Gotowa bibliografia na temat „SEC insertion sequence”

ABSTRACT The cotranslational incorporation of the unusual amino acid selenocysteine (Sec) into both prokaryotic and eukaryotic proteins requires the recoding of a UGA stop codon as one specific for Sec. The recognition of UGA as Sec in mammalian selenoproteins requires a Sec insertion sequence (SECIS) element in the 3′ untranslated region as well as the SECIS binding protein SBP2. Here we report a detailed analysis of SBP2 structure and function using truncation and site-directed mutagenesis. We have localized the RNA binding domain to a conserved region shared with several ribosomal proteins and eukaryotic translation termination release factor 1. We also identified a separate and novel functional domain N-terminal to the RNA binding domain which was required for Sec insertion but not for SECIS binding. Conversely, we showed that the RNA binding domain was necessary but not sufficient for Sec insertion and that the conserved glycine residue within this domain was required for SECIS binding. Using glycerol gradient sedimentation, we found that SBP2 was stably associated with the ribosomal fraction of cell lysates and that this interaction was not dependent on its SECIS binding activity. This interaction also occurred with purified components in vitro, and we present data which suggest that the SBP2-ribosome interaction occurs via 28S rRNA. SBP2 may, therefore, have a distinct function in selecting the ribosomes to be used for Sec insertion.

Cotranslational insertion of selenocysteine (Sec) proceeds by recoding UGA to a sense codon. This recoding is governed by the Sec insertion sequence (SECIS) element, an RNA structure on the mRNA, but size, location, structure determinants, and mechanism differ for Bacteria, Eukarya, and Archaea. For Archaea, the structure–function relation of the SECIS is poorly understood, as only rather laborious experimental approaches are established. Furthermore, these methods do not allow for quantitative probing of Sec insertion. In order to overcome these limitations, we engineered bacterial β-lactamase into an archaeal selenoprotein, thereby establishing a reporter system, which correlates enzyme activity to Sec insertion. Using this system, in vivo Sec insertion depending on the availability of selenium and the presence of a SECIS element was assessed inMethanococcus maripaludis. Furthermore, a minimal SECIS element required for Sec insertion inM. maripaludiswas defined and a conserved structural motif shown to be essential for function. Besides developing a convenient tool for selenium research, converting a bacterial enzyme into an archaeal selenoprotein provides proof of concept that novel selenoproteins can be engineered in Archaea.

Selenium is an essential micronutrient with important functions in human health and relevance to several pathophysiological conditions. The biological effects of selenium are largely mediated by selenium-containing proteins (selenoproteins) that are present in all three domains of life. Although selenoproteins represent diverse molecular pathways and biological functions, all these proteins contain at least one selenocysteine (Sec), a selenium-containing amino acid, and most serve oxidoreductase functions. Sec is cotranslationally inserted into nascent polypeptide chains in response to the UGA codon, whose normal function is to terminate translation. To decode UGA as Sec, organisms evolved the Sec insertion machinery that allows incorporation of this amino acid at specific UGA codons in a process requiring a cis-acting Sec insertion sequence (SECIS) element. Although the basic mechanisms of Sec synthesis and insertion into proteins in both prokaryotes and eukaryotes have been studied in great detail, the identity and functions of many selenoproteins remain largely unknown. In the last decade, there has been significant progress in characterizing selenoproteins and selenoproteomes and understanding their physiological functions. We discuss current knowledge about how these unique proteins perform their functions at the molecular level and highlight new insights into the roles that selenoproteins play in human health.

ABSTRACT Selenocysteine (Sec), the 21st amino acid in protein, is encoded by UGA. The Sec insertion sequence (SECIS) element, which is the stem-loop structure present in 3′ untranslated regions (UTRs) of eukaryotic selenoprotein-encoding genes, is essential for recognition of UGA as a codon for Sec rather than as a stop signal. We now report the identification of a new eukaryotic selenoprotein, designated selenoprotein M (SelM). The 3-kb human SelM-encoding gene has five exons and is located on chromosome 22 but has not been correctly identified by either Celera or the public Human Genome Project. We characterized human and mouse SelM cDNA sequences and expressed the selenoprotein in various mammalian cell lines. The 3" UTR of the human, mouse, and rat SelM-encoding genes lacks a canonical SECIS element. Instead, Sec is incorporated in response to a conserved mRNA structure, in which cytidines are present in place of the adenosines previously considered invariant. Substitution of adenosines for cytidines did not alter Sec incorporation; however, other mutant structures did not support selenoprotein synthesis, demonstrating that this new form of SECIS element is functional. SelM is expressed in a variety of tissues, with increased levels in the brain. It is localized to the perinuclear structures, and its N-terminal signal peptide is necessary for protein translocation.

Selenoproteins contain the 21st amino acid, selenocysteine (Sec), which is incorporated at select UGA codons when a specialized hairpin sequence, the Sec insertion sequence (SECIS) element, is present in the 3’ UTR. Aside from the SECIS, selenoprotein mRNA 3’ UTRs are not conserved between different selenoproteins within a species. In contrast, the 3’-UTR of a given selenoprotein is often conserved across species, which supports the hypothesis that cis-acting elements in the 3’-UTR other than the SECIS exert post-transcriptional control on selenoprotein expression. In order to determine the function of one such SECIS context, we chose to focus on the plasma selenoprotein, SELENOP, which is required to maintain selenium homeostasis as a selenium transport protein that contains 10 Sec residues. It is unique in that its mRNA contains two SECIS elements in the context of a highly conserved 843-nucleotide 3’ UTR. Here we have used RNA affinity chromatography and identified PTBP1 as the major RNA binding protein that specifically interacts with the sequence between the two SECIS elements. We then used CRISPR/Cas9 genome editing to delete two regions surrounding the first SECIS element. We found that these sequences are involved in regulating SELENOP mRNA and protein levels, which are inversely altered as a function of selenium concentrations.

In bacteria, selenocysteine (Sec) is incorporated into proteins via the recoding of a particular codon, the UGA stop codon in most cases. Sec-tRNASec is delivered to the ribosome by the Sec-dedicated elongation factor SelB that also recognizes a Sec-insertion sequence element following the codon on the mRNA. Since the excess of SelB may lead to sequestration of Sec-tRNASec under selenium deficiency or oxidative stress, the expression levels of SelB and tRNASec should be regulated. In this bioinformatic study, I analyzed the Rhizobiales SelB species because they were annotated to have a non-canonical C-terminal extension. I found that the open reading frame (ORF) of diverse Alphaproteobacteria selB genes includes an entire tRNASec sequence (selC) and overlaps with the start codon of the downstream ORF. A remnant tRNASec sequence was found in the Sinorhizobium melilotiselB genes whose products have a shorter C-terminal extension. Similar overlapping traits were found in Gammaproteobacteria and Nitrospirae. I hypothesized that once the tRNASec moiety is folded and processed, the expression of the full-length SelB may be repressed. This is the first report on a nested tRNA gene inside a protein ORF in bacteria.

The elongation of eukaryotic selenoproteins relies on a poorly understood process of interpreting in-frame UGA stop codons as selenocysteine (Sec). We used cryo-electron microscopy to visualize Sec UGA recoding in mammals. A complex between the noncoding Sec-insertion sequence (SECIS), SECIS-binding protein 2 (SBP2), and 40 S ribosomal subunit enables Sec-specific elongation factor eEFSec to deliver Sec. eEFSec and SBP2 do not interact directly but rather deploy their carboxyl-terminal domains to engage with the opposite ends of the SECIS. By using its Lys-rich and carboxyl-terminal segments, the ribosomal protein eS31 simultaneously interacts with Sec-specific transfer RNA (tRNA Sec ) and SBP2, which further stabilizes the assembly. eEFSec is indiscriminate toward l -serine and facilitates its misincorporation at Sec UGA codons. Our results support a fundamentally distinct mechanism of Sec UGA recoding in eukaryotes from that in bacteria.

ABSTRACT The pseudopilin PulG is an essential component of the pullulanase-specific type II secretion system from Klebsiella oxytoca. PulG is the major subunit of a short, thin-filament pseudopilus, which presumably elongates and retracts in the periplasm, acting as a dynamic piston to promote pullulanase secretion. It has a signal sequence-like N-terminal segment that, according to studies with green and red fluorescent protein chimeras, anchors unassembled PulG in the inner membrane. We analyzed the early steps of PulG inner membrane targeting and insertion in Escherichia coli derivatives defective in different protein targeting and export factors. The β-galactosidase activity in strains producing a PulG-LacZ hybrid protein increased substantially when the dsbA, dsbB, or all sec genes tested except secB were compromised by mutations. To facilitate analysis of native PulG membrane insertion, a leader peptidase cleavage site was engineered downstream from the N-terminal transmembrane segment (PrePulG*). Unprocessed PrePulG* was detected in strains carrying mutations in secA, secY, secE, and secD genes, including some novel alleles of secY and secD. Furthermore, depletion of the Ffh component of the signal recognition particle (SRP) completely abolished PrePulG* processing, without affecting the Sec-dependent export of periplasmic MalE and RbsB proteins. Thus, PulG is cotranslationally targeted to the inner membrane Sec translocase by SRP.

The Sec translocon provides a polypeptide-conducting channel, which is insulated from the hydrophobic lipidic environment of the membrane, for translocation of hydrophilic passenger polypeptides. Its lateral gate allows a downstream hydrophobic segment (stop-transfer sequence) to exit the channel laterally for integration into the lipid phase. We note that this channel model only partly accounts for the translocon function. The other essential role of translocon is to facilitate de novo insertion of the N-terminal topogenic segment of a substrate polypeptide into the membrane. Recent structural studies suggest that de novo insertion does not use the polypeptide-conducting channel; instead, it takes place directly at the lateral gate, which is prone to opening. We propose that the de novo insertion process, in concept, is similar to that of insertases (such as YidC in bacteria and EMC3 in eukaryotes), in which an intramembrane surface of the machinery provides the halfway point of insertion.

Mammalian thioredoxin reductase (TR) is a flavoprotein catalysing reduction of oxidized thioredoxin in an NADPH-dependent manner, and contains a selenocysteine (Sec) residue near the C-terminus. We observed that TR activity was decreased in A549 cells by the lowering of the fetal bovine serum content in the culture medium and was recovered by the addition of selenium. To study the role of Sec in TR activity, we have isolated a full-length clone of the rat TR cDNA (3.3 kb) and have expressed it in COS-1 cells in a transient-expression system. TR activities in COS-1 cells expressing rat TR were increased in accordance with supplemented sodium selenite concentrations, whereas levels of TR protein, examined by Western blotting, were not affected by sodium selenite concentrations. We introduced various deletions into the 3ʹ-untranslated region of the TR cDNA to localize and examine the role of a Sec insertion-sequence (SECIS) element in the functional expression of TR. TR activities were observed only in COS-1 cells transfected with the TR cDNAs containing the putative SECIS element located between 1856 and 1915 bp in the correct orientation. We also carried out radiolabelling of proteins by incubation of the cDNA-transfected cells with sodium [75Se]selenite. 75Se was incorporated into the expressed TR protein of the cells transfected with the SECIS element-containing cDNAs, but not into those without the SECIS element or with an inverted SECIS element. These data clearly showed a requirement of selenium for the formation of functional TR protein.

Membrane proteins comprise around 20-30% of a typical proteome and play crucial roles in a wide variety of biochemical pathways. Apart from their general biological significance, membrane proteins are of particular interest to the pharmaceutical industry, being targets for more than half of all available drugs. This thesis focuses on prediction methods for membrane proteins that ultimately rely on their amino acid sequence only. By identifying soluble protein domains in membrane protein sequences, we were able to constrain and improve prediction of membrane protein topology, i.e. what parts of the sequence span the membrane and what parts are located on the cytoplasmic and extra-cytoplasmic sides. Using predicted topology as input to a profile-profile based alignment protocol, we managed to increase sensitivity to detect distant membrane protein homologs. Finally, experimental measurements of the level of membrane integration of systematically designed transmembrane helices in vitro were used to derive a scale of position-specific contributions to helix insertion efficiency for all 20 naturally occurring amino acids. Notably, position within the helix was found to be an important factor for the contribution to helix insertion efficiency for polar and charged amino acids, reflecting the highly anisotropic environment of the membrane. Using the scale to predict natural transmembrane helices in protein sequences revealed that, whereas helices in single-spanning proteins are typically hydrophobic enough to insert by themselves, a large part of the helices in multi-spanning proteins seem to require stabilizing helix-helix interactions for proper membrane integration. Implementing the scale to predict full transmembrane topologies yielded results comparable to the best statistics-based topology prediction methods.

Selenium is an essential trace mineral of fundamental importance to human health. Its beneficial functions are largely attributed to its presence within a group of proteins named selenoproteins in the form of the amino acid selenocysteine (Sec). Recently, it was revealed that the human selenoproteome consists of 25 selenoproteins, and for many of them their function remains unknown. The most prominent known roles of selenoproteins are to maintain the intracellular redox homeostasis, redox regulation of intracellular signalling and thyroid hormone metabolism. Sec incorporation into selenoproteins employs a unique mechanism that involves decoding of the UGA stop codon. The process requires interplay between distinct, intrinsic features such as the Sec Insertion Sequence (SECIS) element, the tRNASec and multiple protein factors. The work presented in this thesis has focused on characterising the regulation of human SECIS binding protein 2, SBP2, a factor central to this process. Experimental approaches combined with bioinformatics analysis revealed that SBP2 is subjected to alternative splicing. A total of nine alternatively spliced transcripts appear to be expressed in cells, potentially encoding five different protein isoforms. The alternative splicing events are restricted to the 5?-region, which is proposed to be dispensable for Sec incorporation. One of the variants identified, contains a mitochondrial targeting sequence that was capable of targetting SBP2 into the mitochondrial compartment. This isoform also appears to be expressed endogenously within the mitochondria in cells. Previous reports have depicted SBP2 as a ribosomal protein, despite the presence of a putative Nuclear Localisation Signal (NLS). In this study it was found that SBP2 subcellular localisation is not restricted to ribosomes. Intrinsic functional NLS and Nuclear Export Signals (NESs), enable SBP2 to shuttle between the nucleus and the cytoplasm via the CRM1 pathway. In addition, the subcellular localisation of SBP2 appears to play an important role in regulating Sec incorporation into selenoproteins. The subcellular localisation of SBP2 is altered by conditions imposing oxidative stress. Several oxidising agents induce the nuclear accumulation of SBP2, which occurs via oxidation of cysteine residues within a novel redox-sensitive cysteine rich domain (CRD). Cysteine residues were to form disulfide bonds and glutathione-mixed disulfides during oxidising conditions, which are efficiently reversed in vitro by the thioredoxin and glutaredoxin systems, respectively. These modifications negatively regulate selenoprotein synthesis. Cells depleted of SBP2 are more sensitive to oxidative stress than control cells, which correlated with a substantial decrease in selenoprotein synthesis after treatment with oxidising agents. These results provide direct evidence that SBP2 is required for Sec incorporation in vivo and suggest that nuclear sequestration of SBP2 under such conditions may represent a mechanism to regulate the expression of selenoproteins. Collectively, these results suggest that SBP2 is regulated at multiple levels: by alternative splicing, changes in subcellar localisation and redox control.

Selenium is an essential trace mineral of fundamental importance to human health. Its beneficial functions are largely attributed to its presence within a group of proteins named selenoproteins in the form of the amino acid selenocysteine (Sec). Recently, it was revealed that the human selenoproteome consists of 25 selenoproteins, and for many of them their function remains unknown. The most prominent known roles of selenoproteins are to maintain the intracellular redox homeostasis, redox regulation of intracellular signalling and thyroid hormone metabolism. Sec incorporation into selenoproteins employs a unique mechanism that involves decoding of the UGA stop codon. The process requires interplay between distinct, intrinsic features such as the Sec Insertion Sequence (SECIS) element, the tRNASec and multiple protein factors. The work presented in this thesis has focused on characterising the regulation of human SECIS binding protein 2, SBP2, a factor central to this process. Experimental approaches combined with bioinformatics analysis revealed that SBP2 is subjected to alternative splicing. A total of nine alternatively spliced transcripts appear to be expressed in cells, potentially encoding five different protein isoforms. The alternative splicing events are restricted to the 5?-region, which is proposed to be dispensable for Sec incorporation. One of the variants identified, contains a mitochondrial targeting sequence that was capable of targetting SBP2 into the mitochondrial compartment. This isoform also appears to be expressed endogenously within the mitochondria in cells. Previous reports have depicted SBP2 as a ribosomal protein, despite the presence of a putative Nuclear Localisation Signal (NLS). In this study it was found that SBP2 subcellular localisation is not restricted to ribosomes. Intrinsic functional NLS and Nuclear Export Signals (NESs), enable SBP2 to shuttle between the nucleus and the cytoplasm via the CRM1 pathway. In addition, the subcellular localisation of SBP2 appears to play an important role in regulating Sec incorporation into selenoproteins. The subcellular localisation of SBP2 is altered by conditions imposing oxidative stress. Several oxidising agents induce the nuclear accumulation of SBP2, which occurs via oxidation of cysteine residues within a novel redox-sensitive cysteine rich domain (CRD). Cysteine residues were to form disulfide bonds and glutathione-mixed disulfides during oxidising conditions, which are efficiently reversed in vitro by the thioredoxin and glutaredoxin systems, respectively. These modifications negatively regulate selenoprotein synthesis. Cells depleted of SBP2 are more sensitive to oxidative stress than control cells, which correlated with a substantial decrease in selenoprotein synthesis after treatment with oxidising agents. These results provide direct evidence that SBP2 is required for Sec incorporation in vivo and suggest that nuclear sequestration of SBP2 under such conditions may represent a mechanism to regulate the expression of selenoproteins. Collectively, these results suggest that SBP2 is regulated at multiple levels: by alternative splicing, changes in subcellar localisation and redox control.
Thesis (PhD Doctorate)
Doctor of Philosophy (PhD)
School of Biomolecular and Biomedical Sciences
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With the significant growth of the amount of biosequence data, it becomes increasingly important to develop new techniques for finding “knowledge” from the data. Pattern discovery is a fundamental operation in such applications. It attempts to find patterns in biosequences that can help scientists to analyze the property of a sequence or predict the function of a new entity. The discovered patterns may also help to classify an unknown sequence, that is, assign the sequence to an existing family. In this chapter, we show how to discover active patterns in a set of protein sequences and classify an unlabeled DNA sequence. We use protein sequences as an example to illustrate our discovery algorithm, though the algorithm applies to sequences of any sort, including both protein and DNA. The patterns we wish to discover within a set of sequences are regular expressions of the form *X1 * X2 * ... . The X1,X2,... are segments of a sequence, that is, subsequences made up of consecutive letters, and * represents a variable length don’t care (VLDC). In matching the expression *X1 * X2 * ... with a sequence S, the VLDCs may substitute for zero or more letters in S at zero cost. The dissimilarity measure used in comparing two sequences is the edit distance, that is, the minimum cost of edit operations used to transform one subsequence to the other after an optimal and zero-cost substitution for the VLDCs, where the edit operations include insertion, deletion, and change of one letter to another (Wagner and Fischer, 1974; K. Zhang et al., 1994). That is, we find a one-to-one mapping from each VLDC to a subsequence of the data sequence and from each pattern subsequence to a subsequence of the data sequence such that the following two conditions are satisfied, (i) The mapping preserves the left-to-right ordering (if a VLDC at position i in the pattern maps to a subsequence starting at position i1 and ending at position i2 in the data sequence, and a VLDC at position j in the pattern maps to a subsequence starting at position j1 and ending at position j2 in the data sequence, and i < j, then i2 < j2).

In this chapter, the authors discuss an efficient and effective index mechanism for search engines to support both conjunctive and disjunctive queries. The main idea behind it is to decompose an inverted list into a collection of disjoint sub-lists. The authors associate each word with an interval sequence, which is created by applying a kind of tree coding to a trie structure constructed over all the word sequences in a database. Then, attach each interval, instead of a word, with an inverted sub-list. In this way, both set intersection and union can be conducted by performing a series of simple interval containment checks. Experiments have been conducted, which shows that the new index is promising. Also, how to maintain indices, when inserting or deleting documents, is discussed in great detail.

In this chapter, we discuss an efficient and effective index mechanism for search engines to support both conjunctive and disjunctive queries. The main idea behind it is to decompose an inverted list into a collection of disjoint sub-lists. We will associate each word with an interval sequence, which is created by applying a kind of tree coding to a trie structure constructed over all the word sequences in a database. Then, attach each interval, instead of a word, with an inverted sub-list. In this way, both set intersection and union can be conducted by performing a series of simple interval containment checks. Experiments have been conducted, which shows that the new index is promising. Also, how to maintain indexes, when inserting or deleting documents, is discussed in great detail.

In the previous chapters, we discussed problems involving an exact match of string patterns. We now turn to problems involving similar but not necessarily exact pattern matches. There are a number of similarity or distance measures, and many of them are special cases or generalizations of the Levenshtein metric. The problem of evaluating the measure of string similarity has numerous applications, including one arising in the study of the evolution of long molecules such as proteins. In this chapter, we focus on the problem of evaluating a longest common subsequence, which is expressively equivalent to the simple form of the Levenshtein distance. The Levenshtein distance is a metric that measures the similarity of two strings. In its simple form, the Levenshtein distance, D(x , y), between strings x and y is the minimum number of character insertions and/or deletions (indels) required to transform string x into string y. A commonly used generalization of the Levenshtein distance is the minimum cost of transforming x into y when the allowable operations are character insertion, deletion, and substitution, with costs δ(λ , σ), δ(σ, λ), and δ(σ1, σ2) , that are functions of the involved character(s). There are direct correspondences between the Levenshtein distance of two strings, the length of the shortest edit sequence from one string to the other, and the length of the longest common subsequence (LCS) of those strings. If D is the simple Levenshtein distance between two strings having lengths m and n, SES is the length of the shortest edit sequence between the strings, and L is the length of an LCS of the strings, then SES = D and L = (m + n — D)/2. We will focus on the problem of determining the length of an LCS and also on the related problem of recovering an LCS. Another related problem, which will be discussed in Chapter 6, is that of approximate string matching, in which it is desired to locate all positions within string y which begin an approximation to string x containing at most D errors (insertions or deletions).

Up to now the three-dimensional structure of t-PA or parts of this enzyme is unknown. Using computer graphical methods the spatial structure of the enzymatic part of t-PA is predicted on the hypothesis, the three-dimensional backbone structure of t-PA being similar to that of other serine proteases. The t-PA model was built up in three steps:1) Alignment of the t-PA sequence with other serine proteases. Comparison of enzyme structures available from Brookhaven Protein Data Bank proved elastase as a basis for modeling.2) Exchange of amino acids of elastase differing from the t-PA sequence. The replacement of amino acids was performed such that backbone atoms overlapp completely and side chains superpose as far as possible.3) Modeling of insertions and deletions. To determine the spatial arrangement of insertions and deletions parts of related enzymes such as chymotrypsin or trypsin were used whenever possible. Otherwise additional amino acid sequences were folded to a B-turn at the surface of the proteine, where all insertions or deletions are located. Finally the side chain torsion angles of amino acids were optimised to prevent close contacts of neigh bouring atoms and to improve hydrogen bonds and salt bridges.The resulting model was used to explain binding of arginine 560 of plasminogen to the active site of t-PA. Arginine 560 interacts with Asp 189, Gly 19 3, Ser 19 5 and Ser 214 of t-PA (chymotrypsin numbering). Furthermore interaction of chromo-genic substrate S 2288 with the active site of t-PA was studied. The need for D-configuration of the hydrophobic amino acid at the N-terminus of this tripeptide derivative could be easily explained.

We have investigated the effect of altering the leader sequence of human factor VII on its biological activity. Factor VII is a vitamin K-dependent blood coagulation protein whose activity depends on the presence of gamma-carboxyglutamic acid (gla) residues in its amino terminal region. Since factor VII and other vitamin K-dependent proteins exhibit structural homology in the propeptide, it has been suggested that the propeptide is involved in gamma-carboxylation. Recently, two factor IX patients were identified with point mutations which prevented the processing of the propeptide and generated a factor IX with greatly reduced biological activity (Diuguid et al., PNAS 83; 5803; Bentley et al., Cell 45: 343). To examine this question using recombinant DNA technology, we altered the sequence of the factor VII propeptide by in vitro mutagenesis of the factor VII cDNA and then expressed the altered genes in baby hamster kidney (BHK) cells. For the 60 and 38 aa leader forms of factor VII, the arg (R) at -1 was changed to ser (S), yielding the sequence HRRRS before the +1 ala. In addition, for the 60 aa leader form, a ser was inserted after the arg at -1, resulting in the sequence HRRRRS before the +1 ala. As determined by ELISA, the mutant proteins were synthesized and secreted by BHK cells at levels comparable to the wild-type forms of factor VII. Analysis by radioimmune precipitation and SDS-PAGE indicated that substitution of arg by ser at -1 prohibits processing of the factor VII propeptide, whereas, insertion of a ser after the four arg's does not. However, all three proteins have reduced biological activity by approximately 5-fold when compared to the wild-type forms with the one-stage clotting assay. All three proteins are also quantitatively precipitated by Ba citrate, indicating they are at least partially gamma-carboxylated. These results suggest that the correct sequence of the propeptide, not just cleavage of the propeptide, is necessary for generating a biologically active molecule. The effect of these sequence alterations on gamma-carboxylation will be evaluated further by analysis of the amino acid sequence and composition of the mutant proteins.

α2-Antiplasmin (α2AP) is the primary physiological plasmin inhibitor in human plasma. The inhibition is rapid (second order rate constants (k1) are expressed as M−1 s−1 ) (k1 = 2 × 107) and occurs as the consequence of an irreversible 1:1 stoichiometric complex formation; the exact nature of and the forces involved in complex formation are not fully understood. In fact, what makes α2AP an inhibitor, rather than simply a substrate remains unresolved. Recently, we deduced the primary structure of α2 AP from the sequence of its cDNA. 95%of this sequence was confirmed by amino acid (aa) sequence analysis of naturalα2 AP (α2 AP)? The 452 aa molecule contains 2 disulfide bonds and 4 glycosylated Asn residues, aa sequence alignment confirmed α2AP's membership in the Serpin family. The reactive site sequence as determined by NH2 - and COOH-terminal aa sequence analysis of the plasmin-modified inhibitor and the released M−r ∼ 8000 peptide is Met362-Ser363-Arg364-Met365-Ser366, P3-P2-P1-P'1-P'2, respectively.Natural and engineered P1 residue substitutions in the Serpin α2 -antitrypsin ( α2 AT) have shown altered specificities and efficiencies. To further examine the role of P and P' residues in determining Serpin specificity, in the present study we have by site-directed mutagenesis, deleted (△) the P'l-Met365 residue of a AP thereby producing a recombinant (r) inhibitor (r α2 AP△Met365) whose putative new reactive site mimics that of antithrombin III (ATIII) and a AT-Pittsburgh (Pl-Arg-P'1-Ser). A second variant was constructed (ra2AP△Arg364) in which the Pl-Arg364 residue was deleted, producing the new sequence Met362-Ser363-Met364-Ser365, containing 2 potential sites analogous to the Pl-P'l, Met-Ser reactive site of α2 AT. The variants and r α2 AP were expressed in CH0 cells, purified and compared with n α2 AP, α2AT and ATIII for the ability to inhibit plasmin, thrombin, trypsin and elastase. n α2 AP and r α2 AP had nearly identical inhibition constants and like ATIII did not inhibit neutrophil elastase. Without heparin both α2 APs and ATIII inhibited thrombin moderately (k1 = 2 to 4× 103 ). Bovine trypsin was neutralized by the α2 APs with k1 = 3 × 106 and by ATIII with k1 = 1 × 105. The α2APs inhibited plasmin (k1 = 2 ×107 ) much more efficiently than ATIII (K1 =2 × 103 ). In contrast, was a highly effective antielastase (k1 = 1 × 107 ), a poor plasmin and thrombin inhibitor ancl inhibited bovine trypsin with = 2 × 10. As reported by others, α2 AT-Pittsburg has greatly reduced antielastase activity and greatly enhanced antithrombin activity. Analysis of ra APAMet365 revealed little change in activity toward plasmin, trypsin and elastase. Thus, α2 AP has no absolute requirement for Met .in the P'l position in order to effectively inhibit plasmin and trypsin. The other P^ subsites appear to be spatially flexible as deletion of the natural P'l residue must displace them. Contrary to prediction a 20-fold decrease in antithrombin activity was observed rather than an enhanced activity. Analysis of rα2 AP△Arg364 showed that it is unreactive with plasmin, trypsin and thrombin, but that it has acquired a significant antielastase activity (k1 = 1.5 × 105). The exact PI residue(s) has not been determined but removal of the bulky basic Arg364 may have resulted in accessibility of the predicted reactive site(s) peptide bond(s) Met362-Ser363 or Met364-Ser365 to the active site cleft of elastase. α2AP'Enschede', a natural mutant with deficient antiplasmin activity, was shown to contain an Ala insertion between aa 353 and 357, 7 to 10 positions NH2-terminal to its reactive site (Holmes et al., this meeting). This mutation results in conversion of α2 AP'Enschede' from an inhibitor to a substrate that retains a high affinity for the active site of plasmin.

Abstract This paper provides an overview of the work completed to design, qualify, manufacture and integrate electrical and optical double barrier penetrators with the Electrically Trace Heated Pipe-in-Pipe (ETH-PiP) as part of the Neptune Energy Fenja Development Project. Typical subsea penetrator systems in the oil and gas industry, such as pumps, compressors and X-trees are designed to be retrievable, to enable periodic refurbishment as well as providing the option for replacement, if required. However, the ETH-PiP architecture makes retrieval of system components complicated and uneconomical. Both the electrical and optical dual barrier penetrator system designs have to comply with a set of ETH-PiP specific criteria, such as to be maintenance free over a 25 years service life, prevent water ingress to the pipeline, provide pressure containment for operational media (in an unlikely scenario where the inner pipe bursts) and guarantee minimum footprint to allow an optimum integration onto the Pipeline End Termination (PLET) structure. In addition, the electrical system has to comply with a medium voltage rating (i.e. 5.0/8.7kV) to ensure a wide range of possible ETH-PiP architectures. The optical system has to maintain insertion loss below 0.5dB and a back reflection below -45dB to comply with the stringent requirements of distributed temperature monitoring sensor system over long distances. The qualification program of the electrical dual barrier penetrator system was performed in accordance with IEC 60502-4 and SEPS-SP-1001. A tailor made sequence had to be developed for the optical system, based on guidance from SEAFOM-TSD-01, considering that the system partly falls outside the associated standard application. The electrical dual barrier penetrator system qualification sequence was developed in two phases; firstly, the electrical transition contacts in the feedthrough chamber were qualified in accordance with IEC 60502-4 and secondly, four electrical double barrier penetrator prototypes were manufactured to allow the completion of the qualification sequence defined as per SEPS-SP-1001. The optical dual barrier penetrator system qualification employed the manufacturing of three prototypes to execute the pre-defined qualification sequence. Following the individual qualification of the electrical and optical dual barrier penetrator systems, subsequent welding and full-scale assembly trials were performed to ensure that the maximum allowable temperatures within the penetrators would not be exceeded during welding to the PLET, and to proof test the assembly procedure. Electrical verification testing was also undertaken during these trials to verify that the integrity of the penetrators had been maintained during the assembly and that the PLET arrangement did not give rise to any electrical stresses that could result in excessive deterioration of the penetrators. Integration of the four electrical and two optical dual barrier penetrator systems to the project PLET was completed in Q1 2020, with the actual subsea installation of the first ETH-PiP section including the PLET in Q3 2020.

MD-805 is a potent thrombin-inhibitor having the structure of tri-pods; Arg skeletone, N-terminal side and C-terminal side. MD-805 showed weaker inhibitory activity to other enzymes than thrombin. In this report, to gather more detailed informations about the structural features of serine enzymes concerning the specificity, we experimentally examined the structure-activity relationship (SAR) of a number of arginine derivatives including MD-805 and theoretically generated a MD-805-trypsin complex model using the results of X-ray crystallography of MD-805 and BPTI-trypsin complex by calculation in principle to minimize van der Waals contacts, and thus we discussed to interpret SAR from the molecular level. SAR of C-terminal side of arginine derivatives was obtained with the inhibitory activity to trypsin, plasmin, and glandular kallikrein and compared with the previous results of thrombin, the followings being indicated: (1) The hydrophobic binding pocket (HBP), which was reported by us to be at least partly similar in stereogeometry between trypsin and thrombin, had the depth corresponded to the length of ethylpiperidine, (2) concerning the site (termed the P site) next to HBP, there were large differences in stereogeometry between trypsin and thrombin; the P site of trypsin could accept propyl and phenyl group attached to 4-position of piperidine, while that of thrombin was unable to accept them and (3) the P sites of plasmin and glandular kallikrein resembled that of trypsin in being able to accept phenyl group. MD-805-trypsin complex model supported the reasonable understanding that the stereogeometrical similarity in HBP between thrombin and trypsin was attributable to the high homology in amino acid sequences in Ser-195 loop and that the dissimilarity in the P sites between thrombin and the others was attributable to 9 amino acids insertion found only in thrombin (Loop B). Furthermore, many dansylarginine derivatives showed very strong inhibition for pseudocholinesterase, however, SAR for C-terminal side of these derivatives revealed the similarity and dissimilarity in HBP and the P site between pseudocholinesterase and the proteases described above.

Introducción: La consolidación del desarrollo urbano es asociada a la movilidad, en mayor medida si se trata de AUTOPISTAS. Involucrando lógicas propias y autónomas, resultan funcionales exclusivamente a sus fines: contener y conducir de modo eficiente el flujo vehicular, partiendo de imponer sus también propios condicionamientos espaciales -estructuras de soporte, intercambiadores, organizadores bajo, sobre y a nivel, puentes, túneles, pantallas visuales, entre otros-. Queda afectado entonces el sector del territorio sobre el que descienden, por una secuencia dominantemente lineal de distintas categorías de impactos, vinculados además al nivel de consolidación urbana. Como producto de la interacción entre Fijos y Flujos -TERRITORIO/ AUTOPISTAS-, es que surgen las Situaciones Intersticiales urbanas, encaradas aquí desde un origen investigativo en donde el Intersticio fue entendido como diferencia entre entidades territoriales anteriores y nuevas superpuestas, redundante en hibridación material o funcional de respectivas condiciones originales, y abordado como producto de acciones y relaciones sociales, temporales y espaciales. Este espacio intersticial, fue considerado entonces información ineludible al abordar operaciones sobre áreas urbanas en correlato con flujos de movilidad autopistas. Objetivos: Se pretende generar un corpus de inferencias conducente a la elaboración de futuros diagnósticos, pautas y estrategias, a fin de “mitigar” los impactos afectantes e irresueltos que producen la sumatoria de situaciones intersticiales y remanentes espaciales, para ser eficientemente incorporados como variables a tomar en cuenta en los estudios del territorio en tanto urbano. Metodología: Los datos emergen de la aplicación de un instrumento de lectura e interpretación sistémica que atiende a la complejidad del tejido urbano y la superposición de estratos físicos y fenomenológicos: Herramienta Intersticio, en situaciones intersticiales “bajo autopista” en un recorte de Región Metropolitana de Buenos Aires/ RMBA – Argentina. Conclusiones: Conclusiones genéricas producto del análisis, evidencian que el Flujo Autopista-AU corta/secciona a la CIUDAD, aceptado como hecho consumado su implantación y descenso en aras de la conectividad del territorio. Los DETERMINANTES ESPACIALES (modulación, soporte, senda-techo entre otros) condicionan las apropiaciones de manera tal que sea cual fuere el carácter del ámbito de inserción, contexto y autopista establecen un vínculo que–rozando en algunos casos la indiferencia-, no incita a contexto y autopista establecen un vínculo que–rozando en algunos casos la indiferencia-, no incita a “pleitos” urbanos verificables… Una adecuación (voluntaria?) que no evita que programa y usos predominantes, incidan desde este status quo en la dinámica del sitio… Aunque en muchos casos no difieran de los propios del sector de pertenencia, domina el conflicto en el escenario y su función de uso, situación que no ocurre cuando el mismo uso tiene una pertenencia al tejido urbano de la ciudad: es que la Autopista deja en su abajo, una suerte de confusión entre lo público y lo privado, que sumada a la incidencia de estructura de soporte y plano superior límite, hacen que se produzcan siempre indefiniciones y/o conflictos -problemas propios de las infraestructuras en el territorio cuando no poseen diagnósticos desde la gestión de pertenencia-. Afrontar específicamente la evaluación de las condiciones de habitabilidad de las situaciones intersticiales estudiadas y/o la determinación de casos pasibles de una optimización -reconfiguración de la situación presente-, conducen a reconocer la necesidad de una proyección del “sobre” y “bajo” autopista de manera conjunta. Sumado a lo anterior, se confirma como necesaria la planificación previa de los intersticios “bajo autopista”, apoyada en el estudio realizado en esta investigación, desde una potencialidad espacial latente y mal aprovechada, así como desde la anarquía evidente que los distintos usos-programas encontrados en esos espacios de muestra, en general con calidad urbana degradada. El ineludible vínculo entre el desarrollo urbano y la movilidad, deberá contar con instrumentos propios que contemplen estos espacios, no como remanente de una intervención, sino de manera sostenible, compatible con la preservación y mejora del medioambiente natural y urbano, contribuyendo por las actividades que induce, directa e indirectamente en la formación de capital social. Introduction: Urban consolidation development is associated to motility, in great part if is referred to HIGHWAYS. Involving own and autonomy logics, they results functionality exclusively to its purpose: to content and conduce in an efficient way vehicular fluxes, starting from impose its own spatial conditions - support structures, organizing under and upper level of bridges panels, tunnels, visuals screens, and so on-. The territorial sector where it happens this descending is affected because of a domineerig lineal sequence of different categories of impacts entailed besides to urban consolidation level. As a product of interaction between Fix and Fluxes -TERRITORY/ HIHGWAYS-, is that appears urban Interstitials Situations, faced here from an investigative origin where Interstice was understood as the difference between previous territorial entities and new ones superposed, redounding in material or functional hybridization of respective original conditions, and boarded as a product of social, and spatial actions and relations. So, this interstitial space, was considered unavoidable information when boarding operations over urban areas in relation with motility fluxes highways. Objectives: The research, presently in development pretends to fix regulations and strategies appointing to a systematically formulation of typological patterns taking in account interstitially space, unavoidable information to face actions over urban areas in relation with motility high-way fluxes and appropriation in the under high-way. Methodologies: Is based in data emerging from the application of a reading and systemic interpretation instrument appointing to the complexity of urban tissue and the superposition of physics and phenomenological layers, -Interstitial Tool-, in interstitials situations “under highway” in a fragment of the Buenos Aires Metropolitan Region / RMBA – Argentina. By other way, digital descriptions are used as the best choice for representing all this process –still unfinished-, by the application of digital methods to board the understanding of the mentioned urban problematic. Conclusions: Generic conclusions as result of the analysis, put in evidence that the motility high-way fluxes cuts/sections the CITY, accepted its implantation and descent as a consummated fact in account of territorial connectivity. SPATIAL DETERMINING (modulation, support, way-cover between others), conditions appropriations in that way, that it doesn’t matter character of the insertion contour; context and highway establishes a nexus; an urban adequation (voluntary or involuntary) that cannot avoid that programs and uses doesn’t fall into a sort of accepted status quo, even if in much cases have no difference from proper uses of the insertion area; conflict takes possession of the scene and its uses, all that because the highway leaves in its “under” a sort of confusion between public and privat activities, adding to this, the incidence of the supporting structure and upper plane that conduces always to not resulted conditions and/or conflicts proper from this kind of infrastructures over territories when there isn’t governmental diagnostics and actions-. The evaluation of specific conditions of habitability of the interstitial situations mentioned, must be boarded to be changed, as well as those cases apt to be optimized, producing a reconfiguration of present situation. There is a responsibility about a simultaneous design of the upper and under highways. The unavoidable bond between urban development and motility must depend on, own instruments that overview those spaces , not as remnants of another intervention but in a sustainability way, compatible with preservation and an natural and urban ambient improvement, contributing to all that makes direct or indirectly to construct the social capital of urban areas.

To cause disease, plant viruses must replicate and spread locally and systemically within the host. Cell-to-cell virus spread is mediated by virus-encoded movement proteins (MPs), which modify the structure and function of plasmodesmata (Pd), trans-wall co-axial membranous tunnels that interconnect the cytoplasm of neighboring cells. Tobacco mosaic virus (TMV) employ a single MP for cell- cell spread and for which CP is not required. The PIs, Beachy (USA) and Epel (Israel) and co-workers, developed new tools and approaches for study of the mechanism of spread of TMV that lead to a partial identification and molecular characterization of the cellular machinery involved in the trafficking process. Original research objectives: Based on our data and those of others, we proposed a working model of plant viral spread. Our model stated that MPᵀᴹⱽ, an integral ER membrane protein with its C-terminus exposed to the cytoplasm (Reichel and Beachy, 1998), alters the Pd SEL, causes the Pd cytoplasmic annulus to dilate (Wolf et al., 1989), allowing ER to glide through Pd and that this gliding is cytoskeleton mediated. The model claimed that in absence of MP, the ER in Pd (the desmotubule) is stationary, i.e. does not move through the Pd. Based on this model we designed a series of experiments to test the following questions: -Does MP potentiate ER movement through the Pd? - In the presence of MP, is there communication between adjacent cells via ER lumen? -Does MP potentiate the movement of cytoskeletal elements cell to cell? -Is MP required for cell-to-cell movement of ER membranes between cells in sink tissue? -Is the binding in situ of MP to RNA specific to vRNA sequences or is it nonspecific as measured in vitro? And if specific: -What sequences of RNA are involved in binding to MP? And finally, what host proteins are associated with MP during intracellular targeting to various subcellular targets and what if any post-translational modifications occur to MP, other than phosphorylation (Kawakami et al., 1999)? Major conclusions, solutions and achievements. A new quantitative tool was developed to measure the "coefficient of conductivity" of Pd to cytoplasmic soluble proteins. Employing this tool, we measured changes in Pd conductivity in epidermal cells of sink and source leaves of wild-type and transgenic Nicotiana benthamiana (N. benthamiana) plants expressing MPᵀᴹⱽ incubated both in dark and light and at 16 and 25 ᵒC (Liarzi and Epel, 2005 (appendix 1). To test our model we measured the effect of the presence of MP on cell-to-cell spread of a cytoplasmic fluorescent probe, of two ER intrinsic membrane protein-probes and two ER lumen protein-probes fused to GFP. The effect of a mutant virus that is incapable of cell-to-cell spread on the spread of these probes was also determined. Our data shows that MP reduces SEL for cytoplasmic molecules, dilates the desmotubule allowing cell-cell diffusion of proteins via the desmotubule lumen and reduces the rate of spread of the ER membrane probes. Replicase was shown to enhance cell-cell spread. The data are not in support of the proposed model and have led us to propose a new model for virus cell-cell spread: this model proposes that MP, an integral ER membrane protein, forms a MP:vRNAER complex and that this ER-membrane complex diffuses in the lipid milieu of the ER into the desmotubule (the ER within the Pd), and spreads cell to cell by simple diffusion in the ER/desmotubule membrane; the driving force for spread is the chemical potential gradient between an infected cell and contingent non-infected neighbors. Our data also suggests that the virus replicase has a function in altering the Pd conductivity. Transgenic plant lines that express the MP gene of the Cg tobamovirus fused to YFP under the control the ecdysone receptor and methoxyfenocide ligand were generated by the Beachy group and the expression pattern and the timing and targeting patterns were determined. A vector expressing this MPs was also developed for use by the Epel lab . The transgenic lines are being used to identify and isolate host genes that are required for cell-to-cell movement of TMV/tobamoviruses. This line is now being grown and to be employed in proteomic studies which will commence November 2005. T-DNA insertion mutagenesis is being developed to identify and isolate host genes required for cell-to-cell movement of TMV.