MUC1, a glycoprotein overexpressed by a variety of human adenocarcinomas, is a type I transmembrane protein (MUC1/TM) that soon after its synthesis undergoes proteolytic cleavage in its extracellular domain. This cleavage generates two subunits, ␣ and ␤, that specifically recognize each other and bind together in a strong noncovalent interaction. Proteolysis occurs within the SEA module, a 120-amino acid domain that is highly conserved in a number of heavily glycosylated mucin-like proteins. Post-translational cleavage of the SEA module occurs at a site similar to that in MUC1 in the glycoproteins IgHepta and MUC3. However, as in the case of other proteins containing the cleaved SEA module, the mechanism of MUC1 proteolysis has not been elucidated. Alternative splicing generates two transmembrane MUC1 isoforms, designated MUC1/Y and MUC1/X. We demonstrated here that MUC1/X, whose extracellular domain is comprised solely of the SEA module in addition to 30 MUC1 N-terminal amino acids, undergoes proteolytic cleavage at the same site as the MUC1/TM protein. In contrast, the MUC1/Y isoform, composed of an N-terminally truncated SEA module, is not cleaved. Cysteine or threonine mutations of the MUC1/X serine residue (Ser-63) immediately C-terminal to the cleavage site generated cleaved proteins, whereas mutation of the Ser-63 residue of MUC1/X to any other of 17 amino acids did not result in cleavage. In vitro incubation of highly purified precursor MUC1/X protein resulted in self-cleavage. Furthermore, addition of hydroxylamine, a strong nucleophile, markedly enhanced cleavage. Both these features are signature characteristics of self-cleaving proteins, and we concluded that MUC1 undergoes autoproteolysis mediated by an N 3 O-acyl rearrangement at the cleavage site followed by hydrolytic resolution of the unstable ester and concomitant cleavage. It is likely that all cleaved SEA module-containing proteins follow a similar route.The MUC1 gene is highly expressed in a number of human epithelial malignancies, including breast, prostate, and colon carcinomas, as well as on the malignant plasma cells of multiple myeloma (1-9). As a well characterized tumor-associated protein, it has generated considerable interest as a tumor marker for disease prognosis (10 -14) as well as a target for tumor cell killing (15-18). Although alternative splicing can generate multiple MUC1 protein forms (19 -23), the most intensively studied MUC1 protein is a type I transmembrane protein comprised of a heavily glycosylated extracellular domain containing a tandem-repeat array, a transmembrane domain, and a cytoplasmic domain (Fig. 1, MUC1/TM) (24 -26). MUC1/TM is proteolytically cleaved soon after its synthesis, generating two subunits, ␣ and ␤, that specifically recognize each other and bind together by a strong noncovalent interaction (27).Cleavage occurs within the SEA module (28 -30), a highly conserved protein module so-called from its initial identification in a sperm protein, in enterokinase, and in agrin (31), that is found in a numb...

We report here syntenic loci in humans and mice incorporating gene clusters coding for secreted proteins each comprising 10 cysteine residues. These conform to three-fingered protein/Ly-6/urokinase-type plasminogen activator receptor (uPAR) domains that shape three-fingered proteins (TFPs). The founding gene is PATE, expressed primarily in prostate and less in testis. We have identified additional human PATElike genes (PATE-M, PATE-DJ, and PATE-B) that co-localize with the PATE locus, code for novel secreted PATE-like proteins, and show selective expression in prostate and/or testis. Anti-PATE-B-specific antibodies demonstrated the presence of PATE-B in the region

e Dengue virus (DENV) causes dengue fever, a major health concern worldwide. We identified an amphipathic helix (AH) in the N-terminal region of the viral nonstructural protein 4A (NS4A). Disruption of its amphipathic nature using mutagenesis reduced homo-oligomerization and abolished viral replication. These data emphasize the significance of NS4A in the life cycle of the dengue virus and demarcate it as a target for the design of novel antiviral therapy. Dengue virus (DENV) infection is a growing public health threat, with more than one-third of the world population at risk (1). DENV is a positive single-strand RNA virus. Its genome is translated into a single polyprotein, which is cleaved to produce structural (components of the mature virus) and nonstructural (NS) proteins. In addition, the NS proteins generate the viral replication complexes (RC) (2). DENV replicates its RNA genome in association with modified intracellular membranes; the details of the assembly of these complexes are incompletely understood.NS4A, a transmembrane endoplasmic reticulum (ER) resident protein, is thought to induce the host membrane modifications that harbor the viral RC (3). A similar function for NS4A was reported in other flaviviruses (4, 5). To further understand the role of NS4A, we analyzed its cytosolic N-terminal region (amino acids 1 to 48) using sequence alignment of the four DENV serotypes. Within this sequence, amino acids that differed in their identity maintained their biochemical properties, suggesting the presence of a conserved structural motif with a potential functional significance (Fig. 1A). Secondary structure algorithms (6) indicated that this segment is predicted to fold into an ␣-helix (Fig. 1B). Helical wheel projections of amino acids 3 to 20 indicated a conserved polar-nonpolar asymmetry indicative of an amphipathic helix (AH) (Fig. 1C). To experimentally examine the conformation of the NS4A N terminus, a recombinant peptide comprising amino acids 1 to 48 was prepared. Codon-optimized DENV2 NS4A 1-48 was cloned into pGEV2 (7) with an N-terminal fusion to the immunoglobulin binding domain of streptococcal protein G (GB1). A tobacco etch virus (TEV) protease cleavage site (ENLYFQ) was introduced into the beginning of the NS4A coding sequence. Due to difficulties in separating NS4A from GB1 after TEV protease cleavage, an N-terminal glutathione S-transferase (GST) affinity tag was added to GB1-TEV-NS4A(1-48) by cloning it into pGEX4T-2. Protein expression and purification of the GST-GB1-NS4A(1-48) fusion were performed as described previously (8), except that an on-column cleavage was performed by adding TEV protease. The flowthrough was concentrated and subjected to size exclusion chromatography (HiLoad 16/60 Superdex 75) yielding pure NS4A(1-48). The resulting peptide was characterized using far-UV circular dichroism (CD) (Fig. 1D). In aqueous buffer, NS4A(1-48) showed limited solubility and a CD spectrum typical of a random coil conformation with an ␣-helix content below 10%. Addition of various memb...

BackgroundDengue virus (DENV) is a mosquito-transmitted positive single strand RNA virus belonging to the Flaviviridae family. DENV causes dengue fever, currently the world's fastest-spreading tropical disease. Severe forms of the disease like dengue hemorrhagic fever and dengue shock syndrome are life-threatening. There is no specific treatment and no anti-DENV vaccines. Our recent data suggests that the amino terminal cytoplasmic region of the dengue virus non-structural protein 4A (NS4A) comprising amino acid residues 1 to 48 forms an amphipathic helix in the presence of membranes. Its amphipathic character was shown to be essential for viral replication. NMR-based structure-function analysis of the NS4A amino terminal region depends on its milligram-scale production and labeling with NMR active isotopes.Methodology/Principal FindingsThis report describes the optimization of a uniform procedure for the expression and purification of the wild type NS4A(1-48) peptide and a peptide derived from a replication-deficient mutant NS4A(1-48; L6E, M10E) with disrupted amphipathic nature. A codon-optimized, synthetic gene for NS4A(1-48) was expressed as a fusion with a GST-GB1 dual tag in E. coli. Tobacco etch virus (TEV) protease mediated cleavage generated NS4A(1-48) peptides without any artificial overhang. Using the described protocol up to 4 milligrams of the wild type or up to 5 milligrams of the mutant peptide were obtained from a one-liter culture. Isotopic labeling of the peptides was achieved and initial NMR spectra were recorded.Conclusions/SignificanceSmall molecules targeting amphipathic helices in the related Hepatitis C virus were shown to inhibit viral replication, representing a new class of antiviral drugs. These findings highlight the need for an efficient procedure that provides large quantities of the amphipathic helix containing NS4A peptides. The double tag strategy presented in this manuscript answers these needs yielding amounts that are sufficient for comprehensive biophysical and structural studies, which might reveal new drug targets.