The major human and mouse granzymes are structurally and functionally divergent

Approximately 2% of mammalian genes encode proteases. Comparative genomics reveals that those involved in immunity and reproduction show the most interspecies diversity and evidence of positive selection during evolution. This is particularly true of granzymes, the cytotoxic proteases of natural killer cells and CD8+ T cells. There are 5 granzyme genes in humans and 10 in mice, and it is suggested that granzymes evolve to meet species-specific immune challenge through gene duplication and more subtle alterations to substrate specificity. We show that mouse and human granzyme B have distinct structural and functional characteristics. Specifically, mouse granzyme B is 30 times less cytotoxic than human granzyme B and does not require Bid for killing but regains cytotoxicity on engineering of its active site cleft. We also show that mouse granzyme A is considerably more cytotoxic than human granzyme A. These results demonstrate that even "orthologous" granzymes have species-specific functions, having evolved in distinct environments that pose different challenges.     

TOR1 and TOR2 are structurally and functionally similar but not identical phosphatidylinositol kinase homologues in yeast.

The Saccharomyces cerevisiae genes TOR1 and TOR2 were originally identified by mutations that confer resistance to the immunosuppressant rapamycin. TOR2 was previously shown to encode an essential 282-kDa phosphatidylinositol kinase (PI kinase) homologue. The TOR1 gene product is also a large (281 kDa) PI kinase homologue, with 67% identity to TOR2. TOR1 is not essential, but a TOR1 TOR2 double disruption uniquely confers a cell cycle (G1) arrest as does exposure to rapamycin; disruption of TOR2 alone is lethal but does not cause a cell cycle arrest. TOR1-TOR2 and TOR2-TOR1 hybrids indicate that carboxy-terminal domains of TOR1 and TOR2 containing a lipid kinase sequence motif are interchangeable and therefore functionally equivalent; the other portions of TOR1 and TOR2 are not interchangeable. The TOR1-1 and TOR2-1 mutations, which confer rapamycin resistance, alter the same potential protein kinase C site in the respective protein's lipid kinase domain. Thus, TOR1 and TOR2 are likely similar but not identical, rapamycin-sensitive PI kinases possibly regulated by phosphorylation. TOR1 and TOR2 may be components of a novel signal transduction pathway controlling progression through G1.     

Brain and egg tubulins from antarctic fishes are functionally and structurally distinct.

The multitubulin hypothesis proposes that chemically distinct tubulins may possess different polymerization properties or may form functionally different microtubules. To test this hypothesis, we have examined the functional properties and the structures of singlet-specific nonneural and neural tubulins from Antarctic fishes. Tubulins were purified from eggs of Notothenia coriiceps neglecta, and from brain tissues of N. coriiceps neglecta or N. gibberifrons, by DEAE ion-exchange chromatography and cycles of microtubule assembly/disassembly. At temperatures between 0 and 20 degrees C, each of these tubulins polymerized efficiently in vitro to yield microtubules of normal morphology. Critical concentrations for polymerization of egg tubulin ranged from 0.057 mg/ml at 3 degrees C to 0.002 mg/ml at 18 degrees C, whereas those for brain tubulin at like temperatures were 4-10-fold larger. Polymerization of both tubulins was entropically driven, but the apparent standard enthalpy and entropy changes for microtubule elongation by egg tubulin (delta Happ0 = +33.9 kcal/mol, delta Sapp0 = +151 entropy units) were significantly greater than values observed for brain tubulin (delta Happ0 = +26.5 kcal/mol, delta Sapp0 = +121 entropy units). Egg tubulin was composed of approximately six alpha and two beta chains and lacked the beta III isotype, whereas brain tubulin was more complex (greater than or equal to 10 of each chain type). Furthermore, egg alpha tubulins were more basic, and their carboxyl termini more resistant to cleavage by subtilisin, than were the alpha chains of brain. We conclude that brain and egg tubulins from the Antarctic fishes are functionally distinct in vitro, due either to qualitative or quantitative differences in isotypic composition, to differential posttranslational modification of shared isotypes, or to both.     

Smooth muscle and brain inositol 1,4,5-trisphosphate receptors are structurally and functionally similar

Inositol 1,4,5-trisphosphate (InsP3) mediates smooth muscle contraction by mobilizing intracellular calcium release. In this study we provide a direct comparison of the smooth muscle and brain InsP3 receptors in terms of InsP3 binding and primary structure. The KD for InsP3 binding for both receptors was found to be essentially the same. Sequences from 11 bovine smooth muscle receptor tryptic peptides (120 amino acids) were identified in the mouse brain receptor with two substitutions attributable to species differences. A cDNA (approximately 1-kilobase) encoding a portion of the mouse smooth muscle InsP3 receptor was cloned and found to be identical to that reported for the brain receptor. This cDNA was used as a probe to demonstrate that the approximately 10-kilobase InsP3 receptor mRNA is detected in brain, smooth muscle, heart, liver, and kidney but was not detected in skeletal muscle or skin.     

Dissociation of human alphaB-crystallin aggregates by thiocyanate is structurally and functionally reversible

Conformational modifications and changes in the aggregation state of human B-crystallin were investigated at different concentrations of SDS, KBr, urea, and NH₄SCN and at different temperatures. Intrinsic fluorescence measurements indicated complete and reversible unfolding of the protein at 2 M NH₄SCN, whereas the concentration of urea required for complete and irreversible unfolding was 6 M. Gel permeation chromatography indicated almost complete dissociation of the micelle-like aggregate of B-crystallin in 2 M NH₄SCN, but only partial dissociation into large-sized aggregates in 6 M urea. Thiocyanate-treated B-crystallin recovered its chaperone-like activity upon dilution of the dissociating agent, whereas the urea-treated protein did not.     

Major components of a sea urchin block to polyspermy are structurally and functionally conserved

One sperm fusing with one egg is requisite for successful fertilization; additional sperm fusions are lethal to the embryo. Because sperm usually outnumber eggs, evolution has selected for mechanisms that prevent this polyspermy by immediately modifying the egg extracellular matrix. We focus here on the contribution of cortical granule contents in the sea urchin block to polyspermy to begin to understand how well this process is conserved. We identified each of the major constituents of the fertilization envelope in two species of seaurchins, Strongylocentrotus purpuratus and Lytechinus variegatus, that diverged 30 to 50 million years ago. Our results show that the five major structural components of the fertilization envelope, derived from the egg cortical granules, are semiconserved. Most of these orthologs share sequence identity and encode multiple low-density lipoprotein receptor type A repeats or CUB domains but at least two contain radically different carboxy-terminal repeats. Using a new association assay, we also show that these major structural components are functionally conserved during fertilization envelope construction. Thus, it seems that this population of female reproductive proteins has retained functional motifs while gaining significant sequence diversity-two opposing paths that may reflect cooperativity among the proteins that compose the fertilization envelope.     

Staufen- and FMRP-containing neuronal RNPs are structurally and functionally related to somatic P bodies

Local control of mRNA translation modulates neuronal development, synaptic plasticity, and memory formation. A poorly understood aspect of this control is the role and composition of ribonucleoprotein (RNP) particles that mediate transport and translation of neuronal RNAs. Here, we show that staufen- and FMRP-containing RNPs in Drosophila neurons contain proteins also present in somatic "P bodies," including the RNA-degradative enzymes Dcp1p and Xrn1p/Pacman and crucial components of miRNA (argonaute), NMD (Upf1p), and general translational repression (Dhh1p/Me31B) pathways. Drosophila Me31B is shown to participate (1) with an FMRP-associated, P body protein (Scd6p/trailer hitch) in FMRP-driven, argonaute-dependent translational repression in developing eye imaginal discs; (2) in dendritic elaboration of larval sensory neurons; and (3) in bantam miRNA-mediated translational repression in wing imaginal discs. These results argue for a conserved mechanism of translational control critical to neuronal function and open up new experimental avenues for understanding the regulation of mRNA function within neurons.     

Vitronectin exists in two structurally and functionally distinct forms in human plasma

Vitronectin (serum spreading factor, S-protein or epibolin) is a plasma glycoprotein implicated in cell adhesion, as well as in the regulation of complement-mediated cytolysis and antithrombin III function. Vitronectin was found to exist in fresh human plasma as a heterogeneous mixture consisting of 2% heparin-binding form and the remainder as a non-binding species. Heparin-binding vitronectin consisted of 6.5 S aggregates with a Stokes radius of 5.6 nm, which was enriched in the 65 kDa polypeptide, with a high content of molecules and a putative unfolded conformation. In contrast, non-heparin-binding vitronectin was a 4.2 S monomer with a Stokes radius of 3.9 nm, which appeared to be in a folded conformation with an immunologically cryptic site. Both vitronectins displayed similar activities in mediating the spreading of BHK fibroblastic cells on substrates. During blood coagulation, 5% more of the non-heparin-binding vitronectin was converted into the heparin-binding form, producing a greater than 3.5-fold increase in this species. Our results indicate that vitronectin normally exists in circulating blood in at least two structurally and functionally distinct forms which may serve different functions.     

Redox sensitive cysteine residues in calbindin D28k are structurally and functionally important

Human calbindin D(28k) is a Ca(2+) binding protein that has been implicated in the protection of cells against apoptosis. In this study, the structural and functional significance of the five cysteine residues present in this protein have been investigated through a series of cysteine-to-serine mutations. The mutants were studied under relevant physiological redox potentials in which conformational changes were monitored using ANS binding. Urea-induced denaturations, as monitored by intrinsic tryptophan fluorescence, were also carried out to compare their relative stability. It was shown that the two N-terminal cysteine residues undergo a redox-driven structural change consistent with disulfide bond formation. The other cysteine residues are not by themselves sufficient at inducing structural change, but they accentuate the disulfide-dependent conformational change in a redox-dependent manner. Mass spectrometry data show that the three C-terminal cysteine residues can be modified by glutathione. Furthermore, under oxidizing conditions, the data display additional species consistent with the conversion of cysteine thiols to sulfenic acids and disulfides to disulfide-S-monoxides. The biological function of calbindin D(28k) appears to be tied to the redox state of the cysteine residues. The two N-terminal cysteine residues are required for activation of myo-inositol monophosphatase, and enzyme activation is enhanced under conditions in which these residues are oxidized. Last, oxidized calbindin D(28k) binds Ca(2+) with lower affinity than does the reduced protein.     

A human glioma cell line secretes three structurally and functionally different dimeric forms of platelet-derived growth factor

A human malignant glioma cell line, U-343 MGa Cl2:6, has previously been shown to secrete platelet-derived-growth-factor(PDGF)-like activity [Nistér, M., Heldin, C.-H., Wasteson, A. and Westermark, B. (1984) Proc. Natl Acad. Sci. USA 81, 926-930]. We report here that this activity consists of three different molecules separable by reversed-phase chromatography and immobilized-metal-ion affinity chromatography. HPLC reversed-phase chromatography resolved two peaks of activity, which were denoted glioma-derived growth factor-I (GDGF-I) and GDGF-II. GDGF-I was purified to greater than 90% purity; in SDS gel electrophoresis, it appeared as a 31-kDa component which by reduction was converted to 17 kDa. Immunoprecipitation of radiolabeled, reduced and alkylated GDGF-I with antisera made against peptides from the A and B chains of PDGF, gave a specific signal only with antiserum against the A chain. Furthermore, when reduced and alkylated GDGF-I was analyzed by reversed-phase HPLC, it eluted at the position of PDGF A chains. We conclude that GDGF-I is a homodimer of a polypeptide similar to the A chain of PGDF. GDGF-II was found to have higher mitogenic activity than GDGF-I. Analysis by immunoprecipitation with PDGF-chain-specific antisera revealed that GDGF-II contained a polypeptide similar to the B chain of PDGF. Immobilized-metal-ion affinity chromatography revealed that 95% of the mitogenic activity of GDGF-II consisted of a heterodimer of one A and one B chain, whereas 5% consisted of a B-chain homodimer. Thus, U-343 MGa Cl 2:6 cells secrete all three possible dimeric forms of PDGF.     

Dog and human acid beta-D-galactosidases are structurally similar.

The purification of dog liver acid beta-galactosidase is described. The dog enzyme migrated as a single major band on polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate, with a molecular weight of 60000. Antiserum raised against purified human liver acid beta-galactosidase cross-reacted with beta-galactosidase from dog liver, but not with those from cat liver or Escherichia coli. Tryptic peptide maps of the dog and human acid beta-galactosidases indicate that 21 of the 24 peptides observed were homologous; a similar result was obtained after chymotryptic peptide mapping. We conclude that dog and human acid beta-galactosidases are structurally similar, and that canine GM1 gangliosidosis (acid beta-galactosidase deficiency) is an excellent model for the same disease in man.     

Specialized 'dauciform' roots of Cyperaceae are structurally distinct, but functionally analogous with 'cluster' roots

When grown in nutrient solutions of extremely low [P] (相似文献   

Malignant hyperthermia mutation sites in the Leu2442-Pro2477 (DP4) region of RyR1 (ryanodine receptor 1) are clustered in a structurally and functionally definable area

To explain the mechanism of pathogenesis of channel disorder in MH (malignant hyperthermia), we have proposed a model in which tight interactions between the N-terminal and central domains of RyR1 (ryanodine receptor 1) stabilize the closed state of the channel, but mutation in these domains weakens the interdomain interaction and destabilizes the channel. DP4 (domain peptide 4), a peptide corresponding to residues Leu2442-Pro2477 of the central domain, also weakens the domain interaction and produces MH-like channel destabilization, whereas an MH mutation (R2458C) in DP4 abolishes these effects. Thus DP4 and its mutants serve as excellent tools for structure-function studies. Other MH mutations have been reported in the literature involving three other amino acid residues in the DP4 region (Arg2452, Ile2453 and Arg2454). In the present paper we investigated the activity of several mutants of DP4 at these three residues. The ability to activate ryanodine binding or to effect Ca2+ release was severely diminished for each of the MH mutants. Other substitutions were less effective. Structural studies, using NMR analysis, revealed that the peptide has two a-helical regions. It is apparent that the MH mutations are clustered at the C-terminal end of the first helix. The data in the present paper indicates that mutation of residues in this region disrupts the interdomain interactions that stabilize the closed state of the channel.     

Hypomorphic variants of lactase-phlorizin hydrolase in congenital lactase deficiency are trafficking incompetent and functionally inactive

Patients with the rare autosomal recessive disorder congenital lactase deficiency (CLD) present with severe, potentially life-threatening symptoms shortly after birth.Several variants have been characterized within the gene for lactase-phlorizin hydrolase (LCT) that are associated with CLD. Here, we analyze at the biochemical and cellular levels LCT mutants harboring the genetic variants p.Y1390*, p.E1612*, p.S1150Pfs*19, p.S1121L, p.R1587H, and p.S688P.Our data unequivocally demonstrate that these mutants are absolutely transport incompetent, some of which are readily degraded, and are enzymatically inactive. The current study contributes to and expands our understanding on the pathogenesis of CLD at the molecular level.     

The amino terminal sequences of bovine and human chromogranin A and secretory protein I are identical

The amino terminal sequences of bovine and human adrenal medullary chromogranin A have been determined. Their sequences are identical and also identical to the published sequence of secretory protein I from the parathyroid gland. This data indicates that the previously published sequence of chromogranin A is incorrect at residues 2 and 19. These data confirm earlier observations of a substantial similarity between secretory protein I and chromogranin A and, in fact, strongly suggest that they are identical.