Elastase-mediated Activation of the Severe Acute Respiratory Syndrome Coronavirus Spike Protein at Discrete Sites within the S2 Domain

Proteolytic priming is a common method of controlling the activation of membrane fusion mediated by viral glycoproteins. The severe acute respiratory syndrome coronavirus spike protein (SARS-CoV S) can be primed by a variety of host cell proteases, with proteolytic cleavage occurring both as the S1/S2 boundary and adjacent to a fusion peptide in the S2 domain. Here, we studied the priming of SARS-CoV S by elastase and show an important role for residue Thr⁷⁹⁵ in the S2 domain. A series of alanine mutants were generated in the vicinity of the S2 cleavage site, with the goal of examining elastase-mediated cleavage within S2. Both proteolytic cleavage and fusion activation were modulated by altering the cleavage site position. We propose a novel mechanism whereby SARS-CoV fusion protein function can be controlled by spatial regulation of the proteolytic priming site, with important implications for viral pathogenesis.     

Arylsulfatase K,a Novel Lysosomal Sulfatase

The human sulfatase family has 17 members, 13 of which have been characterized biochemically. These enzymes specifically hydrolyze sulfate esters in glycosaminoglycans, sulfolipids, or steroid sulfates, thereby playing key roles in cellular degradation, cell signaling, and hormone regulation. The loss of sulfatase activity has been linked to severe pathophysiological conditions such as lysosomal storage disorders, developmental abnormalities, or cancer. A novel member of this family, arylsulfatase K (ARSK), was identified bioinformatically through its conserved sulfatase signature sequence directing posttranslational generation of the catalytic formylglycine residue in sulfatases. However, overall sequence identity of ARSK with other human sulfatases is low (18–22%). Here we demonstrate that ARSK indeed shows desulfation activity toward arylsulfate pseudosubstrates. When expressed in human cells, ARSK was detected as a 68-kDa glycoprotein carrying at least four N-glycans of both the complex and high-mannose type. Purified ARSK turned over p-nitrocatechol and p-nitrophenyl sulfate. This activity was dependent on cysteine 80, which was verified to undergo conversion to formylglycine. Kinetic parameters were similar to those of several lysosomal sulfatases involved in degradation of sulfated glycosaminoglycans. An acidic pH optimum (∼4.6) and colocalization with LAMP1 verified lysosomal functioning of ARSK. Further, it carries mannose 6-phosphate, indicating lysosomal sorting via mannose 6-phosphate receptors. ARSK mRNA expression was found in all tissues tested, suggesting a ubiquitous physiological substrate and a so far non-classified lysosomal storage disorder in the case of ARSK deficiency, as shown before for all other lysosomal sulfatases.     

Optimization of Glycoalkaloid Analysis for Use in Industrial Potato Fruit Juice Downstreaming

Annually, within the European Union about 1.7 million tons of starch is produced by processing over 8 million tons of potato tubers, Solanum tuberosum. In recent years, the potato protein content has gained tremendous industrial interest, since these proteins have excellent nutritional value. As naturally occurring, secondary plant metabolites steroidal potato glycoalkaloids are formed in potatoes. The two major glycoalkaloids in potatoes are α‐solanine and α‐chaconine. Because of the significant toxicity of the glycoalkaloids for human and for animal nutrition it was essential to develop efficient extraction processes. The need for an easy, fast, sensitive and reliable glycoalkaloid assay at the very beginning of the production chain is obvious. In this study an efficient analytical assay for potato glycoalkaloids from powdery protein samples under industrially relevant conditions is described: sample extraction, analyte pre‐purification, and final HPLC analysis. An acetic acid extraction/homogenization process was used for glycoalkaloid extraction from potato protein samples. The extracts were purified by means of solid phase extraction cartridges using the different washing steps developed in this study. The final determination was performed through an HPLC method using a Reprosil‐Pur NH₂ column. The limit of detection was 5 μg/mL for α‐solanine and α‐chaconine, respectively, corresponding to concentrations of 20 ppm in potato protein powder.     

Dna2 Is Involved in CA Strand Resection and Nascent Lagging Strand Completion at Native Yeast Telomeres

Post-replicational telomere end processing involves both extension by telomerase and resection to produce 3′-GT-overhangs that extend beyond the complementary 5′-CA-rich strand. Resection must be carefully controlled to maintain telomere length. At short de novo telomeres generated artificially by HO endonuclease in the G₂ phase, we show that dna2-defective strains are impaired in both telomere elongation and sequential 5′-CA resection. At native telomeres in dna2 mutants, GT-overhangs do clearly elongate during late S phase but are shorter than in wild type, suggesting a role for Dna2 in 5′-CA resection but also indicating significant redundancy with other nucleases. Surprisingly, elimination of Mre11 nuclease or Exo1, which are complementary to Dna2 in resection of internal double strand breaks, does not lead to further shortening of GT-overhangs in dna2 mutants. A second step in end processing involves filling in of the CA-strand to maintain appropriate telomere length. We show that Dna2 is required for normal telomeric CA-strand fill-in. Yeast dna2 mutants, like mutants in DNA ligase 1 (cdc9), accumulate low molecular weight, nascent lagging strand DNA replication intermediates at telomeres. Based on this and other results, we propose that FEN1 is not sufficient and that either Dna2 or Exo1 is required to supplement FEN1 in maturing lagging strands at telomeres. Telomeres may be among the subset of genomic locations where Dna2 helicase/nuclease is essential for the two-nuclease pathway of primer processing on lagging strands.     

Autoactivation of Mouse Trypsinogens Is Regulated by Chymotrypsin C via Cleavage of the Autolysis Loop

Chymotrypsin C (CTRC) is a proteolytic regulator of trypsinogen autoactivation in humans. CTRC cleavage of the trypsinogen activation peptide stimulates autoactivation, whereas cleavage of the calcium binding loop promotes trypsinogen degradation. Trypsinogen mutations that alter these regulatory cleavages lead to increased intrapancreatic trypsinogen activation and cause hereditary pancreatitis. The aim of this study was to characterize the regulation of autoactivation of mouse trypsinogens by mouse Ctrc. We found that the mouse pancreas expresses four trypsinogen isoforms to high levels, T7, T8, T9, and T20. Only the T7 activation peptide was cleaved by mouse Ctrc, causing negligible stimulation of autoactivation. Surprisingly, mouse Ctrc poorly cleaved the calcium binding loop in all mouse trypsinogens. In contrast, mouse Ctrc readily cleaved the Phe-150–Gly-151 peptide bond in the autolysis loop of T8 and T9 and inhibited autoactivation. Mouse chymotrypsin B also cleaved the same peptide bond but was 7-fold slower. T7 was less sensitive to chymotryptic regulation, which involved slow cleavage of the Leu-149–Ser-150 peptide bond in the autolysis loop. Modeling indicated steric proximity of the autolysis loop and the activation peptide in trypsinogen, suggesting the cleaved autolysis loop may directly interfere with activation. We conclude that autoactivation of mouse trypsinogens is under the control of mouse Ctrc with some notable differences from the human situation. Thus, cleavage of the trypsinogen activation peptide or the calcium binding loop by Ctrc is unimportant. Instead, inhibition of autoactivation via cleavage of the autolysis loop is the dominant mechanism that can mitigate intrapancreatic trypsinogen activation.     

Faster Protein Splicing with the Nostoc punctiforme DnaE Intein Using Non-native Extein Residues

Inteins are naturally occurring intervening sequences that catalyze a protein splicing reaction resulting in intein excision and concatenation of the flanking polypeptides (exteins) with a native peptide bond. Inteins display a diversity of catalytic mechanisms within a highly conserved fold that is shared with hedgehog autoprocessing proteins. The unusual chemistry of inteins has afforded powerful biotechnology tools for controlling enzyme function upon splicing and allowing peptides of different origins to be coupled in a specific, time-defined manner. The extein sequences immediately flanking the intein affect splicing and can be defined as the intein substrate. Because of the enormous potential complexity of all possible flanking sequences, studying intein substrate specificity has been difficult. Therefore, we developed a genetic selection for splicing-dependent kanamycin resistance with no significant bias when six amino acids that immediately flanked the intein insertion site were randomized. We applied this selection to examine the sequence space of residues flanking the Nostoc punctiforme Npu DnaE intein and found that this intein efficiently splices a much wider range of sequences than previously thought, with little N-extein specificity and only two important C-extein positions. The novel selected extein sequences were sufficient to promote splicing in three unrelated proteins, confirming the generalizable nature of the specificity data and defining new potential insertion sites for any target. Kinetic analysis showed splicing rates with the selected exteins that were as fast or faster than the native extein, refuting past assumptions that the naturally selected flanking extein sequences are optimal for splicing.     

Membrane Association via an Amino-terminal Amphipathic Helix Is Required for the Cellular Organization and Function of RNase II

The subcellular localization of the exoribonuclease RNase II is not known despite the advanced biochemical characterization of the enzyme. Here we report that RNase II is organized into cellular structures that appear to coil around the Escherichia coli cell periphery and that RNase II is associated with the cytoplasmic membrane by its amino-terminal amphipathic helix. The helix also acts as an autonomous transplantable membrane binding domain capable of directing normally cytoplasmic proteins to the membrane. Assembly of the organized cellular structures of RNase II required the RNase II amphipathic membrane binding domain. Co-immunoprecipitation of the protein from cell extracts indicated that RNase II interacts with itself. The RNase II self-interaction and the ability of the protein to assemble into organized cellular structures required the membrane binding domain. The ability of RNase II to maintain cell viability in the absence of the exoribonuclease polynucleotide phosphorylase was markedly diminished when the RNase II cellular structures were lost due to changes in the amphipathicity of the amino-terminal helix, suggesting that membrane association and assembly of RNase II into organized cellular structures play an important role in the normal function of the protein within the bacterial cell.