), mitogen-activated protein kinase (MAPK)1/3 and MAPK14 signaling, and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and signal transducer and activator of transcription 3 (Stat-3), and chemical inhibitors targeting these pathways suppressed induction of inflammatory mediators. Calcium mobilization studies showed that the purified protease activated protease-activated receptors (PAR) F2R, F2RL1, F2RL2, F2RL3, and F2R and F2RL1 knockdown suppressed the induction of inflammatory mediators. Intranasal instillation of purified protease increased lung chemokine (C-X-C motif) ligand (CXCL)1, interleukin (IL)-6, and tumor necrosis factor (TNF) levels in mice. Our studies have shown that chicken trypsin is a proinflammatory constituent of poultry organic dust, and induces lung inflammatory mediators via increased ROS and PAR activation in a cell signaling pathway involving PKC, MAPK1/3 and MAPK14, and NF-κB and Stat-3.NEW & NOTEWORTHY Inhalation of dust in industrial agricultural operations is linked to the development of lung diseases. Our studies have isolated for the first time a trypsin protease from poultry farm dust and have shown that it stimulates lung inflammation. The protease stimulates the production of oxidants and cell signaling pathways to increase inflammatory mediator production. Targeting trypsin protease in poultry farm environment may be a useful strategy to counter the harmful effects of dust.

Structurally Different Yet Functionally Similar: Aptamers Specific for the Ebola Virus Soluble Glycoprotein and GP1,2 and Their Application in Electrochemical Sensing.

The Ebola virus glycoprotein (GP) gene templates several mRNAs that produce either the virion-associated transmembrane protein or one of two secreted glycoproteins. Soluble glycoprotein (sGP) is the predominant product. GP1 and sGP share an amino terminal sequence of 295 amino acids but differ in quaternary structure, with GP1 being a heterohexamer with GP2 and sGP a homodimer. Two structurally different DNA aptamers were selected against sGP that also bound GP1,2. These DNA aptamers were compared with a 2'FY-RNA aptamer for their interactions with the Ebola GP gene products. The three aptamers have almost identical binding isotherms for sGP and GP1,2 in solution and on the virion. They demonstrated high affinity and selectivity for sGP and GP1,2. Furthermore, one aptamer, used as a sensing element in an electrochemical format, detected GP1,2 on pseudotyped virions and sGP with high sensitivity in the presence of serum, including from an Ebola-virus-infected monkey. Our results suggest that the aptamers interact with sGP across the interface between the monomers, which is different from the sites on the protein bound by most antibodies. The remarkable similarity in functional features of three structurally distinct aptamers suggests that aptamers, like antibodies, have preferred binding sites on proteins.

A Capsid Protein Fragment of a Fusagra-like Virus Found in Carica papaya Latex Interacts with the 50S Ribosomal Protein L17.

Papaya sticky disease is caused by the association of a fusagra-like and an umbra-like virus, named papaya meleira virus (PMeV) and papaya meleira virus 2 (PMeV2), respectively. Both viral genomes are encapsidated in particles formed by the PMeV ORF1 product, which has the potential to encode a protein with 1563 amino acids (aa). However, the structural components of the viral capsid are unknown. To characterize the structural proteins of PMeV and PMeV2, virions were purified from Carica papaya latex. SDS-PAGE analysis of purified virus revealed two major proteins of ~40 kDa and ~55 kDa. Amino-terminal sequencing of the ~55 kDa protein and LC-MS/MS of purified virions indicated that this protein starts at aa 263 of the deduced ORF1 product as a result of either degradation or proteolytic processing. A yeast two-hybrid assay was used to identify Arabidopsis proteins interacting with two PMeV ORF1 product fragments (aa 321-670 and 961-1200). The 50S ribosomal protein L17 (AtRPL17) was identified as potentially associated with modulated translation-related proteins. In plant cells, AtRPL17 co-localized and interacted with the PMeV ORF1 fragments. These findings support the hypothesis that the interaction between PMeV/PMeV2 structural proteins and RPL17 is important for virus-host interactions.

Crucial role of non-hydrophobic residues in H-region signal peptide on secretory production of l-asparaginase II in Escherichia coli

Protein secretion into the periplasmic space requires several interactions between the amino-terminal signal sequence of the protein and secretion machinery components. Therefore, modification of the components of amino-terminal sequence can be used as a powerful strategy to improve secretion efficiency. The hydrophobic region is an important domain for signal peptide function due to interaction with different components of the secretion apparatus. In this study, to evaluate the effect of hydrophobicity level and secondary structure of the h-domain signal peptide on the secretion efficiency, a series of missense mutations were constructed in the hydrophobic domain of the l-asparaginase II signal peptide. The h-region hydrophobicity level of mutants G8L, T16L, and G8L/T16L was increased compared with the wild-type. In addition, the amino acid glycine as a helix-breaker residue was substituted with leucine (G8L), forming a stable and extended α-helix structure in the h-domain. The effect of introducing an aromatic residue in this region was also investigated by mutant G8F. Our mutagenesis studies showed that increasing the hydrophobicity levels, extending the α-helical conformation, and the introduction of an aromatic residue within the h-region signal sequence reduced the secretion level of asparaginase. These results imply a vital role of non-hydrophobic residues in the H-region.

Bacterial Signal Peptides- Navigating the Journey of Proteins.

In 1971, Blobel proposed the first statement of the Signal Hypothesis which suggested that proteins have amino-terminal sequences that dictate their export and localization in the cell. A cytosolic binding factor was predicted, and later the protein conducting channel was discovered that was proposed in 1975 to align with the large ribosomal tunnel. The 1975 Signal Hypothesis also predicted that proteins targeted to different intracellular membranes would possess distinct signals and integral membrane proteins contained uncleaved signal sequences which initiate translocation of the polypeptide chain. This review summarizes the central role that the signal peptides play as address codes for proteins, their decisive role as targeting factors for delivery to the membrane and their function to activate the translocation machinery for export and membrane protein insertion. After shedding light on the navigation of proteins, the importance of removal of signal peptide and their degradation are addressed. Furthermore, the emerging work on signal peptidases as novel targets for antibiotic development is described.

Proteolytic Cleavage of the Extracellular Domain Affects Signaling of Parathyroid Hormone 1 Receptor.

Parathyroid hormone 1 receptor (PTH1R) is a member of the class B family of G protein-coupled receptors, which are characterized by a large extracellular domain required for ligand binding. We have previously shown that the extracellular domain of PTH1R is subject to metalloproteinase cleavage in vivo that is regulated by ligand-induced receptor trafficking and leads to impaired stability of PTH1R. In this work, we localize the cleavage site in the first loop of the extracellular domain using amino-terminal protein sequencing of purified receptor and by mutagenesis studies. We further show, that a receptor mutant not susceptible to proteolytic cleavage exhibits reduced signaling to Gs and increased activation of Gq compared to wild-type PTH1R. These findings indicate that the extracellular domain modulates PTH1R signaling specificity, and that its cleavage affects receptor signaling.

A new thermostable rhizopuspepsin: Purification and biochemical characterisation

The new thermostable fungal aspartic protease was produced through solid-state fermentation from Rhizopus azygosporus (MTCC 10195). The protease was purified by a three-tandem steps of ammonium sulfate precipitation (25−65 % saturation), ion exchange (DEAE sepharose CL 6B) chromatography, and gel filtration (Sephacryl S 200) chromatography. The optimum pH and temperature for protease activity were 4 and 52 ± 1.8 °C, respectively. The enzyme was unusually thermostable, as it retained 50 % of its activity beyond 85 °C after 20 min of incubation. The apparent molecular weight was 33.2 ± 2.3 kDa with a final specific activity of 86.6 U/mg. The enzyme was rich in β sheets (63 %) and was inhibited by pepstatin A, indicating that it is an aspartic protease. MS/MS analysis revealed the enzyme is an endopeptidase cleaving the C-terminus of Phe, Leu, Lys, Val, His, Glu, Met, and Tyr. Amino-terminal sequencing, coupled with in-gel trypsin digestion and MS analysis revealed that the enzyme was being reported for the first time with “experimental evidence at protein-level”.

Recent Progress of Small Molecule Menin-MLL Interaction Inhibitors as Therapeutic Agents for Acute Leukemia.

Mixed lineage leukemia (MLL) gene rearrangements are associated with acute leukemia. The protein menin is regarded as a critical oncogenic cofactor of the resulting MLL fusion proteins in acute leukemia. A direct interaction between menin and the MLL amino terminal sequences is necessary for MLL fusion protein-mediated leukemogenesis. Thus, inhibition of the interaction between menin and MLL has emerged as a novel therapeutic strategy. Recent improvements in structural biology and chemical reactivity have promoted the design and development of selective and potent menin-MLL interaction inhibitors. In this Perspective, different classes of menin-MLL interaction inhibitors are comprehensively summarized. Further research potential, challenges, and opportunities in the field are also discussed.

Multifunctionality of prostatic acid phosphatase in prostate cancer pathogenesis.

The role of human prostatic acid phosphatase (PAcP, P15309|PPAP_HUMAN) in prostate cancer was investigated using a new proteomics tool termed signal sequence swapping (replacement of domains from the native cleaved amino terminal signal sequence of secretory/membrane proteins with corresponding regions of functionally distinct signal sequence subtypes). This manipulation preferentially redirects proteins to different pathways of biogenesis at the endoplasmic reticulum (ER), magnifying normally difficult to detect subsets of the protein of interest. For PAcP, this technique reveals three forms identical in amino acid sequence but profoundly different in physiological functions, subcellular location, and biochemical properties. These three forms of PAcP can also occur with the wildtype PAcP signal sequence. Clinical specimens from patients with prostate cancer demonstrate that one form, termed PLPAcP, correlates with early prostate cancer. These findings confirm the analytical power of this method, implicate PLPAcP in prostate cancer pathogenesis, and suggest novel anticancer therapeutic strategies.

A new Kunitz trypsin inhibitor from Erythrina poeppigiana exhibits antimicrobial and antibiofilm properties against bacteria

Erythrina poeppigiana belongs to Fabaceae family (subfamily Papillionoideae) and is commonly found in tropical and subtropical regions in Brazil. Herein, we described the purification and characterization of a new Kunitz-type inhibitor, obtained from E. poeppigiana seeds (EpTI). EpTI is composed by three isoforms of identical amino-terminal sequences with a molecular weight ranging from 17 to 20 kDa. The physicochemical features showed by EpTI are common to Kunitz inhibitors, including the dissociation constant (13.1 nM), stability against thermal (37–100 °C) and pH (2–10) ranging, and the presence of disulfide bonds stabilizing its reactive site. Furthermore, we investigated the antimicrobial, anti-adhesion, and anti-biofilm properties of EpTI against Gram-positive and negative bacteria. The inhibitor showed antimicrobial activity with a minimum inhibitory concentration (MIC, 5–10 µM) and minimum bactericidal concentration (MBC) of 10 µM for Enterobacter aerogenes, Enterobacter cloacae, Klebsiella pneumoniae, Staphylococcus aureus, and Staphylococcus haemolyticus. The combination of EpTI with ciprofloxacin showed a marked synergistic effect, reducing the antibiotic concentration by 150%. The increase in crystal violet uptake for S. aureus and K. pneumoniae strains was approximately 30% and 50%, respectively, suggesting that the bacteria plasma membrane is targeted by EpTI. Treatment with EpTI at 1x and 10 x MIC significantly reduced the biofilm formation and prompted the disruption of a mature biofilm. At MIC/2, EpTI decreased the bacterial adhesion to polystyrene surface within 2 h. Finally, EpTI showed low toxicity in animal model Galleria mellonella. Given its antimicrobial and anti-biofilm properties, the EpTI sequence might be used to design novel drug prototypes.

Multifunctionality of Prostatic Acid Phosphatase in Prostate Cancer Pathogenesis.

The role of human prostatic acid phosphatase (PAcP, P15309|PPAP_HUMAN) in prostate cancer was investigated using a new proteomic tool termed signal sequence swapping (replacement of domains from the native cleaved amino terminal signal sequence of secretory/membrane proteins with corresponding regions of functionally distinct signal sequence subtypes). This manipulation preferentially redirects proteins to different pathways of biogenesis at the endoplasmic reticulum, magnifying normally difficult to detect subsets of the protein of interest. For PAcP this technique reveals three forms identical in amino acid sequence but profoundly different in physiological functions, subcellular location, and biochemical properties. These three forms of PAcP can also occur with the wild-type PAcP signal sequence. Clinical specimens from patients with prostate cancer demonstrate that one form, termed PLPAcP, correlates with early prostate cancer. These findings confirm the analytical power of this method, implicate PLPAcP in prostate cancer pathogenesis, and suggest novel anticancer therapeutic strategies.

Preparing mFc- and hFc-Fusion Proteins from Mammalian Cells.

Fc-fusion proteins are composed of an immunoglobulin Fc domain that is directly linked to the antigen of interest. Typically, these vectors will contain an amino-terminal signal sequence that permits trafficking to the cell surface and secretion into the media and a carboxy-terminal Fc receptor that enables purification on Protein A-Sepharose. Fc-fusion proteins have several applications in protein microarrays, oncological therapies, and vaccine and antibody development. Presence of the Fc domain significantly increases the plasma life of the fusion partner, which prolongs therapeutic activity. Furthermore, the Fc domain enhances the solubility and stability of the partner molecule. Because Fc-fusion proteins are secreted into the culture medium, purification by affinity chromatography is relatively easy and cost-effective. Immunizing a murine host with mFc-fusion protein generates an antigen-specific immune response because the Fc domain is recognized as "self" by the host.

Looking for a safe haven: tail-anchored proteins and their membrane insertion pathways.

Insertion of membrane proteins into the lipid bilayer is a crucial step during their biosynthesis. Eukaryotic cells face many challenges in directing these proteins to their predestined target membrane. The hydrophobic signal peptide or transmembrane domain (TMD) of the nascent protein must be shielded from the aqueous cytosol and its target membrane identified followed by transport and insertion. Components that evolved to deal with each of these challenging steps range from chaperones to receptors, insertases, and sophisticated translocation complexes. One prominent translocation pathway for most proteins is the signal recognition particle (SRP)-dependent pathway which mediates co-translational translocation of proteins across or into the endoplasmic reticulum (ER) membrane. This textbook example of protein insertion is stretched to its limits when faced with secretory or membrane proteins that lack an amino-terminal signal sequence or TMD. Particularly, a large group of so-called tail-anchored (TA) proteins that harbor a single carboxy-terminal TMD require an alternative, post-translational insertion route into the ER membrane. In this review, we summarize the current research in TA protein insertion with a special focus on plants, address challenges, and highlight future research avenues.

DAEH N-terminal sequence of avian serum albumins as catalytic center of Cu (II)-dependent organophosphorus hydrolyzing A-esterase activity

O-hexyl O-2,5-dichlorophenyl phosphoramidate (HDCP) induces delayed neuropathy. The R (+)-HDCP inhibits and caused the so call “aging reaction” on inhibited-NTE. This enantiomer is not hydrolyzed by Ca(II)-dependent A-esterases in mammal tissues but is hydrolyzed by Cu(II)-dependent chicken serum albumin (CSA). With the aim of identifying HDCP hydrolysis by other vertebrate albumins, we incubated albumin with 400 μM racemic HDCP in the presence of 100 μM copper sulfate. HDCPase activity was assessed by measurement of HDCP with chiral chromatography. Human, sheep, dog, pig, lamprey or cobra serum albumin did not show a significant activity (~10%). Rabbit and bovine albumins hydrolyzed both enantiomers of HDCP (25% and 50% respectively). Turkey serum albumin had more HDCPase activity (~80 μM remaining) than the chicken albumin (~150 μM remaining). No animal albumins other than chicken showed stereoselective hydrolysis. Preincubation of chicken albumin with 1 mM the histidine modifying agents, 100 μM N-bromosuccinimide (NBS) and Zn(II), inhibited its Cu(II)-dependent R (+)-HDCPase activity, where as other mM amino acids modifiers had no inhibitory effects. . These results confirm that the stereoselective hydrolysis of (+)-HDCP is a specific A-esterase catalytic property of chicken albumin. The higher HDCPase activity by turkey albumin suggests the amino-terminal sequence of avian albumins (DAEHK) is the active center of this Cu(II)-dependent A-esterase activity.

Conformational Structural Alteration of NCS1 Protein Through Pb2+and Mn2+ Association

NCS1 (Frequenin) is a member of neuronal calcium sensor (NCS) family. This globular protein contains four EF-hand motifs and an amino-terminal myristylation sequence. Pb2+has been widely viewed as a health concern for its anthropogenic toxicity. It has been observed that Pb2+ modulates interactions between calcium-binding protein calmodulin (CaM) and its binding partners through binding in the EF-hands of CaM and it may play a significant role in Pb2+-induced neurotoxicity. Mn is a highly abundant metal in nature which has been found in human brain and associated with the several diseases like multiple sclerosis, Alzheimer's and Parkinson's disease and is considered as potential toxic element. Previous studies from our group showed Pb2+ and Cd2+ binding to DREAM protein that can contribute to Pb2+/Cd2+ induced neurotoxicity. Here we showed that Pb2+ binds to EF-hands in apo-NCS1with a high affinity (Kd = 7.0±1.5 nM) whereas Cd2+ did not show any binding with NCS1. For the first time we investigated the Mn2+ association to NCS1 protein. This cation binds with an apparent equilibrium dissociation constant of 340±67 µM. Pb2+ and Mn2+binding trigger secondary and tertiary structural rearrangements of NCS1 that are analogous to those observed for Ca2+ association. In addition, Pb2+ as well as Mn2+ promote NCS1 interaction with hydrophobic molecule 1,8-ANS. Interestingly,Ca2+NCS1:1,8-ANS complex exhibits a co-operative binding of Pb2+ with Kd = 103±23 µM whereas Mn2+ showed apparent Kd value 924±185 µM with Ca2+NCS1:1,8-ANS complex. These results suggest allosteric interactions between the 1,8-ANS binding site and Pb2+ binding EF-hands. Data provided here points towards ability of EF-hands in NCS-1 to interact with various metals and a possible role of NCS1 in Pb2+/Mn2+ neurotoxicity.