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PubMed1988

Antigen processing for presentation to T lymphocytes: function, mechanisms, and implications for the T-cell repertoire.

Antigen processing encompasses the metabolic events that a protein antigen must undergo in or on the antigen-presenting cell before it can be recognized by the T lymphocyte. It appears that a primary goal of these events is to unfold the protein to expose residues that are buried in the native conformation, which is designed to be soluble in water. The APC usually accomplishes this task by proteolytic cleavage of the protein, but we have found that artificial unfolding without proteolysis is sufficient. The purpose of unfolding may be to allow different faces of the antigenic site to bind simultaneously to the T-cell receptor and the MHC molecule on the APC, or to interact with other structures on the membrane of the APC. This requirement for unfolding appears to apply to everything from small peptides to large multimeric proteins. We have found that the way the antigen is processed and the structure of the fragments produced can greatly affect the availability of antigenic sites. For instance, some antigenic sites are not recognized when the native protein is used as immunogen, despite the fact that immunization with a small peptide corresponding to that site reveals both the ability of the site to bind to MHC molecules of the animal in question and the presence of a T-cell repertoire specific for that site. The antigenic site is not destroyed by processing, since it can be presented by the same F1 APC to T cells of another MHC type. Similarly, cross-reactivity between homologous epitopes of related proteins may occur at the peptide level even though the native proteins do not crossreact for the same T-cell clone. Since these events occur with monoclonal T cells, they cannot be due to suppressor cells specific for other sites on the native molecule. The best explanation is that the products of natural processing of the protein are larger than the peptides corresponding to the minimal antigenic sites, and contain hindering structures that interfere with binding to some MHC molecules and not others, or to some T-cell receptors and not others. Thus, antigen processing is a third factor that can lead to apparent Ir gene defects - in addition to MHC specificity and holes in the T-cell repertoire - and can significantly influence which antigenic sites are immunodominant.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1979

Purification and preparation of antibody to RNA polymerase II stimulatory factors from Ehrlich ascites tumor cells.

An improved method was developed for purification of the protein termed S-II that specifically stimulates RNA polymerase II of Ehrlich ascites tumor cells. The specific activity of the final preparation was 400 000 units/mg of protein, which is about 30-fold higher than that of the previous preparation [Sekimizu, K., et al. (1976) Biochemistry 15, 5064]. The final preparation gave a single band on both sodium dodecyl sulfate and nondenaturing gel electrophoresis, and the protein extracted from the band on nondenaturing gel had stimulatory activity. S-II is a basic protein with a molecular weight of 40 500. The fundamental characteristics of S-II determined with the previous preparation were confirmed with completely purified S-II. A specific antibody to S-II was prepared. This antibody inhibited only the stimulatory activity of S-II and did not affect the activity of RNA polymerase II itself. Thus, S-II is probably not a component of the multimeric proteins of RNA polymerase II.

Animals↗
PubMed1975

Genetic evidence on the organization and action of the qa-1 gene product: a protein regulating the induction of three enzymes in quinate catabolism in Neurospora crassa.

The first three reactions in the catabolism of qainic acid in Neurospora crassa are under the genetic control of the qa gene cluster. This cluster consists of three structural genes encoding three inducible enzymes plus a regulatory gene (qa-1+) whose diffusible product apparently acts in a positive fashion to initiate coordinate synthesis of the three enzymes when an appropriate inducer is present. Genetic and biochemical evidence for both complementing and temperature-sensitive qa-1 alleles indicates that the product of the qa-1+ gene is an oligomeric (multimeric) protein. On the basis of cis-trans tests of appropriate double mutants (plus genetic mapping data for temperature-sensitive mutants), at least certain constitutive mutants (which produce all three qa enzymes in the absence of an inducer) are mutants in the regulatory gene and not in controlling elements such as initiators. The detection of stable (non-revertible) qa-1 intralocus deletion (multisite)mutants provides additional evidence for positive regulation in the qa system. Extensive genetic recombination data provide evidence that the two types of qa-1 mutants--slow-complementing (qa-1-s) and fast-complementing (qa-1-f)--map in discrete, non-overlapping segments of the qa-1 locus. These two distinct types of mutants are hypothesized to produce altered regulatory protein molecules that fail to interact either with a DNA initiator site (qa-1-s types) or with an inducer (qa-1-f types). The striking similarities between the qa system in this lower eukaryote and certain prokaryote operon systems are discussed.

Alcohol Oxidoreductases↗
PubMed1988

Distinctive charge configurations in proteins of the Epstein-Barr virus and possible functions.

The protein products of several open reading frames (ORFs) of the Epstein-Barr virus (EBV) are remarkable in their distribution of charged residues. The nuclear antigen proteins EBNA1-EBNA4 of the EBV latent state contain separate significant clusters of charge of each sign. They (excepting EBNA4) also feature distinctive periodic charge patterns [e.g., (+, O)8, (O, -, -)7] and significant tandem repeats. None of the other ORFs (about 80) of the genome possess the conjunction of these properties. Only the protein encoded from BMLF1, the first immediate early transactivator protein, contains significant multiple charge clusters and periodic charge patterns. All proteins that contain significant repeats also contain at least one significant charge cluster of a single sign. These include EBNA5 and LYDMA produced during latency and BZLF1, whose expression terminates latency and initiates productive growth. It is reasonable to conclude that these aggregate significant charge configurations and repeats are important functionally for the latent existence and for the initiation of the lytic cycle and may be characteristic of these conditions. We discuss how large multimeric protein structures bound together by clusters of unlike charge may provide a mechanism for regulation of the expression of these proteins.

Amino Acid Sequence↗
PubMed1986

In vivo cloning ad characterization of mutations of the regulatory locus ompR of Escherichia coli K12.

The product of the ompR gene of E. coli K12 is a positive regulatory protein, which is needed for the expression of the major outer membrane proteins OmpC and OmpF in E. coli K12. A simple in vivo technique was used to transfer three ompR mutations (ompR101, ompR472, ompR4) onto a multicopy plasmid carrying the wild-type ompR gene. The resulting clones were transformed into wild type and corresponding mutant backgrounds to analyze their effects on ompC and ompF expression. All of the cloned ompR mutant alleles exhibited a dominant OmpC- phenotype in an ompR+ background. In addition negative complementation of ompF expression was observed between chromosomal ompR4 and multicopy ompR101 alleles. The results suggest an interaction between different OmpR molecules and thereby support the idea that OmpR can exist as a multimeric protein.

Alleles↗
PubMed1992

Sec23p and a novel 105-kDa protein function as a multimeric complex to promote vesicle budding and protein transport from the endoplasmic reticulum.

A cell-free protein transport reaction has been used to monitor the purification of a functional form of the Sec23 protein, a SEC gene product required for the formation or stability of protein transport vesicles that bud from the endoplasmic reticulum (ER). Previously, we reported that Sec23p is an 84-kDa peripheral membrane protein that is released from a sedimentable fraction by vigorous mechanical agitation of yeast cells and is required for ER to Golgi transport assayed in vitro. We have purified soluble Sec23p by complementation of an in vitro ER to Golgi transport reaction reconstituted with components from sec23 mutant cells. Sec23p overproduced in yeast exists in two forms: a monomeric species and a species that behaves as a 250- to 300-kDa complex that contains Sec23p and a distinct 105-kDa polypeptide (p105). Sec23p purified from cells containing one SEC23 gene exists solely in the large multimeric form. A stable association between Sec23p and p105 is confirmed by cofractionation of the two proteins throughout the purification. p105 is a novel yeast protein involved in ER to Golgi transport. Like Sec23p, it is required for vesicle budding from the ER because p105 antiserum completely inhibits transport vesicle formation in vitro.

Animals↗
PubMed1986

Altered translation initiation factor 2 in the cold-sensitive ssyG mutant affects protein export in Escherichia coli.

The Escherichia coli gene secY (pr1A) codes for an integral membrane protein that plays an essential role in protein export. We previously isolated cold-sensitive mutations (ssy) as extragenic suppressors of temperature-sensitive secY24 mutation. Now we show that the ssyG class of mutations are within infB coding for the translation initiation factor IF2. The mutants produce altered forms of IF2 with a cold-sensitive in vitro activity to form a translation initiation complex. The mutation suppresses not only secY24 but also other secretion-defective mutations such as secA51 and rp10215. The beta-galactosidase enzyme activity of the MalE-LacZ 72-47 hybrid protein is strikingly reduced in the ssyG mutant at the permissive high temperature, while the hybrid protein itself is normally synthesized. This effect, which was observed only for the hybrid protein with a functional signal sequence, may result from some alteration in the cellular localization of the protein. These results suggest that IF2 or the translation initiation step can modulate protein export reactions. The isolation of cold-sensitive ssyG mutations in infB provides genetic evidence that IF2 is indeed essential for normal growth of E. coli cells.

Bacterial Proteins↗
PubMed1992

Role of gizzard myosin light chains in calcium binding.

The contraction of molluscan and vertebrate smooth muscles is regulated by myosin. Although the myosin and its associated two subunits, the regulatory light chain and the essential light chain, constitute the Ca2+ regulatory system in both types of muscles, the mechanisms by which Ca2+ signal is transduced are quite different. In molluscan muscles, the direct binding of Ca2+ to the regulatory system triggers muscle contraction. In vertebrate smooth muscles, however, phosphorylation of the regulatory light chain is the major triggering mechanism. We measured Ca2+ binding in gizzard myosin and in hybrids of scallop myosin containing gizzard regulatory light chain or in hybrids of scallop regulatory domain containing gizzard essential light chain. Isolated chicken gizzard myosin did not bind Ca2+ in the range of pCa 8.0 to 5.0 in the presence of 2 mM MgCl2, supporting the lack of the specific Ca(2+)-binding site in gizzard myosin. Phosphorylation of the regulatory light chain did not generate a specific (Ca2+)-binding site. The hybrid scallop myosin containing gizzard regulatory light chain showed a similar Ca2+ binding as native scallop myosin with a one to one stoichiometry of Ca2+ to myosin head saturating at about pCa 6.0 at pH 7.6. In contrast, the hybrid scallop regulatory domain containing gizzard essential light chain did not bind Ca2+ either at pCa 6.0 or at pCa 8.0. Control preparations reconstituted with scallop essential light chains bound 0.69 mol per mol Ca2+ at pCa 6.0 with no binding at pCa 8.0.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1990

Phosphorylase b kinase deficiency in man: a review.

Phosphorylase b kinase is involved in the activation of glycogen phosphorylase and is thus involved in the breakdown of glycogen. The enzyme exists as several tissue specific isoenzymes of which the muscle enzyme (rabbit) has been most characterized. It is a multimeric protein composed of four subunits, alpha, beta, gamma and delta. The four subunits are coded on different chromosomes, the alpha, beta and gamma subunit genes being on the X, 16 and 17 chromosomes respectively. The delta subunit is a calmodulin and confers calcium sensitivity on phosphorylase b kinase. Tissue specificity of the enzyme is conferred, at least in some cases, by variation in the gamma subunit. Seven different clinical types of phosphorylase b kinase deficiency have been described. The most common type is X-linked and affects the liver only; other types affect liver, muscle and liver, muscle or heart and have an autosomal recessive mode of inheritance, while in some types the mode of inheritance is not clear. Diagnosis based on the study of erythrocytes or leukocytes can be misleading due to the tissue specific nature of the enzyme, and liver or muscle biopsies may be required.

Diagnosis↗
PubMed2024

Early Events in β2AR Dimer Dynamics Mediated by Activation-Related Microswitches.

G-Protein-Coupled Receptors (GPCRs) make up around 3-4% of the human genome and are the targets of one-third of FDA-approved drugs. GPCRs typically exist as monomers but also aggregate to form higher-order oligomers, including dimers. β2AR, a pharmacologically relevant GPCR, is known to be targeted for the treatment of asthma and cardiovascular diseases. The activation of β2AR at the dimer level remains under-explored. In the current study, molecular dynamics (MD) simulations have been performed to understand activation-related structural changes in β2AR at the dimer level. The transition from inactive to active and vice versa has been studied by starting the simulations in the apo, agonist-bound, and inverse agonist-bound β2AR dimers for PDB ID: 2RH1 and PDB ID: 3P0G, respectively. A cumulative total of around 21-μs simulations were performed. Residue-based distances, RMSD, and PCA calculations suggested that either of the one monomer attained activation-related features for the apo and agonist-bound β2AR dimers. The TM5 and TM6 helices within the two monomers were observed to be in significant variation in all the simulations. TM5 bulge and proximity of TM2 and TM7 helices may be contributing to one of the early events in activation. The dimeric interface between TM1 and helix 8 were observed to be well maintained in the apo and agonist-bound simulations. The presence of inverse agonists favored inactive features in both the monomers. These key features of activation known for monomers were observed to have an impact on β2AR dimers, thereby providing an insight into the oligomerization mechanism of GPCRs.

Receptors, Adrenergic, beta-2↗
PubMed1990

Nuclear Ah receptor from mouse hepatoma cells: effect of partial proteolysis on relative molecular mass and DNA-binding properties.

The nuclear Ah receptor from mouse hepatoma (Hepa-1c1c9) cells is a 176-kDa multimeric protein which is stable under conditions of up to 1 M KCl. Under denaturing conditions, the Hepa-1 nuclear receptor can be dissociated into a ligand-binding subunit of Mr approximately 91,000. The identity of subunits that compose the nuclear Ah receptor is currently unknown. We used partial proteolysis under nondenaturing conditions as an approach to study the domain organization of the nuclear form of Ah receptor from Hepa-1c1c9 cells treated with [3H]2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in culture. Low concentrations of trypsin (0.5 microgram/mg nuclear protein) generated heterogeneous fragments with the main fragment having a Stokes radius (Rs) approximately 6 nm. More discrete ligand-binding fragments of Mr approximately 84,000 (Rs approximately 4 nm/approximately 5 S) and Mr approximately 16,000 (Rs approximately 2 nm/approximately 2 S) could be generated using higher concentrations of trypsin (5 micrograms/mg nuclear protein). The relative concentration of the 84 and 16-kDa fragment was dependent on duration of protease treatment; formation of the 16-kDa fragment was accompanied by some loss in [3H]TCDD binding. Treatment of nuclear Ah receptor with alpha-chymotrypsin (1 microgram/mg nuclear protein) generated a single, apparently homogeneous ligand-binding fragment of Mr approximately 101,000 (Rs approximately 5 nm/approximately 5 S). When analyzed by DNA-cellulose chromatography, the chymotryptic fragment eluted at a significantly higher KCl concentration (462 mM) compared to native untreated nuclear Ah receptor (385 mM). Despite this increased affinity for DNA-cellulose columns, the ligand-binding fragment generated by chymotrypsin treatment was unable to interact with a dioxin responsive element in a gel retardation assay. DNA-cellulose binding ability, therefore, does not appear to be a reliable indicator of specific DNA interactions for these protease-modified fragments.

Animals↗
PubMed1988

Developmental genetics of the gastrulation defective locus in Drosophila melanogaster.

The fs(1)gastrulation defective (dg) locus is one of the dorsal-group genes of Drosophila. Maternal expression of this gene is required for gastrulation movements and the differentiation of structures along the embryonic dorso-ventral axis. Twelve alleles of gd displayed a complex pattern of complementation, suggesting a direct interaction between subunits of a multimeric protein. Essential expression of the gd locus was strictly maternal with no zygotic contribution by the paternally derived allele. Clonal analysis revealed that expression of the gd locus was required in the germ line and that extreme dorsalization represented the null gd phenotype. Temperature-sensitive (ts) alleles displayed a ts period that included the last 4-5 hr of oogenesis and the first 1.5-2 hr of embryogenesis. Eggs from one ts allelic combination displayed reduced hatching when retained in the ovary at permissive temperatures, suggesting the loss of a labile egg component. This lability may also be responsible for the variable phenotypes displayed by offspring from individual females.

Alleles↗
PubMed1991

Design of site-specific protein drugs.

The ability to clone and express proteins at high levels of abundance and purity has led to their increasing consideration as therapeutic agents. The use of various protein engineering and recombinant DNA techniques has enabled progression to where proteins can be tailored so that they have not only the ability to interact with a unique (extra)cellular component in order to produce a pharmacological effect, but their structure can be altered so as to achieve a selective biological disposition. This ability to control the biological dispersion of proteins is becoming a key element in the design of therapeutic polypeptides and proteins, finding application in modalities intended for treating HIV infections, accessible tumor masses, and diseases of the hematopoietic system, etc. For recombinant site-specific systems to be successfully introduced into the clinic, radically different approaches are required during their development and clinical testing. These relate not only to the pharmacokinetics and receptor availability of the drug, but also its pharmacodynamics, its analytics and its safety pharmacology. This contribution examines how the control of the biological disposition of proteins may be achieved using both site-directed mutagenesis, sequence re-ordering, hybrid protein formation, protein resurfacing, as well as synthetic adduction to polymers.

Chromosome Deletion↗
PubMed1987

Distinct sequence determinants direct intracellular sorting and modification of a yeast vacuolar protease.

We have mapped a sequence determinant in the vacuolar glycoprotein carboxypeptidase Y (CPY) that directs intracellular sorting of this enzyme. Through the study of hybrid proteins, consisting of amino-terminal segments of CPY fused to the secretory enzyme invertase, we have found that the N-terminal 50 amino acids of CPY are sufficient to direct delivery of a CPY-Inv hybrid protein to the yeast vacuole. Our data suggest that this 50 amino acid segment of CPY contains two distinct functional domains; an N-terminal signal peptide followed by a segment of 30 amino acids that contains the vacuolar sorting signal. Deletion of this putative vacuole sorting signal from an otherwise wild-type CPY protein leads to missorting of CPY. Furthermore, examination of the Asn-linked oligosaccharides present on CPY and CPY-Inv hybrid proteins suggests that an additional determinant in CPY specifies the extent to which these proteins are glycosylated in the Golgi complex.

Amino Acid Sequence↗
PubMed1987

The location of sulphydryl groups in alpha-crystallin.

The microenvironments of the sulphydryl groups in the multimeric protein, alpha-crystallin, were studied by examining: the rate of the reaction of the groups with DTNB; the effect of increasing urea concentrations on their accessibilities; and the quenching of a fluorescent probe. In foetal bovine alpha-crystallin (1 SH/alpha A subunit) both kinetic and quenching studies indicated that over 90% of the sulphydryl groups fell into a single buried class; the remainder was exposed. In the human protein (2 SH/alpha A subunit), half of the groups were buried and the other half exposed. Accessible sulphydryl groups increased gradually as the urea concentration was increased, with complete exposure at about 4.0 M. Sedimentation velocity analyses revealed that no significant dissociation of the aggregates into subunits occurred below 3.5 M urea, at which point over 80% of the sulphydryl groups were exposed. An age-dependent increase (3-35%) was found in the proportion of exposed sulphydryl groups in bovine alpha-crystallin and a decrease in the urea concentration required to expose the remainder. It was concluded that the single cysteine is buried in the newly synthesized protein, but becomes solvent-exposed as a result of age-related conformational changes. Our observations are consistent with a quaternary structure in which all alpha A subunits occupy equivalent sites.

Aging↗
PubMed1987

Influence of gamma-chain amino-terminal acetylation on subunit assembly of human fetal hemoglobin.

A human hemoglobin F subunit recombination study was performed to determine the relative efficiency of recombination of amino-terminally acetylated gamma-chains and non-acetylated chains with alpha-chains. The results of this work suggested that the acetylated gamma Ic-chains combined more readily with the alpha-chains than the non-acetylated gamma o-chains. An important factor in the function and assembly of multi-subunit macromolecules is the interaction of the unlike subunits. A model system for the study of such interactions has been the protein hemoglobin. With respect to the hemoglobin molecule, it has been noted that relative affinities of normal and mutant subunits for the unlike subunits can have a significant influence on hemoglobin synthesis at the post-translation level, i.e., subunit assembly [1,2]. A similar mechanism may control the formation of human Hb FIc, a hemoglobin with NH2-terminally acetylated gamma- (gamma Ic)-chains. In this instance acetylation may occur on the ribosomes before subunit assembly. A previous report from our laboratory showed a slight increase in the relative proportion of Hb FIc in cord blood samples with over 5% Hb Bart's [3]. The data were interpreted to be due to an influence of alpha-chain deficiency (alpha-thalassemia) on the formation of Hb FIc and Hb Fo tetramers by a preference of alpha-chains for gamma Ic-chains over gamma o- (non-acetylated gamma)-chains. The present study involves an examination of the relative affinity of the gamma Ic- and gamma o-chains for the normal alpha-chains in an in vitro recombination system.

Acetylation↗
PubMed1990

The amino-acid sequence of multiple lectins of the acorn barnacle Megabalanus rosa and its homology with animal lectins.

The amino-acid sequence of a lectin isolated from the coelomic fluid of the acorn barnacle Megabalanus rosa has been determined. The lectin (Mr 140,000) is a multimeric protein whose subunit consists of 173 amino acids and one carbohydrate chain attached to Asn-39. The amino-acid sequence was determined by the manual sequencing of peptides derived from the protein by digestion with Staphylococcus aureus V8 proteinase, lysine endopeptidase and chymotrypsin, as well as fragments produced by cleavage with cyanogen bromide. The amino-acid sequence of the lectin was compared with the sequence of one (Mr 64,000) of the multiple lectins of M. rosa. They are distinct molecules in spite of a significant homology in their amino-acid sequences. The amino-acid sequence includes some regions homologous to those in other invertebrate lectins, such as sea urchin and flesh fly lectins, and vertebrate lectins. This is the first report to show the amino-acid sequence of multiple lectins isolated from an invertebrate.

Amino Acid Sequence↗