Additional N-terminal Amino Acids Research Articles

On the Variativity of Cell Adhesive Response under the Action of Related Short Peptides

Analysis of the participation of short peptides GER and FGER containing common tripeptide fragment in the regulation of adhesive response of CHO-K1 cells was conducted. Both peptides stimulated cell adhesion both to untreated plastic and to gelatin-coated plastic, but did not change cell attachment to poly-L-lysine-coated plastic. Tripeptide GER had larger stimulation effect on cell adhesion to untreated plastic. Peptide FGER increased the rate of cell attachment to gelatin in a wider range of concentrations as compared to adhesion to untreated plastic. Variativity of cell spreading to different substrates under peptide action was demonstrated. On untreated plastic both investigated peptides practically in equal extent stimulated cell spreading. On gelatin peptide FGER kept the stimulation effect on cell spreading, but peptide GER partly inhibited cell spreading as compared to cell spreading on untreated plastic. It was established that insertion of additional N-terminal hydrophobic amino acid residue Phe to tripeptide fragment GER changes the regulatory activity of peptide at the cell adhesion model depending on the stage of cell connection with substrate and/or on substrate properties. The structural-functional activity of investigated short peptides on the instance of different structural components of adhesive structures is discussed.

Automated Synthesis of 68Ga-Labeled DOTA-MGS8 and Preclinical Characterization of Cholecystokinin-2 Receptor Targeting.

The new minigastrin analog DOTA-MGS8 targeting the cholecystokinin-2 receptor (CCK2R) used in this study displays the combination of two site-specific modifications within the C-terminal receptor binding sequence together with an additional N-terminal amino acid substitution preventing fast metabolic degradation. Within this study, the preparation of 68Ga-labeled DOTA-MGS8 was validated using an automated synthesis module, describing the specifications and analytical methods for quality control for possible clinical use. In addition, preclinical studies were carried out to characterize the targeting potential. [68Ga]Ga-DOTA-MGS8 showed a high receptor-specific cell internalization into AR42J rat pancreatic cells (~40%) with physiological expression of rat CCK2R as well as A431-CCK2R cells transfected to stably express human CCK2R (~47%). A favorable biodistribution profile was observed in BALB/c nude mice xenografted with A431-CCK2R cells and mock-transfected A431 cells as control. The high tumor uptake of ~27% IA/g together with low background activity and limited uptake in non-target tissue confirms the potential for high-sensitivity positron emission tomography of stabilized MG analogs in patients with MTC and other CCK2R-related malignancies.

Structure and Activity of a Selective Antibiofilm Peptide SK-24 Derived from the NMR Structure of Human Cathelicidin LL-37.

The deployment of the innate immune system in humans is essential to protect us from infection. Human cathelicidin LL-37 is a linear host defense peptide with both antimicrobial and immune modulatory properties. Despite years of studies of numerous peptides, SK-24, corresponding to the long hydrophobic domain (residues 9–32) in the anionic lipid-bound NMR structure of LL-37, has not been investigated. This study reports the structure and activity of SK-24. Interestingly, SK-24 is entirely helical (~100%) in phosphate buffer (PBS), more than LL-37 (84%), GI-20 (75%), and GF-17 (33%), while RI-10 and 17BIPHE2 are essentially randomly coiled (helix%: 7–10%). These results imply an important role for the additional N-terminal amino acids (likely E16) of SK-24 in stabilizing the helical conformation in PBS. It is proposed herein that SK-24 contains the minimal sequence for effective oligomerization of LL-37. Superior to LL-37 and RI-10, SK-24 shows an antimicrobial activity spectrum comparable to the major antimicrobial peptides GF-17 and GI-20 by targeting bacterial membranes and forming a helical conformation. Like the engineered peptide 17BIPHE2, SK-24 has a stronger antibiofilm activity than LL-37, GI-20, and GF-17. Nevertheless, SK-24 is least hemolytic at 200 µM compared with LL-37 and its other peptides investigated herein. Combined, these results enabled us to appreciate the elegance of the long amphipathic helix SK-24 nature deploys within LL-37 for human antimicrobial defense. SK-24 may be a useful template of therapeutic potential.

A LIN28B Tumor-Specific Transcript in Cancer

The diversity and complexity of the cancer transcriptome may contain transcripts unique to the tumor environment. Here, we report a LIN28B variant, LIN28B-TST, which is specifically expressed in hepatocellular carcinoma (HCC) and many other cancer types. Expression of LIN28B-TST is associated withsignificantly poor prognosis in HCC patients. LIN28B-TST initiates from a de novo alternative transcription initiation site that harbors a strong promoter regulated by NFYA but not c-Myc. Demethylation of the LIN28B-TST promoter might be a prerequisite for its transcription and transcriptional regulation. LIN28B-TST encodes a protein isoform with additional N-terminal amino acids and is critical for cancer cell proliferation and tumorigenesis. Our findings reveal a mechanism of LIN28B activation in cancer and the potential utility of LIN28B-TST for clinical purposes.

Mass spectrometric revival of an l-rhamnose– and d-galactose–specific lectin from a lost strain of Streptomyces

Blood type B-specific Streptomyces sp. 27S5 hemagglutinin (SHA) was discovered and characterized in the 1970s. Although strain 27S5 has been lost, the purified SHA protein survived intact under frozen conditions and retained its activity. Using modern techniques, here we further characterized SHA. Fourier-transform ion cyclotron resonance MS analysis determined the average molecular mass of SHA as 13,314.67 Da. MS of digested SHA peptides, Streptomyces genomic database matching, and N-terminal sequencing solved the 131-residue amino acid sequence of SHA. We found that SHA is homologous to N-terminally truncated hypothetical proteins encoded by the genomes of Streptomyces lavendulae, Streptomyces sp. Mg1, and others. The gene of the closest homologue in S. lavendulae, a putative polysaccharide deacetylase (PDSL), encodes 68 additional N-terminal amino acids, and its C terminus perfectly matched the SHA sequence, except for a single Ala-to-Glu amino acid difference. We expressed recombinant SHA(PDSL-A108E) (rSHA) as an enzymatically cleavable fusion protein in Escherichia coli, and glycan microarray analyses indicated that refolded rSHA exhibits the blood type B- and l-rhamnose-specific characteristics of authentic SHA, confirming that rSHA is essentially identical with SHA produced by Streptomyces sp. 27S5. We noted that SHA comprises three similar domains, representing 70% of the protein, and that these SHA domains partially overlap with annotated clostridial hydrophobic with conserved W domains. Furthermore, examination of GFP-tagged SHA revealed binding to microbial surfaces. rSHA may be useful both for studying the role of SHA/clostridial hydrophobic with conserved W domains in carbohydrate binding and for developing novel diagnostics and therapeutics for l-rhamnose-containing microorganisms.

Insight into the molecular recognition mechanism of the coactivator NCoA1 by STAT6

Crucial for immune and anti-inflammatory cellular responses, signal transducer and activator of transcription 6 (STAT6) regulates transcriptional activation in response to interleukin-4 and -13 -induced tyrosine phosphorylation by direct interaction with coactivators. The interaction of STAT6 with nuclear coactivator 1 (NCoA1) is mediated by a short region of the STAT6 transactivation domain that includes the motif LXXLL and interacts with the PAS-B domain of NCoA1. Despite the availability of an X-ray structure of the PAS-B domain/ Leu794-Gly814-STAT6 complex, the mechanistic details of this interaction are still poorly understood. Here, we determine the structure of the NCoA1257–385/STAT6783–814 complex using Nuclear Magnetic Resonance (NMR) and X-ray crystallography. The STAT6783–814 peptide binds with additional N-terminal amino acids to NCoA1257–385, compared to the STAT6794–814 peptide, explaining its higher affinity. Secondary and tertiary structures existing in the free peptide are more highly populated in the complex, suggesting binding by conformational selection.

Evidence of Alternative Cystatin C Signal Sequence Cleavage Which Is Influenced by the A25T Polymorphism.

Cystatin C (Cys C) is a small, potent, cysteine protease inhibitor. An Ala25Thr (A25T) polymorphism in Cys C has been associated with both macular degeneration and late-onset Alzheimer’s disease. Previously, studies have suggested that this polymorphism may compromise the secretion of Cys C. Interestingly, we found that untagged A25T, A25T tagged C-terminally with FLAG, or A25T FLAG followed by green fluorescent protein (GFP), were all secreted as efficiently from immortalized human cells as their wild-type (WT) counterparts (e.g., 112%, 100%, and 88% of WT levels from HEK-293T cells, respectively). Supporting these observations, WT and A25T Cys C variants also showed similar intracellular steady state levels. Furthermore, A25T Cys C did not activate the unfolded protein response and followed the same canonical endoplasmic reticulum (ER)-Golgi trafficking pathway as WT Cys C. WT Cys C has been shown to undergo signal sequence cleavage between residues Gly26 and Ser27. While the A25T polymorphism did not affect Cys C secretion, we hypothesized that it may alter where the Cys C signal sequence is preferentially cleaved. Under normal conditions, WT and A25T Cys C have the same signal sequence cleavage site after Gly26 (referred to as ‘site 2’ cleavage). However, in particular circumstances when the residues around site 2 are modified (such as by the presence of an N-terminal FLAG tag immediately after Gly26, or by a Gly26Lys (G26K) mutation), A25T has a significantly higher likelihood than WT Cys C of alternative signal sequence cleavage after Ala20 (‘site 1’) or even earlier in the Cys C sequence. Overall, our results indicate that the A25T polymorphism does not cause a significant reduction in Cys C secretion, but instead predisposes the protein to be cleaved at an alternative signal sequence cleavage site if site 2 is hindered. Additional N-terminal amino acids resulting from alternative signal sequence cleavage may, in turn, affect the protease inhibition function of Cys C.

The 1.59 Å resolution structure of the minor pseudopilin EpsH of Vibrio cholerae reveals a long flexible loop

The type II secretion complex exports folded proteins from the periplasm to the extracellular milieu. It is used by the pathogenic bacterium Vibrio cholerae to export several proteins, including its major virulence factor, cholera toxin. The pseudopilus is an essential component of the type II secretion system and likely acts as a piston to push the folded proteins across the outer membrane through the secretin pore. The pseudopilus is composed of the major pseudopilin, EpsG, and four minor pseudopilins, EpsH, EpsI, EpsJ and EpsK. We determined the x-ray crystal structure of the head domain of EpsH at 1.59Å resolution using molecular replacement with the previously reported EpsH structure, 2qv8, as the template. Three additional N-terminal amino acids present in our construct prevent an artifactual conformation of residues 160–166, present in one of the two monomers of the 2qv8 structure. Additional crystal contacts stabilize a long flexible loop comprised of residues 104–135 that is more disordered in the 2qv8 structure but is partially observed in our structure in very different positions for the two EpsH monomers in the asymmetric unit. In one of the conformations the loop is highly extended. Modeling suggests the highly charged loop is capable of contacting EpsG and possibly secreted protein substrates, suggesting a role in specificity of pseudopilus assembly or secretion function.

Translation Initiation from Two In-Frame AUGs Generates Mitochondrial and Cytoplasmic Forms of the p43 Component of the Multisynthetase Complex

In humans, nine aminoacyl-tRNA synthetases form a stable multiprotein complex with the three auxiliary proteins p18, p38, and p43. The N-terminal moiety of p43 is involved in its anchoring to the complex, and its C-terminal moiety has a potent tRNA binding capacity. The p43 component of the complex is also the precursor of p43(ARF), an apoptosis-released factor, and of p43(EMAPII), the endothelial-monocyte activating polypeptide II. Here we identified a new translation product of the gene of p43, which contains nine additional N-terminal amino acid residues. This gene product is targeted to the mitochondria and accounts for 2% of p43 expressed in human cells. The cytoplasmic and mitochondrial species of p43 are produced from the same mRNA by a mechanism of leaky scanning of the AUG codon at position -27, which is in an unfavorable sequence context for translation initiation. The finding that a mitochondrial species of p43 exists in human cells further exemplifies the multifaceted implications of p43 and opens new perspectives for the understanding of the role of p43 in the apoptotic cell.

The XLMR gene ACSL4 plays a role in dendritic spine architecture

ACSL4 is a gene involved in non-syndromic X-linked mental retardation. It encodes for a ubiquitous protein that adds coenzyme A to long-chain fatty acids, with a high substrate preference for arachidonic acid. It presents also a brain-specific isoform deriving from an alternative splicing and containing 41 additional N-terminal amino acids. To start to unravelling the link between ACSL4 and mental retardation, we have performed molecular and cell biological studies. By retro-transcription polymerase chain reaction analyses we identified a new transcript with a shorter 5′-UTR region. By immunofluorescence microscopy in embryonic rat hippocampal neurons we report that ACSL4 is associated preferentially to endoplasmic reticulum tubules. ACSL4 knockdown by siRNAs in hippocampal neurons indicated that this protein is largely dispensable for these cells' gross architectural features (i.e. axonal and dendritic formation and final length) yet it is required for the presence of normal spines. In fact, reduced levels of ACSL4 led to a significant reduction in dendritic spine density and an alteration in spine/filopodia distribution. The possible mechanisms behind this phenotype are discussed.

Cardiac Troponin Assays

Many clinical chemistry laboratories perform troponin testing to aid in the diagnosis of acute coronary syndrome (ACS). Laboratories must interact with clinicians to establish diagnostic cut points, achieve acceptable assay performance, and troubleshoot questionable findings. Here we present 3 hypothetical scenarios related to troponin testing in the clinical chemistry laboratory. Case Presentation 1: J.P. is an obese 55-year-old male with a history of gastroesophageal reflux disease who presents to the emergency department with substernal chest pain that he attributes to a “large meal.” J.P. is pale and diaphoretic. An ECG shows tachycardia with normal sinus rhythm. J.P.’s troponin levels (decision limit >0.04 μg/L) at presentation and at 6 and 12 hours after presentation are 0.09, 4.41, and 19.62 μg/L, respectively. Cardiac troponin (cTn) has established itself firmly as the “gold standard” in the diagnosis of ACS. cTn should be measured in all patients presenting with symptoms suggestive of ACS, in conjunction with physical examination and ECG.1 Because of the specificity of cTn for myocardial damage, a single cTn above the decision limit, along with clinical evidence, is indicative of myocardial injury. However, serial testing can be useful, depending on clinical presentation and onset of symptoms. cTn is a protein complex, located along the thin filaments of myofibrils, that regulates the contraction of cardiac muscle. It is composed of 3 distinct gene products. cTnC binds calcium, cTnT attaches to tropomyosin on thin filaments, and cTnI inhibits actomyosin ATPase. Expression of each subunit is cardiac restricted. The cardiac isoforms of cTnI and cTnT have additional unique N-terminal amino acid sequences, which allows for specific antibody and assay development and detection of each component in the bloodstream. The diagnostic utilities of cTnI and cTnT are comparable. Most cTn assays are sandwich immunoassays that use either monoclonal or polyclonal antibodies. It is essential …

Characterization of p43(ARF), a Derivative of the p43 Component of Multiaminoacyl-tRNA Synthetase Complex Released during Apoptosis

In human, nine aminoacyl tRNA synthetases are associated with the three auxiliary proteins, p18, p38, and p43, to form a stable multiprotein complex. The p43 component, which has a potent tRNA binding capacity, is associated to the complex via its N-terminal moiety. This protein is also the precursor of the endothelial monocyte-activating polypeptide II (p43(EMAPII), corresponding to the C-terminal moiety of p43), a cytokine generated during apoptosis. Here we examined the cellular pathway that, starting from the p43 subunit of the complex, leads to this extracellular cytokine. We identified a new intermediate in this pathway, named p43(ARF) for Apoptosis-released Factor. This intermediate is produced in cellulo by proteolytic cleavage of endogenous p43 and is rapidly recovered in the culture medium. This p43 derivative was purified from the medium of human U937 cells subjected to serum starvation. It contains 40 additional N-terminal amino acid residues as compared with the cytokine p43(EMAPII) and may be generated by a member of the matrix metalloproteinase family. Recombinant p43(ARF) is a monomer in solution and binds tRNA with a Kd of approximately 6 nM, 30-fold lower than that of p43. Highly purified p43(ARF) or p43(EMAPII) do not stimulate the expression of E-selectin by human umbilical vein endothelial cells. Our results suggest that the cleavage of p43 and its cellular delocalization, and thus the release of this tRNA binding subunit from the complex, is one of the molecular mechanisms leading to the shut down of protein synthesis in apoptosis.

Multiple forms of α-glucosidase in rice seeds ( Oryza sativa L., var Nipponbare)

Two isoforms of α-glucosidases (ONG2-I and ONG2-II) were purified from dry rice seeds ( Oryza sativa L., var Nipponbare). Both ONG2-I and ONG2-II were the gene products of ONG2 mRNA expressed in ripening seeds. Each enzyme consisted of two components of 6 kDa-peptide and 88 kDa-peptide encoded by this order in ONG2 cDNA ( ong2), and generated by post-translational proteolysis. The 88 kDa-peptide of ONG2-II had 10 additional N-terminal amino acids compared with the 88 kDa-peptide of ONG2-I. The peptides between 6 kDa and 88 kDa components (26 amino acids for ONG2-I and 16 for ONG2-II) were removed by post-translational proteolysis. Proteolysis induced changes in adsorption and degradation of insoluble starch granules. We also obtained three α-glucosidase cDNAs ( ong1, ong3, and ong4) from ripening seeds. The ONG1, ONG2, and ONG4 genes were situated in distinct locus of rice genome. The transcripts encoding ONG2 and ONG3 were generated by alternative splicing. Members of α-glucosidase multigene family are differentially expressed during ripening and germinating stages in rice.

Structure of Escherichia coli uracil-DNA glycosylase and its complexes with nonhydrolyzable substrate analogues in solution studied by synchrotron small-angle X-ray scattering

The structure of native and modified uracil DNA glycosylase from E. coli in solution was studied by synchrotron small-angle X-ray scattering. The modified enzyme (6His-uracyl DNA glycosylase) differs from the native one by the presence of an additional N-terminal 11-meric sequence amino acid residues including a block of six His residues. It was found that the conformations of these enzymes in solution at moderate ionic strength (60 mM NaCI) substantially differ in spite of minimal differences in the amino acid sequences and functional activity. The structure of native uracil DNA glycosylase in solution is close to that in crystal, showing a tendency for association. The interaction of this enzyme with nonhydrolyzable analogues of DNA ligands causes a partial dissociation of associates and a compactization of protein structure. At the same time, 6His-uracyl DNA glycosylase has a compact structure essentially different from the crystal one. A decrease in the ionic strength of solution results in a partial disruption of compact structure of the modified protein, without changes in its functional activity.

The pseudorabies virus serine/threonine kinase Us3 contains mitochondrial, nuclear and membrane localization signals.

The serine/threonine kinase encoded by the Us3 gene is conserved amongst all known alphaherpesviruses. Us3 has been reported to function in a variety of aspects of the virus lifecycle including protection of cells from virus-induced apoptosis, de-envelopment of enveloped virus particles from the perinuclear space and cell-to-cell spread of virus infection. In this report, we examined the sub-cellular localization of the pseudorabies virus (PRV) Us3 homolog. The PRV Us3 gene encodes two proteins termed Us3a and Us3b. Us3a differs from Us3b in that it contains 54 additional N-terminal amino acids. In transfected cells, Us3a localized predominantly to the plasma membrane whereas the Us3b protein localized predominantly to the nucleus. To explore the differences in the localization of the Us3a and Us3b proteins, we fused the amino-terminal 54 amino acids of Us3a to the amino-terminus of the enhanced green fluorescent protein (EGFP). Surprisingly, this fusion protein localized exclusively to mitochondria in transfected cells. Analysis of mutated Us3-EGFP fusion proteins in transfected cells revealed that the carboxy-terminal 101 amino acids of Us3a and Us3b comprises a membrane/vesicular localization domain, and that the N-terminal 102 amino acids of Us3b comprises a nuclear localization domain. We provide a model to rationalize the complex localization of Us3a and Us3b in transfected cells and hypothesize that the mitochondrial, nuclear and membrane localization motifs function in the reported anti-apoptotic, egress and cell-to-cell spread functions of Us3.

Mammalian DSCAMs: roles in the development of the spinal cord, cortex, and cerebellum?

Central nervous system (CNS) development involves neural patterning, neuronal and axonal migrations, and synapse formation. DSCAM, a chromosome 21 axon guidance molecule, is expressed by CNS neurons during development and throughout adult life. We now report that DSCAM and its chromosome 11 paralog DSCAML1 exhibit inverse ventral–dorsal expression patterns in the developing spinal cord and distinct, partly inverse, expression patterns in the developing cortex, beginning in the Cajal–Retzius cells. In the adult cortex, DSCAM predominates in layer 3/5 pyramidal cells and DSCAML1 predominates in layer 2 granule cells. In the cerebellum, DSCAM is stronger in the Purkinje cells and DSCAML1 in the granule cells. Finally, we find that the predicted DSCAML1 protein contains 60 additional N-terminal amino acids which may contribute to its distinct expression pattern and putative function. We propose that the DSCAMs comprise novel elements of the pathways mediating dorsal–ventral patterning and cell-fate specification in the developing CNS.

Expression and Refolding of Recombinant Human α-Tocopherol Transfer Protein Capable of Specific α-Tocopherol Binding

alpha-Tocopherol transfer protein (alpha-TTP) is a cytosolic protein found predominantly in mammalian liver that is proposed to be responsible for the stereoselective uptake of alpha-tocopherol from the diet. Although recombinant alpha-TTP has been reported previously, little detail has been provided about the yields and competency of the recovered protein at binding tocopherols and other ligands. In this work, we report the successful expression and refolding of a recombinant human alpha-TTP. Ligation-independent cloning generated a construct in pET-30 encoding an alpha-TTP fusion protein (pET-30/ttp) containing a six-histidine tag and an S-tag, each cleavable by a separate protease upon expression in Escherichia coli. Overexpression of the protein led to the formation of inclusion bodies that were solubilized in 8 M urea and purified by metal chelate affinity chromatography. Another construct in pET-28b (pET-28b/ttp) provided a soluble protein product after expression that contained a 40-amino-acid N-terminal extension, which can be reduced to 21 amino acids by cleavage with thrombin. The success of different refolding experiments was assessed using a Lipidex gel-based tocopherol binding assay. The best recovery of refolded recombinant alpha-TTP fusion capable of binding alpha-tocopherol was provided by matrix-assisted refolding in the presence of 0.5 M arginine. Cleavage of the fusion protein with Factor Xa successfully generated the full-length wild-type protein with no additional N-terminal amino acids. The resulting purification scheme provides recombinant alpha-TTP in good yield and purity for investigation of both its structure and its binding affinities for different ligands including natural and synthetic tocols.

Structure and Regulation of the Mouse ing1 Gene: THREE ALTERNATIVE TRANSCRIPTS ENCODE TWO PHD FINGER PROTEINS THAT HAVE OPPOSITE EFFECTS ON p53 FUNCTION

The human ING1 gene encodes nuclear protein p33(ING1), previously shown to cooperate with p53 in cell growth control (Garkavtsev, I., Grigorian, I. A., Ossovskaya, V. S., Chernov, M. V., Chumakov, P. M., and Gudkov, A. V. (1998) Nature 391, 295-298). p33(ING1) belongs to a small family of proteins from human, mouse, and yeast of approximately the same size that show significant similarity to one another within the C-terminal PHD finger domain and also contain an additional N-terminal region with subtle but reliably detectable sequence conservation. Mouse ing1 is transcribed from three differently regulated promoters localized within a 4-kilobase pair region of genomic DNA. The resulting transcripts share a long common region encoded by a common exon and differ in their 5'-exon sequences. Two transcripts are translated into the same protein of 185 amino acids, the mouse equivalent of the human p33(ING1), while the third transcript encodes a longer protein that has 94 additional N-terminal amino acids. Overexpression of the longer protein interferes with the accumulation of p53 protein and activation of p53-responsive promoters after DNA damage. Between the two products of ing1, only the longer one forms a complex with p53 detectable by immunoprecipitation. These results indicate that a single gene, ing1, encodes both p53-suppressing and p53-activating proteins that are regulated by alternative promoters.

Differential T-cell recognition of native and recombinant Mycobacterium tuberculosis GroES.

Mycobacterium tuberculosis GroES was purified from culture filtrate, and its identity was confirmed by immunoblot analysis and N-terminal sequencing. Comparing the immunological recognition of native and recombinant GroES, we found that whereas native GroES elicited a strong proliferative response and release of gamma interferon-gamma by peripheral blood mononuclear cells from healthy tuberculin reactors, the recombinant protein failed to do so. The same difference in immunological recognition was observed in a mouse model of TB infection. Both the native and recombinant preparations were recognized by mice immunized with the recombinant protein. Biochemical characterization including sodium dodecyl sulfate-polyacrylamide gel electrophoresis, two-dimensional electrophoresis, and mass spectrometry analysis of both proteins demonstrated no differences between the native and recombinant forms of GroES except for the eight additional N-terminal amino acids derived from the fusion partner in recombinant GroES. The recombinant fusion protein, still tagged with the maltose binding protein, was recognized by T cells isolated from TB-infected mice if mixed with culture filtrate before affinity purification on an amylose column. The maltose binding protein treated in the same manner as a control preparation was not recognized. Based on the data presented, we suggest that the association of biologically active molecules from culture filtrate with the chaperone GroES may be responsible for the observed T-cell recognition of the native preparation.

A novel human estrogen receptor β: identification and functional analysis of additional N-terminal amino acids

A novel human estrogen receptor β (hER β) was cloned from human testis mRNA, ovary and thymus cDNA utilizing PCR and 5′ RACE methods. The 5′ end of hER β contained an additional open reading frame, in-frame and upstream of the published clones. hER β encodes a protein of 530 amino acids with an approximate molecular weight of 63 kDa and is larger than the previously reported rat, mouse and human protein. To determine the functional role of additional N-terminal amino acids, we compared the transcription functions of receptor lacking (hER β T) and receptor containing (hER β L) this N-terminal extension. hER β L is more active than hER β T in transactivating ERE-based reporter genes. hER β L, but not hER β T, attenuated cytokine mediated NF κB activation. Taken together, the additional N-terminal amino acids appear to play a role in modulating estrogen responsive gene expression in vitro.