Biosynthetic control of molecular weight in the polymerization of the octasaccharide subunits of succinoglycan, a symbiotically important exopolysaccharide of Rhizobium meliloti

Succinoglycan, a symbiotically important exopolysaccharide of Rhizobium meliloti, is composed of polymerized octasaccharide subunits, each of which consists of one galactose and seven glucoses with succinyl, acetyl, and pyruvyl modifications. Production of specific low molecular weight forms of R. meliloti exported and surface polysaccharides, including succinoglycan, appears to be important for nodule invasion. In a previous study of the roles of the various exo gene products in succinoglycan biosynthesis, exoP, exoQ, and exoT mutants were found to synthesize undecaprenol-linked fully modified succinoglycan octasaccharide subunits, suggesting possible roles for their gene products in polymerization or transport. Using improved techniques for analyzing succinoglycan biosynthesis by these mutants, we have obtained evidence indicating that R. meliloti has genetically separable systems for the synthesis of high molecular weight succinoglycan and the synthesis of a specific class of low molecular weight oligosaccharides consisting of dimers and trimers of the octasaccharide subunit. Models to account for our unexpected findings are discussed. Possible roles for the ExoP, ExoQ, and ExoT proteins are compared and contrasted with roles that have been suggested on the basis of homologies to key proteins involved in the biosynthesis of O-antigens and of certain exported or capsular cell surface polysaccharides.

Meis1 and pKnox1 bind DNA cooperatively with Pbx1 utilizing an interaction surface disrupted in oncoprotein E2a-Pbx1

E2a-Pbx1 is a chimeric transcription factor oncoprotein produced by the t(1;19) translocation in human pre-B cell leukemia. Class I Hox proteins bind DNA cooperatively with both Pbx proteins and oncoprotein E2a-Pbx1, suggesting that leukemogenesis by E2a-Pbx1 and Hox proteins may alter transcription of cellular genes regulated by Pbx–Hox motifs. Likewise, in murine myeloid leukemia, transcriptional coactivation of Meis1 with HoxA7/A9 suggests that Meis1–HoxA7/9 heterodimers may evoke aberrant gene transcription. Here, we demonstrate that both Meis1 and its relative, pKnox1, dimerize with Pbx1 on the same TGATTGAC motif selected by dimers of Pbx proteins and unidentified partner(s) in nuclear extracts, including those from t(1;19) pre-B cells. Outside their homeodomains, Meis1 and pKnox1 were highly conserved only in two motifs required for cooperativity with Pbx1. Like the unidentified endogenous partner(s), both Meis1 and pKnox1 failed to dimerize significantly with E2a-Pbx1. The Meis1/pKnox1-interaction domain in Pbx1 resided predominantly in a conserved N-terminal Pbx domain deleted in E2a-Pbx1. Thus, the leukemic potential of E2a-Pbx1 may require abrogation of its interaction with members of the Meis and pKnox families of transcription factors, permitting selective targeting of genes regulated by Pbx–Hox complexes. In addition, because most motifs bound by Pbx–Meis1/pKnox1 were not bound by Pbx1–Hox complexes, the leukemic potential of Meis1 in myeloid leukemias may involve shifting Pbx proteins from promoters containing Pbx–Hox motifs to those containing Pbx–Meis motifs.

Enhancement of DNA repair in human skin cells by thymidine dinucleotides: Evidence for a p53-mediated mammalian SOS response

Thymidine dinucleotide (pTpT) stimulates melanogenesis in mammalian pigment cells and intact skin, mimicking the effects of UV irradiation and UV-mimetic DNA damage. Here it is shown that, in addition to tanning, pTpT induces a second photoprotective response, enhanced repair of UV-induced DNA damage. This enhanced repair results in a 2-fold increase in expression of a UV-damaged chloramphenicol acetyltransferase expression vector transfected into pTpT-treated skin fibroblasts and keratinocytes, compared with diluent-treated cells. Direct measurement of thymine dimers and (6–4) photoproducts by immunoassay demonstrates faster repair of both of these UV-induced photoproducts in pTpT-treated fibroblasts. This enhanced repair capacity also improves cell survival and colony-forming ability after irradiation. These effects of pTpT are accomplished, at least in part, by the up-regulation of a set of genes involved in DNA repair (ERCC3 and GADD45) and cell cycle inhibition (SDI1). At least two of these genes (GADD45 and SDI1) are known to be transcriptionally regulated by the p53 tumor suppressor protein. Here we show that pTpT activates p53, leading to nuclear accumulation of this protein, and also increases the specific binding of this transcription factor to its DNA consensus sequence.

Structural and Functional Analysis of a Novel Coiled-Coil Protein Involved in Ypt6 GTPase-regulated Protein Transport in Yeast

The yeast transport GTPase Ypt6p is dispensable for cell growth and secretion, but its lack results in temperature sensitivity and missorting of vacuolar carboxypeptidase Y. We previously identified four yeast genes (SYS1, 2, 3, and 5) that on high expression suppressed these phenotypic alterations. SYS3 encodes a 105-kDa protein with a predicted high α-helical content. It is related to a variety of mammalian Golgi-associated proteins and to the yeast Uso1p, an essential protein involved in docking of endoplasmic reticulum–derived vesicles to the cis-Golgi. Like Uso1p, Sys3p is predominatly cytosolic. According to gel chromatographic, two-hybrid, and chemical cross-linking analyses, Sys3p forms dimers and larger protein complexes. Its loss of function results in partial missorting of carboxypeptidase Y. Double disruptions of SYS3 and YPT6 lead to a significant growth inhibition of the mutant cells, to a massive accumulation of 40- to 50-nm vesicles, to an aggravation of vacuolar protein missorting, and to a defect in α-pheromone processing apparently attributable to a perturbation of protease Kex2p cycling between the Golgi and a post-Golgi compartment. The results of this study suggest that Sys3p, like Ypt6p, acts in vesicular transport (presumably at a vesicle-docking stage) between an endosomal compartment and the most distal Golgi compartment.

Assembly of a Novel Cartilage Matrix Protein Filamentous Network: Molecular Basis of Differential Requirement of von Willebrand Factor A Domains

Cartilage matrix protein (CMP) is the prototype of the newly discovered matrilin family, all of which contain von Willebrand factor A domains. Although the function of matrilins remain unclear, we have shown that, in primary chondrocyte cultures, CMP (matrilin-1) forms a filamentous network, which is made up of two types of filaments, a collagen-dependent one and a collagen-independent one. In this study, we demonstrate that the collagen-independent CMP filaments are enriched in pericellular compartments, extending directly from chondrocyte membranes. Their morphology can be distinguished from that of collagen filaments by immunogold electron microscopy, and mimicked by that of self-assembled purified CMP. The assembly of CMP filaments can occur from transfection of a wild-type CMP transgene alone in skin fibroblasts, which do not produce endogenous CMP. Conversely, assembly of endogenous CMP filaments by chondrocytes can be inhibited specifically by dominant negative CMP transgenes. The two A domains within CMP serve essential but different functions during network formation. Deletion of the A2 domain converts the trimeric CMP into a mixture of monomers, dimers, and trimers, whereas deletion of the A1 domain does not affect the trimeric configuration. This suggests that the A2 domain modulates multimerization of CMP. Absence of either A domain from CMP abolishes its ability to form collagen-independent filaments. In particular, Asp22 in A1 and Asp255 in A2 are essential; double point mutation of these residues disrupts CMP network formation. These residues are part of the metal ion–dependent adhesion sites, thus a metal ion–dependent adhesion site–mediated adhesion mechanism may be applicable to matrilin assembly. Taken together, our data suggest that CMP is a bridging molecule that connects matrix components in cartilage to form an integrated matrix network.

Enzyme plus light therapy to repair DNA damage in ultraviolet-B-irradiated human skin

Ultraviolet-B (UVB) (290–320 nm) radiation-induced cyclobutane pyrimidine dimers within the DNA of epidermal cells are detrimental to human health by causing mutations and immunosuppressive effects that presumably contribute to photocarcinogenesis. Conventional photoprotection by sunscreens is exclusively prophylactic in nature and of no value once DNA damage has occurred. In this paper, we have therefore assessed whether it is possible to repair UVB radiation-induced DNA damage through topical application of the DNA-repair enzyme photolyase, derived from Anacystis nidulans, that specifically converts cyclobutane dimers into their original DNA structure after exposure to photoreactivating light. When a dose of UVB radiation sufficient to induce erythema was administered to the skin of healthy subjects, significant numbers of dimers were formed within epidermal cells. Topical application of photolyase-containing liposomes to UVB-irradiated skin and subsequent exposure to photoreactivating light decreased the number of UVB radiation-induced dimers by 40–45%. No reduction was observed if the liposomes were not filled with photolyase or if photoreactivating exposure preceded the application of filled liposomes. The UVB dose administered resulted in suppression of intercellular adhesion molecule-1 (ICAM-1), a molecule required for immunity and inflammatory events in the epidermis. In addition, in subjects hypersensitive to nickel sulfate, elicitation of the hypersensitivity reaction in irradiated skin areas was prevented. Photolyase-induced dimer repair completely prevented these UVB radiation-induced immunosuppressive effects as well as erythema and sunburn-cell formation. These studies demonstrate that topical application of photolyase is effective in dimer reversal and thereby leads to immunoprotection.

tRNAVal-heterodimeric maxizymes with high potential as geneinactivating agents: Simultaneous cleavage at two sites in HIV-1 tat mRNA in cultured cells

It has been demonstrated that shortened forms of (stem II-deleted) hammerhead ribozymes with low intrinsic activity form very active dimers with a common stem II (very active short ribozymes capable of forming dimers were designated maxizymes). Intracellular activities of heterodimeric maxizymes and conventional ribozymes, under the control of a human tRNAVal-promoter, were compared against the cleavage of HIV-1 tat mRNA. The pol III-driven maxizymes formed very active heterodimers, and they successfully cleaved HIV-1 tat mRNA in mammalian cells at two sites simultaneously. The cleaved fragments were identified directly by Northern blotting analysis. Despite the initial concerns that a complicated dimerization process and formation of inactive homodimers were involved in addition to the process of association with the target, the overall intracellular activities of tRNAVal-driven maxizymes were significantly higher in mammalian cells than those of two sets of independent, conventional hammerhead ribozymes that were targeted at the same two sites within HIV-1 tat mRNA. Because the tRNAVal-driven maxizymes tested to date have been more effective than tRNAVal-driven “standard” hammerhead ribozymes, the tRNAVal-driven heterodimeric maxizymes appear to have potential utility as gene-inactivating agents.

Fibroblast growth factors 1 and 2 are distinct in oligomerization in the presence of heparin-like glycosaminoglycans

Fibroblast growth factor (FGF) 1 and FGF-2 are prototypic members of the FGF family, which to date comprises at least 18 members. Surprisingly, even though FGF-1 and FGF-2 share more than 80% sequence similarity and an identical structural fold, these two growth factors are biologically very different. FGF-1 and FGF-2 differ in their ability to bind isoforms of the FGF receptor family as well as the heparin-like glycosaminoglycan (HLGAG) component of proteoglycans on the cell surface to initiate signaling in different cell types. Herein, we provide evidence for one mechanism by which these two proteins could differ biologically. Previously, it has been noted that FGF-1 and FGF-2 can oligomerize in the presence of HLGAGs. Therefore, we investigated whether FGF-1 and FGF-2 oligomerize by the same mechanism or by a different one. Through a combination of matrix-assisted laser desorption ionization mass spectrometry and chemical crosslinking, we show here that, under identical conditions, FGF-1 and FGF-2 differ in the degree and kind of oligomerization. Furthermore, an extensive analysis of FGF-1 and FGF-2 uncomplexed and HLGAG complexed crystal structures enables us to readily explain why FGF-2 forms sequential oligomers whereas FGF-1 forms only dimers. FGF-2, which possesses an interface capable of protein association, forms a translationally related oligomer, whereas FGF-1, which does not have this interface, forms only a symmetrically related dimer. Taken together, these data show that FGF-1 and FGF-2, despite their sequence homology, differ in their mechanism of oligomerization.

Native display of complete foreign protein domains on the surface of hepatitis B virus capsids

The nucleocapsid of hepatitis B virus (HBV), or HBcAg, is a highly symmetric structure formed by multiple dimers of a single core protein that contains potent T helper epitopes in its 183-aa sequence. Both factors make HBcAg an unusually strong immunogen and an attractive candidate as a carrier for foreign epitopes. The immunodominant c/e1 epitope on the capsid has been suggested as a superior location to convey high immunogenicity to a heterologous sequence. Because of its central position, however, any c/e1 insert disrupts the core protein’s primary sequence; hence, only peptides, or rather small protein fragments seemed to be compatible with particle formation. According to recent structural data, the epitope is located at the tips of prominent surface spikes formed by the very stable dimer interfaces. We therefore reasoned that much larger inserts might be tolerated, provided the individual parts of a corresponding fusion protein could fold independently. Using the green fluorescent protein (GFP) as a model insert, we show that the chimeric protein efficiently forms fluorescent particles; hence, all of its structurally important parts must be properly folded. We also demonstrate that the GFP domains are surface-exposed and that the chimeric particles elicit a potent humoral response against native GFP. Hence, proteins of at least up to 238 aa can be natively displayed on the surface of HBV core particles. Such chimeras may not only be useful as vaccines but may also open the way for high resolution structural analyses of nonassembling proteins by electron microscopy.

Gβ5 prevents the RGS7-Gαo interaction through binding to a distinct Gγ-like domain found in RGS7 and other RGS proteins

The G protein β subunit Gβ5 deviates significantly from the other four members of Gβ-subunit family in amino acid sequence and subcellular localization. To detect the protein targets of Gβ5 in vivo, we have isolated a native Gβ5 protein complex from the retinal cytosolic fraction and identified the protein tightly associated with Gβ5 as the regulator of G protein signaling (RGS) protein, RGS7. Here we show that complexes of Gβ5 with RGS proteins can be formed in vitro from the recombinant proteins. The reconstituted Gβ5-RGS dimers are similar to the native retinal complex in their behavior on gel-filtration and cation-exchange chromatographies and can be immunoprecipitated with either anti-Gβ5 or anti-RGS7 antibodies. The specific Gβ5-RGS7 interaction is determined by a distinct domain in RGS that has a striking homology to Gγ subunits. Deletion of this domain prevents the RGS7-Gβ5 binding, although the interaction with Gα is retained. Substitution of the Gγ-like domain of RGS7 with a portion of Gγ1 changes its binding specificity from Gβ5 to Gβ1. The interaction of Gβ5 with RGS7 blocked the binding of RGS7 to the Gα subunit Gαo, indicating that Gβ5 is a specific RGS inhibitor.

Free energy of burying hydrophobic residues in the interface between protein subunits

We have obtained an experimental estimate of the free energy change associated with variations at the interface between protein subunits, a subject that has raised considerable interest since the concept of accessible surface area was introduced by Lee and Richards [Lee, B. & Richards, F. M. (1971) J. Mol. Biol. 55, 379–400]. We determined by analytical ultracentrifugation the dimer–tetramer equilibrium constant of five single and three double mutants of human Hb. One mutation is at the stationary α1β1 interface, and all of the others are at the sliding α1β2 interface where cleavage of the tetramer into dimers and ligand-linked allosteric changes are known to occur. A surprisingly good linear correlation between the change in the free energy of association of the mutants and the change in buried hydrophobic surface area was obtained, after corrections for the energetic cost of losing steric complementarity at the αβ dimer interface. The slope yields an interface stabilization free energy of −15 ± 1.2 cal/mol upon burial of 1 Å2 of hydrophobic surface, in very good agreement with the theoretical estimate given by Eisenberg and McLachlan [Eisenberg, D. & McLachlan, A. D. (1986) Nature (London) 319, 199–203].

The interaction of Arp2/3 complex with actin: Nucleation, high affinity pointed end capping, and formation of branching networks of filaments

The Arp2/3 complex is a stable assembly of seven protein subunits including two actin-related proteins (Arp2 and Arp3) and five novel proteins. Previous work showed that this complex binds to the sides of actin filaments and is concentrated at the leading edges of motile cells. Here, we show that Arp2/3 complex purified from Acanthamoeba caps the pointed ends of actin filaments with high affinity. Arp2/3 complex inhibits both monomer addition and dissociation at the pointed ends of actin filaments with apparent nanomolar affinity and increases the critical concentration for polymerization at the pointed end from 0.6 to 1.0 μM. The high affinity of Arp2/3 complex for pointed ends and its abundance in amoebae suggest that in vivo all actin filament pointed ends are capped by Arp2/3 complex. Arp2/3 complex also nucleates formation of actin filaments that elongate only from their barbed ends. From kinetic analysis, the nucleation mechanism appears to involve stabilization of polymerization intermediates (probably actin dimers). In electron micrographs of quick-frozen, deep-etched samples, we see Arp2/3 bound to sides and pointed ends of actin filaments and examples of Arp2/3 complex attaching pointed ends of filaments to sides of other filaments. In these cases, the angle of attachment is a remarkably constant 70 ± 7°. From these in vitro biochemical properties, we propose a model for how Arp2/3 complex controls the assembly of a branching network of actin filaments at the leading edge of motile cells.

A meanfield approach to the thermodynamics of a protein-solvent system with application to the oligomerization of the tumor suppressor p53

The thermodynamic stability and oligomerization status of the tumor suppressor p53 tetramerization domain have been studied experimentally and theoretically. A series of hydrophilic mutations at Met-340 and Leu-344 of human p53 were designed to disrupt the hydrophobic dimer–dimer interface of the tetrameric oligomerization domain of p53 (residues 325–355). Meanfield calculations of the free energy of the solvated mutants as a function of interdimer distance were compared with experimental data on the thermal stability and oligomeric state (tetramer, dimer, or equilibrium mixture of both) of each mutant. The calculations predicted a decreasing stability and oligomeric state for the following amino acids at residue 340: Met (tetramer) > Ser Asp, His, Gln, > Glu, Lys (dimer), whereas the experimental results showed the following order: Met (tetramer) > Ser > Gln > His, Lys > Asp, Glu (dimers). For residue 344, the calculated trend was Leu (tetramer) > Ala > Arg, Gln, Lys (dimer), and the experimental trend was Leu (tetramer) > Ala, Arg, Gln, Lys (dimer). The discrepancy for the lysine side chain at residue 340 is attributed to the dual nature of lysine, both hydrophobic and charged. The incorrect prediction of stability of the mutant with Asp at residue 340 is attributed to the fact that within the meanfield approach, we use the wild-type backbone configuration for all mutants, but low melting temperatures suggest a softening of the α-helices at the dimer–dimer interface. Overall, this initial application of meanfield theory toward a protein-solvent system is encouraging for the application of the theoretical model to more complex systems.

A nucleated assembly mechanism of Alzheimer paired helical filaments

Alzheimer’s disease is characterized by two types of fibrous aggregates in the affected brains, the amyloid fibers (consisting of the Aβ-peptide, generating the amyloid plaques), and paired helical filaments (PHFs; made up of tau protein, forming the neurofibrillary tangles). Hence, tau protein, a highly soluble protein that normally stabilizes microtubules, becomes aggregated into insoluble fibers that obstruct the cytoplasm of neurons and cause a loss of microtubule stability. We have developed recently a rapid assay for monitoring PHF assembly and show here that PHFs arise from a nucleated assembly mechanism. The PHF nucleus comprises about 8–14 tau monomers. A prerequisite for nucleation is the dimerization of tau because tau dimers act as effective building blocks. PHF assembly can be seeded by preformed filaments (made either in vitro or isolated from Alzheimer brain tissue). These results suggest that dimerization and nucleation are the rate-limiting steps for PHF formation in vivo.

Studies on the enzymatic and transcriptional activity of the dimerization cofactor for hepatocyte nuclear factor 1

The relationship between the enzymatic and the transcriptional activity of the bifunctional protein pterin-4a-carbinolamine dehydratase/dimerization cofactor for hepatocyte nuclear factor 1 (DCoH) has been elucidated by site-directed mutagenesis. DCoH dimers harbor a binding site for hepatocyte nuclear factor 1 (HNF1), two active centers that bind pterins, and a saddle-shaped surface that resembles nucleic acid binding domains. Two domains of the protein have been selectively targeted to determine if a change in one activity affects the other. No strong correlation has been found, supporting the idea that carbinolamine dehydratase activity is not required for HNF1 binding in vitro or transcriptional coactivation in vivo. Double mutations in the active center, however, influence the in vivo transcriptional activity but not HNF1 binding. This finding suggests that some active center residues also are used during transcription, possibly for binding of another (macro)molecule. Several mutations in the saddle led to a surprising increase in transcription, therefore linking this domain to transcriptional regulation as well. The transcriptional function of DCoH therefore is composed of two parts, HNF1 binding and another contributing effect that involves the active site and, indirectly, the saddle.