Hepatic lipase: a member of a family of structurally related lipases

Partial amino acid sequence of rat hepatic lipase was obtained by gas-phase microsequence analysis of proteolytic fragments. Sequence comparison to bovine lipoprotein lipase and porcine pancreatic lipase reveals a highly conserved region existing among these three physiologically distinct lipolytic enzymes. In a stretch of 36 amino acid residues previously reported for pancreatic lipase (De Caro, J., Boudouard, M., Bonicel, J., Guidoni, A., Desnuelle, P. and Rovery, M. (1981) Biochim. Biophys. Acta 671, 129-138), nineteen residues are identical for all three enzymes, whereas 27 of 36 are identical in rat hepatic lipase and bovine lipoprotein lipase. The fact that this primary structural conservation extends to three different animal species emphasizes the conclusion that these lipolytic enzymes comprise a protein family originating from a common ancestral gene.     

The yeast ribosome synthesis factor Emg1 is a novel member of the superfamily of alpha/beta knot fold methyltransferases

Emg1 was previously shown to be required for maturation of the 18S rRNA and biogenesis of the 40S ribosomal subunit. Here we report the determination of the crystal structure of Emg1 at 2 Å resolution in complex with the methyl donor, S-adenosyl-methionine (SAM). This structure identifies Emg1 as a novel member of the alpha/beta knot fold methyltransferase (SPOUT) superfamily. In addition to the conserved SPOUT core, Emg1 has two unique domains that form an extended surface, which we predict to be involved in binding of RNA substrates. A point mutation within a basic patch on this surface almost completely abolished RNA binding in vitro. Three point mutations designed to disrupt the interaction of Emg1 with SAM each caused>100-fold reduction in SAM binding in vitro. Expression of only Emg1 with these mutations could support growth and apparently normal ribosome biogenesis in strains genetically depleted of Emg1. We conclude that the catalytic activity of Emg1 is not essential and that the presence of the protein is both necessary and sufficient for ribosome biogenesis.     

Translation termination in eukaryotes: polypeptide release factor eRF1 is composed of functionally and structurally distinct domains

Class-1 polypeptide chain release factors (RFs) trigger hydrolysis of peptidyl-tRNA at the ribosomal peptidyl transferase center mediated by one of the three termination codons. In eukaryotes, apart from catalyzing the translation termination reaction, eRF1 binds to and activates another factor, eRF3, which is a ribosome-dependent and eRF1-dependent GTPase. Because peptidyl-tRNA hydrolysis and GTP hydrolysis could be uncoupled in vitro, we suggest that the two main functions of eRF1 are associated with different domains of the eRF1 protein. We show here by deletion analysis that human eRF1 is composed of two physically separated and functionally distinct domains. The "core" domain is fully competent in ribosome binding and termination-codon-dependent peptidyl-tRNA hydrolysis, and encompasses the N-terminal and middle parts of the polypeptide chain. The C-terminal one-third of eRF1 binds to eRF3 in vivo in the absence of the core domain, but both domains are required to activate eRF3 GTPase in the ribosome. The calculated isoelectric points of the core and C domains are 9.74 and 4.23, respectively. This highly uneven charge distribution between the two domains implies that electrostatic interdomain interaction may affect the eRF1 binding to the ribosome and eRF3, its activity in the termination reaction and activation of eRF3 GTPase. The positively charged core of eRF1 may interact with negatively charged rRNA and peptidyl-tRNA phosphate backbones at the ribosomal eRF1 binding site and exhibit RNA-binding ability. The structural and functional dissimilarity of the core and eRF3-binding domains implies that evolutionarily eRF1 originated as a product of gene fusion.     

Ribosome-associated factor Y adopts a fold resembling a double-stranded RNA binding domain scaffold.

Escherichia coli protein Y (pY) binds to the small ribosomal subunit and stabilizes ribosomes against dissociation when bacteria experience environmental stress. pY inhibits translation in vitro, most probably by interfering with the binding of the aminoacyl-tRNA to the ribosomal A site. Such a translational arrest may mediate overall adaptation of cells to environmental conditions. We have determined the 3D solution structure of a 112-residue pY and have studied its backbone dynamic by NMR spectroscopy. The structure has a betaalphabetabetabetaalpha topology and represents a compact two-layered sandwich of two nearly parallel alpha helices packed against the same side of a four-stranded beta sheet. The 23 C-terminal residues of the protein are disordered. Long-range angular constraints provided by residual dipolar coupling data proved critical for precisely defining the position of helix 1. Our data establish that the C-terminal region of helix 1 and the loop linking this helix with strand beta2 show significant conformational exchange in the ms- micro s time scale, which may have relevance to the interaction of pY with ribosomal subunits. Distribution of the conserved residues on the protein surface highlights a positively charged region towards the C-terminal segments of both alpha helices, which most probably constitutes an RNA binding site. The observed betaalphabetabetabetaalpha topology of pY resembles the alphabetabetabetaalpha topology of double-stranded RNA-binding domains, despite limited sequence similarity. It appears probable that functional properties of pY are not identical to those of dsRBDs, as the postulated RNA-binding site in pY does not coincide with the RNA-binding surface of the dsRBDs.     

The laminin-binding domain of agrin is structurally related to N-TIMP-1

Agrin is the key organizer of postsynaptic differentiation at the neuromuscular junction. This organization activity requires the binding of agrin to the synaptic basal lamina. Binding is conferred by the N-terminal agrin (NtA) domain, which mediates a high-affinity interaction with the coiled coil domain of laminins. Here, we report the crystal structure of chicken NtA at 1.6 A resolution. The structure reveals that NtA harbors an oligosaccharide/oligonucleotide-binding fold with several possible sites for the interaction with different ligands. A high structural similarity of NtA with the protease inhibition domain in tissue inhibitor of metalloproteinases-1 (TIMP-1) supports the idea of additional functions of agrin besides synaptogenic activity.     

Ig alpha and Ig beta are functionally homologous to the signaling proteins of the T-cell receptor.

Signal transduction by antigen receptors and some Fc receptors requires the activation of a family of receptor-associated transmembrane accessory proteins. One common feature of the cytoplasmic domains of these accessory molecules is the presence is at least two YXXA repeats that are potential sites for interaction with Src homology 2 domain-containing proteins. However, the degree of similarity between the different receptor-associated proteins varies from that of T-cell receptor (TCR) zeta and Fc receptor RIIIA gamma chains, which are homologous, to the distantly related Ig alpha and Ig beta proteins of the B-cell antigen receptor. To determine whether T- and B-cell antigen receptors are in fact functionally homologous, we have studied signal transduction by chimeric immunoglobulins bearing the Ig alpha or Ig beta cytoplasmic domain. We found that Ig alpha and Ig beta cytoplasmic domains were able to activate Ca2+ flux, interleukin-2 secretion, and phosphorylation of the same group of cellular substrates as the TCR in transfected T cells. Chimeric proteins were then used to examine the minimal requirements for activation of the Fyn, Lck, and ZAP kinases in T cells. Both Ig alpha and Ig beta were able to trigger Fyn, Lck, and ZAP directly without involvement of TCR components. Cytoplasmic tyrosine residues in Ig beta were required for recruitment and activation of ZAP-70, but these amino acids were not essential for the activation of Fyn and Lck. We conclude that Fyn and Lck are able to recognize a clustered nonphosphorylated immune recognition receptor, but activation of these kinases is not sufficient to induce cellular responses such as Ca2+ flux and interleukin-2 secretion. In addition, the molecular structures involved in antigen receptor signaling pathways are conserved between T and B cells.     

The gene for a novel member of the whey acidic protein family encodes three four-disulfide core domains and is asynchronously expressed during lactation

Secretion of whey acidic protein (WAP) in milk throughout lactation has previously been reported for a limited number of species, including the mouse, rat, rabbit, camel, and pig. We report here the isolation of WAP from the milk of a marsupial, the tammar wallaby (Macropus eugenii). Tammar WAP (tWAP) was isolated by reverse-phase HPLC and migrates in SDS-polyacrylamide gel electrophoresis at 29.9 kDa. tWAP is the major whey protein, but in contrast to eutherians, secretion is asynchronous and occurs only from approximately days 130 through 240 of lactation. The full-length cDNA codes for a mature protein of 191 amino acids, which is comprised of three four-disulfide core domains, contrasting with the two four-disulfide core domain arrangement in all other known WAPs. A three-dimensional model for tWAP has been constructed and suggests that the three domains have little interaction and could function independently. Analysis of the amino acid sequence suggests the protein belongs to a family of protease inhibitors; however, the predicted active site of these domains is dissimilar to the confirmed active site for known protease inhibitors. This suggests that any putative protease ligand may be unique to either the mammary gland, milk, or gut of the pouch young. Examination of the endocrine regulation of the tWAP gene showed consistently that the gene is prolactin-responsive but that the endocrine requirements for induction and maintenance of tWAP gene expression are different during lactation.     

Cartilage-inducing factor-B is a unique protein structurally and functionally related to transforming growth factor-beta

Cartilage-inducing factors-A (CIF-A) and -B (CIF-B), purified from bovine bone on the basis of their ability to induce the cartilage phenotype in vitro, are proteins with molecular weights of 26,000 composed of two apparently identical disulfide-linked chains. CIF-A is apparently identical to TGF-beta from human platelets (Seyedin S. M., Thompson, A. Y., Bentz, H., Rosen, D. M., McPherson, J. M., Conti, A., Siegel, N. R., Galluppi, G. R., and Piez, K. A. (1986) J. Biol. Chem. 261, 5693-5695). We have now found that, like CIF-A and TGF-beta, CIF-B induces anchorage-independent proliferation of NRK-49F cells when these cells are simultaneously treated with epidermal growth factor. Furthermore, CIF-B competes with CIF-A for the same cell membrane receptors in NRK-49F cells. Partial amino acid sequencing reveals that CIF-B is a distinct molecule with extensive homology to CIF-A/TGF-beta. These results show that CIF-B and TGF-beta are structurally and functionally similar molecules, but differ more from each other than does TGF-beta from different species.     

The actin binding protein, fesselin, is a member of the synaptopodin family

Fesselin is a natively unfolded protein that is abundant in avian smooth muscle. Like many natively unfolded proteins, fesselin has multiple binding partners including actin, myosin, calmodulin and α-actinin. Fesselin accelerates actin polymerization and bundles actin. These and other observations suggest that fesselin is a component of the cytoskeleton. We have now cloned fesselin and have determined the cDNA derived amino acid sequence. We verified parts of the sequence by Edman analysis and by mass spectroscopy. Our results confirmed fesselin is homologous to human synaptopodin 2 and belongs to the synaptopodin family of proteins.     

Binding of a sequence-specific single-stranded DNA-binding factor to the simian virus 40 core origin inverted repeat domain is cell cycle regulated.

The inverted repeat domain (IR domain) within the simian virus 40 origin of replication is the site of initial DNA melting prior to the onset of DNA synthesis. The domain had previously been shown to be bound by a cellular factor in response to DNA damage. We demonstrate that two distinct cellular components bind opposite strands of the IR domain. Replication protein A (RPA), previously identified as a single-stranded DNA binding protein required for origin-specific DNA replication in vitro, is shown to have a preference for the pyrimidine-rich strand. A newly described component, IR factor B (IRF-B), specifically recognizes the opposite strand. IRF-B binding activity in nuclear extract varies significantly with cell proliferation and the cell cycle, so that binding of IRF-B to the IR domain is negatively correlated with the onset of DNA synthesis. Loss of IRF-B binding from the nucleus also occurs in response to cellular DNA damage. UV cross-linking indicates that the core binding component of IRF-B is a protein of ca. 34 kDa. We propose that RPA and IRF-B bind opposite strands of the IR domain and together may function in the regulation of origin activation.     

Identification and cloning of a novel IL-15 binding protein that is structurally related to the alpha chain of the IL-2 receptor.

Interleukin-15 (IL-15) is a novel cytokine of the four-helix bundle family which shares many biological activities with IL-2, probably due to its interaction with the IL-2 receptor beta and gamma (IL-2R beta and gamma c) chains. We report here the characterization and molecular cloning of a distinct murine IL-15R alpha chain. IL-15R alpha alone displays an affinity of binding for IL-15 equivalent to that of the heterotrimeric IL-2R for IL-2. A biologically functional heteromeric IL-15 receptor complex capable of mediating IL-15 responses was generated through reconstruction experiments in a murine myeloid cell line. IL-15R alpha is structurally similar to IL-2R alpha; together they define a new cytokine receptor family. The distribution of IL-15 and IL-15R alpha mRNA suggests that IL-15 may have biological activities distinct from IL-2.