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PubMed1996

Sedimentation analyses of the salt- and divalent metal ion-induced oligomerization of nucleolar protein B23.

Protein B23 is a major nonribosomal nucleolar protein and putative ribosome assembly factor that has been demonstrated to form oligomers. Sedimentation velocity and equilibrium analyses were used to examine the oligomerization properties of recombinant proteins B23.1 and B23.2. Under low ionic strength conditions protein B23.1 was predominantly a 2.1S monomer with small amounts of a 7.1S oligomer. At NaCl concentrations of 0.1 M and above the protein was almost exclusively the 7.1S oligomer. The oligomer remained the predominant species in NaCl concentrations as high as 1 M, suggesting that oligomers are not stabilized by electrostatic interactions. Low concentrations of divalent metal ions (0.1 - 1mM Ca2+ or Mg2+) also promoted oligomerization. Reducing agents had no effect on oligomerization, indicating that disulfide bridges are not important in oligomer formation. Protein B23.2, the carboxyl-terminal truncated isoform, had sedimentation characteristics similar to that of protein B23.1, suggesting that the carboxyl-terminal end of protein B23.1 is not essential for oligomerization. Protein B23.1 was previously shown to bind nucleic acids [Wang, D., Baumann, A., Szebeni, A., & Olson, M. O. J.(1995) J. Biol. Chem. 269, 30994-30998]. The effect of protein B23.1 oligomerization on its interaction with a 230 base pair DNA fragment was examined by sedimentation analyses. Under conditions where significant amounts of monomer were present, protein B23.1 was capable of binding DNA, whereas conditions that strongly favored oligomerization caused a nearly complete abolition of DNA binding activity. These studies suggest that protein B23 exists in an equilibrium between monomer and oligomer and that the quaternary structure of the protein may regulate its DNA binding properties.

Animals↗
PubMed1998

Intestinal absorption of nutrients is not influenced by soy fiber and does not differ between oligomeric and polymeric enteral diets.

Enteric feeding is often associated with diarrhea. To avoid this side effect, isoosmotic and fiber-supplemented enteral diets are recommended. The aims of this study were to determine whether supplementing enteral diets with soy fiber influences nutrient absorption and whether in enteric feeding absorption of nutrients and water fluxes differ between hyperosmotic oligomeric and isoosmotic polymeric diets. In mini pigs intestinal absorption and water fluxes were measured by perfusing a 150-cm length of jejunum. Six noncommercial iso- and hyperosmotic oligomeric and polymeric diets and six commercial polymeric diets, either fiber-free or supplemented with soy fiber, were used. Pancreatic enzymes were infused concomitantly with the polymeric diets. The absorption of nutrients and energy did not differ between oligomeric and polymeric diets. Oligomeric diets of high energy density produced a pronounced secretion of water. Despite lower initial osmolality, polymeric diets produced a similar secretion of water due to rapid pancreatic hydrolysis. Supplementing diets with largely insoluble soy fiber increased viscosity only between 4.6 and 14.5 mPa x sec. Soy fiber did not influence absorption of nutrients and energy and had also no effects on luminal transit and flow rate. The lack of effects was not due to dilution of chyme by intestinal secretion of water because no differences existed between isoosmotic and hyperosmotic oligomeric diets. In conclusion, supplementing enteral diets with soy fiber does not impair the absorption of nutrients. Enteric feeding with isoosmotic polymeric diets provides no advantage compared with hyperosmotic oligomeric diets with respect to absorption of nutrients and secretion of water.

Animals↗
PubMed2003

Thermally induced disintegration of the oligomeric structure of alphaB-crystallin mutant F28S is associated with diminished chaperone activity.

alphaB-crystallin, a member of the small heat-shock protein (hsp) family of proteins, is able to function as a molecular chaperone by protecting other proteins from stress-induced aggregation by recognizing and binding to partially unfolded species of damaged proteins. The present work has investigated the role of phenylalanine-28 (F28) of the 22RLFDQFF28 region of alphaB-crystallin in maintaining chaperone function and oligomeric structure under physiological condition and under thermal stress. Bovine alphaB-crystallin was cloned for the first time and the cDNA sequence revealed greater than 90% homology to that of human, rat and mouse alphaB-crystallins. F28 was mutated to a serine followed by expression of the mutant F28S and the wild-type alphaB (alphaB-wt) in E. coli and subsequent purification of the protein by size-exclusion chromatography. Secondary and tertiary structure analyses showed some structural changes in the mutant. Chaperone activity and oligomeric size of the mutant was unchanged at 37 degrees C whereas at 58 degrees C the chaperone activity was significantly decreased and the oligomeric size ranged from low molecular weight to high molecular weight showing disintegration of the oligomeric structure. The data support the idea that the participation of large oligomeric structure rather than smaller units is required to have optimal chaperone activity and the hydrophobic F28 residue is needed for maintaining the native oligomeric structure under thermal stress.

Amino Acid Sequence↗
PubMed2000

Oligomerization of beta-amyloid of the Alzheimer's and the Dutch-cerebral-haemorrhage types.

A novel ELISA has been developed which detects oligomerization of beta-amyloid (A beta). Oligomerization, fibrillization and neurotoxicity of native A beta associated with Alzheimer's disease (AD) type has been compared with E22Q A beta (amyloid beta-protein containing residues 1--40 with the native Glu at residue 22 changed to Gln) implicated in Dutch cerebral haemorrhage disease. Solutions of A beta rapidly yield soluble oligomers in a concentration-dependent manner, which are detected by the ELISA, and by size-exclusion gel chromatography. Conformational changes from disordered to beta-sheet occur more slowly than oligomerization, and fibrils are produced after prolonged incubation. The E22Q A beta oligomerizes, changes conformation and fibrillizes more rapidly than the native form and produces shorter stubbier fibrils. Aged fibrillar preparations of E22Q A beta are more potent than aged fibrils of native A beta in inducing apoptotic changes and toxic responses in human neuroblastoma cell lines, whereas low-molecular-mass oligomers in briefly incubated solutions are much less potent. The differences in the rates of oligomerization of the two A beta forms, their conformational behaviour over a range of pH values, and NMR data reported elsewhere, are consistent with a molecular model of oligomerization in which strands of A beta monomers initially overcome charge repulsion to form dimers in parallel beta-sheet arrangement, stabilized by intramolecular hydrophobic interactions, with amino acids of adjacent chains in register.

Alzheimer Disease↗
PubMed2000

Oligomerization of the chromatin-structuring protein H-NS.

H-NS is a major component of the bacterial nucleoid, involved in condensing and packaging DNA and modulating gene expression. The mechanism by which this is achieved remains unclear. Genetic data show that the biological properties of H-NS are influenced by its oligomerization properties. We have applied a variety of biophysical techniques to study the structural basis of oligomerization of the H-NS protein from Salmonella typhimurium. The N-terminal 89 amino acids are responsible for oligomerization. The first 64 residues form a trimer dominated by an alpha-helix, likely to be in coiled-coil conformation. Extending this polypeptide to 89 amino acids generated higher order, heterodisperse oligomers. Similarly, in the full-length protein no single, defined oligomeric state is adopted. The C-terminal 48 residues do not participate in oligomerization and form a monomeric, DNA-binding domain. These N- and C-terminal domains are joined via a flexible linker which enables them to function independently within the context of the full-length protein. This novel mode of oligomerization may account for the unusual binding properties of H-NS.

Amino Acid Sequence↗
PubMed1999

Oligomerization of the 17-kDa peptide-binding domain of the molecular chaperone HSC70.

Crystallographic and biochemical studies have indicated that the peptide-binding site of the molecular chaperone HSC70 is located in a small subdomain comprising a beta-sheet motif followed by a helical region, and there is some evidence of the involvement of this site in oligomerization of the protein. To determine the structure of this subdomain in solution and examine its involvement in oligomerization of HSC70, a 17-kDa protein (residues 385-540 of HSC70) consisting mainly of the peptide-binding site was constructed and analyzed for oligomerization properties. This small domain was found to bind peptides and to form oligomers in solution, probably tetramers, which dissociated into monomers on peptide binding in a manner comparable with that observed for the whole protein. Furthermore, in the 60-kDa fragment of HSC70, which is composed of the 17-kDa domain and the 44-kDa ATPase domain, not only were the oligomerization properties conserved, but dissociation of multimeric species into monomers on ATP binding also occurred and peptide stimulation of ATPase activity was restored. These results indicate that the isolated 17-kDa peptide-binding domain is necessary and sufficient for oligomerization of the whole protein, suggesting that the peptide-binding site may be involved in the oligomerization process.

Adenosine Triphosphatases↗
PubMed1999

Hetero-oligomeric assembly of P2X receptor subunits. Specificities exist with regard to possible partners.

P2X receptors are a distinct family of ligand-gated ion channels activated by extracellular ATP. Each of the seven identified subunit proteins (P2X1 through P2X7) has been reported to form functional homo-oligomeric channels when expressed in heterologous systems. Functional studies of native receptors, together with patterns of subunit gene expression, suggest that hetero-oligomeric assembly among members of this family may also occur. This prediction is supported by reports describing hetero-oligomeric assembly for three different recombinant subunit combinations. In this report, we systematically examined the ability of all members of the P2X receptor family to interact using a co-immunoprecipitation assay. The seven P2X receptor subunits were differentially epitope-tagged and expressed in various combinations in human embryonic kidney 293 cells. It was found that six of the seven subunits formed homo-oligomeric complexes, the exception being P2X6. When co-assembly between pairs of subunits was examined, all were able to form hetero-oligomeric assemblies with the exception of P2X7. Whereas P2X1, P2X2, P2X5, and P2X6 were able to assemble with most subunits, P2X3 and P2X4 presented a more restricted pattern of co-association. These results suggest that hetero-oligomeric assembly might underlie functional discrepancies observed between P2X responses seen in the native and recombinant settings, while providing for an increased diversity of signaling by ATP.

Adenosine Triphosphate↗
PubMed1999

Subunit oligomerization, and topology of the inositol 1,4, 5-trisphosphate receptor.

The inositol 1,4,5-trisphosphate receptor (InsP(3)R) is a tetrameric assembly of highly conserved subunits that contain multiple membrane-spanning sequences in the C-terminal region of the protein. In studies aimed at investigating the oligomerization and transmembrane topology of the type-1 InsP(3)R, a series of membrane-spanning region truncation and deletion plasmids were constructed. These plasmids were transiently transfected in COS-1 cells, and the resulting expression products were analyzed for the ability to assemble into tetrameric structures. The topology of the membrane-spanning region truncations and the full-length receptor was determined by immunocytochemical analysis of transfected COS-1 cells using complete or selective permeabilization strategies. Our results are the first to experimentally define the presence of six membrane-spanning regions. These results are consistent with the current model for the organization of the InsP(3)R in the endoplasmic reticulum and show that the truncation mutants are properly targeted and oriented in the endoplasmic reticulum membrane, thus making them amenable reagents to study receptor subunit oligomerization. Fractionation of soluble and membrane protein components revealed that the first two membrane-spanning regions were necessary for membrane targeting of the receptor. Sedimentation and immunoprecipitation experiments show that assembly of the receptor subunits was an additive process as the number of membrane-spanning regions increased. Immunoprecipitations from cells co-expressing the full-length receptor and carboxyl-terminal truncations reveal that constructs expressing the first two or more membrane-spanning domains were capable of co-assembling with the full-length receptor. Inclusion of the fifth membrane-spanning segment significantly enhanced the degree of oligomerization. Furthermore, a deletion construct containing only membrane-spanning regions 5 and 6 oligomerized to a similar extent as that of the wild type protein. Membrane-spanning region deletion constructions that terminate with the receptor's 145 carboxyl-terminal amino acids were found to have enhanced assembly characteristics and implicate the carboxyl terminus as a determinant in oligomerization. Our results reveal a process of receptor assembly involving several distinct yet additive components and define the fifth and sixth membrane spanning regions as the key determinants in receptor oligomerization.

Amino Acid Sequence↗
PubMed2002

Constitutive agonist-independent CCR5 oligomerization and antibody-mediated clustering occurring at physiological levels of receptors.

Although homo-oligomerization has been reported for several G protein-coupled receptors, this phenomenon was not studied at low concentrations of receptors. Furthermore, it is not clear whether homo-oligomerization corresponds to an intrinsic property of nascent receptors or if it is a consequence of receptor activation. Here CCR5 receptor oligomerization was studied by bioluminescence resonance energy transfer (BRET) in cells expressing physiological levels of receptors. A strong energy transfer could be observed, in the absence of ligands, in whole cells and in both endoplasmic reticulum and plasma membrane subfractions, supporting the hypothesis of a constitutive oligomerization that occurs early after biosynthesis. No change in BRET was observed upon agonist binding, indicating that the extent of oligomerization is unrelated to the activation state of the receptor. In contrast, a robust increase of BRET, induced by a monoclonal antibody known to promote receptor clustering, suggests that microaggregation of preformed receptor homo-oligomers can occur. Taken together, our data indicate that constitutive receptor homo-oligomerization has a biologically relevant significance and might be involved in the process of receptor biosynthesis.

Biopolymers↗
PubMed2002

Ligand-dependent inhibition of oligomerization at the human thyrotropin receptor.

Recently, several studies have reported oligomerization of G protein-coupled receptors, although the functional implications of this phenomenon are still unclear. Using fluorescence resonance energy transfer (FRET) and coimmunoprecipitation (COIP), we previously reported that the human thyrotropin (TSH) receptor tagged with green fluorescent protein (TSHR(GFP)) and expressed in a heterologous system was present as oligomeric complexes on the cell surface. Here, we have extended this biophysical and biochemical approach to study the regulation of such oligomeric complexes. Co-expression of TSHR(GFP) and TSHR(Myc) constructs in Chinese hamster ovary cells resulted in FRET-positive cells. The specificity of the FRET signal was verified by the absence of energy transfer in individually transfected TSHR(GFP) and TSHR(Myc):Cy3 cells cultured together and also by acceptor photobleaching. Occupation of the receptor molecule by the ligand (TSH) resulted in a dose-dependent decrease in the FRET index from 20% in the absence of TSH to <1% with 10(3) microunits/ml of TSH. Such reduction in oligomeric forms was also confirmed by coimmunoprecipitation. Exposure of TSHR(GFP/Myc) cells to forskolin or cytochalasin D caused no change in the FRET index, confirming that the decrease in the oligomeric complexes was a receptor-dependent phenomenon and free of energy or microtuble requirements. The TSH-induced decrease in TSHR oligomers was found to be secondary to dissociation of the TSHR complexes as evidenced by an increase in fluorescent intensity of photobleached spots of GFP fluorescence with 10(3) microunits/ml of TSH. These data indicated that the less active conformation of the TSHR was comprised of receptor complexes and that such complexes were dissociated on the binding of ligand. Such observations support the concept of a constitutively active TSHR dimer or monomer that is naturally inhibited by the formation of higher order complexes. Inhibition of these oligomeric forms by ligand binding returns the TSHR to an activated state.

Animals↗
PubMed2003

Elucidation of primary structure elements controlling early amyloid beta-protein oligomerization.

Assembly of monomeric amyloid beta-protein (A beta) into oligomeric structures is an important pathogenetic feature of Alzheimer's disease. The oligomer size distributions of aggregate-free, low molecular weight A beta 40 and A beta 42 can be assessed quantitatively using the technique of photo-induced cross-linking of unmodified proteins. This approach revealed that low molecular weight A beta 40 is a mixture of monomer, dimer, trimer, and tetramer, in rapid equilibrium, whereas low molecular weight A beta 42 preferentially exists as pentamer/hexamer units (paranuclei), which self-associate to form larger oligomers. Here, photo-induced cross-linking of unmodified proteins was used to evaluate systematically the oligomerization of 34 physiologically relevant A beta alloforms, including those containing familial Alzheimer's disease-linked amino acid substitutions, naturally occurring N-terminal truncations, and modifications altering the charge, the hydrophobicity, or the conformation of the peptide. The most important structural feature controlling early oligomerization was the length of the C terminus. Specifically, the side-chain of residue 41 in A beta 42 was important both for effective formation of paranuclei and for self-association of paranuclei into larger oligomers. The side-chain of residue 42, and the C-terminal carboxyl group, affected paranucleus self-association. A beta 40 oligomerization was particularly sensitive to substitutions of Glu22 or Asp23 and to truncation of the N terminus, but not to substitutions of Phe19 or Ala21. A beta 42 oligomerization, in contrast, was largely unaffected by substitutions at positions 22 or 23 or by N-terminal truncations, but was affected significantly by substitutions of Phe19 or Ala21. These results reveal how specific regions and residues control A beta oligomerization and show that these controlling elements differ between A beta 40 and A beta 42.

Alzheimer Disease↗
PubMed2003

Oligomerization of the alpha 1a- and alpha 1b-adrenergic receptor subtypes. Potential implications in receptor internalization.

We combined biophysical, biochemical, and pharmacological approaches to investigate the ability of the alpha 1a- and alpha 1b-adrenergic receptor (AR) subtypes to form homo- and hetero-oligomers. Receptors tagged with different epitopes (hemagglutinin and Myc) or fluorescent proteins (cyan and green fluorescent proteins) were transiently expressed in HEK-293 cells either individually or in different combinations. Fluorescence resonance energy transfer measurements provided evidence that both the alpha 1a- and alpha 1b-AR can form homo-oligomers with similar transfer efficiency of approximately 0.10. Hetero-oligomers could also be observed between the alpha 1b- and the alpha 1a-AR subtypes but not between the alpha 1b-AR and the beta2-AR, the NK1 tachykinin, or the CCR5 chemokine receptors. Oligomerization of the alpha 1b-AR did not require the integrity of its C-tail, of two glycophorin motifs, or of the N-linked glycosylation sites at its N terminus. In contrast, helix I and, to a lesser extent, helix VII were found to play a role in the alpha 1b-AR homo-oligomerization. Receptor oligomerization was not influenced by the agonist epinephrine or by the inverse agonist prazosin. A constitutively active (A293E) as well as a signaling-deficient (R143E) mutant displayed oligomerization features similar to those of the wild type alpha 1b-AR. Confocal imaging revealed that oligomerization of the alpha1-AR subtypes correlated with their ability to co-internalize upon exposure to the agonist. The alpha 1a-selective agonist oxymetazoline induced the co-internalization of the alpha 1a- and alpha 1b-AR, whereas the alpha 1b-AR could not co-internalize with the NK1 tachykinin or CCR5 chemokine receptors. Oligomerization might therefore represent an additional mechanism regulating the physiological responses mediated by the alpha 1a- and alpha 1b-AR subtypes.

Amino Acid Sequence↗
PubMed2003

Membrane localization, oligomerization, and phosphorylation are required for optimal raf activation.

Activation of the serine/threonine kinase c-Raf-1 requires membrane localization, phosphorylation, and oligomerization. To study these mechanisms of Raf activation more precisely, we have used a membrane-localized fusion protein, myr-Raf-GyrB, which can be activated by coumermycin-induced oligomerization in NIH3T3 transfectants. By introducing a series of point mutations into the myr-Raf-GyrB kinase domain (S338A, S338A/Y341F, Y340F/Y341F, and T491A/S494A) we can separately study the role that membrane localization, phosphorylation, and oligomerization play in the process of Raf activation. We find that phosphorylation of Ser-338 plays a critical role in Raf activation and that this requires membrane localization but not oligomerization of Raf. Mutation of Tyr-341 had a limited effect, whereas mutation of both Ser-338 and Tyr-341 resulted in a synergistic loss of Raf activation following coumermycin-induced dimerization. Importantly, we found that membrane localization and phosphorylation of Ser-338 were not sufficient to activate Raf in the absence of oligomerization. Thus, our studies suggest that three key steps are required for optimal Raf activation: recruitment to the plasma membrane by GTP-bound Ras, phosphorylation via membrane-resident kinases, and oligomerization.

Aminocoumarins↗
PubMed2004

Subunits of a yeast oligomeric G protein-coupled receptor are activated independently by agonist but function in concert to activate G protein heterotrimers.

G protein-coupled receptors (GPCRs) form dimeric or oligomeric complexes in vivo. However, the function of oligomerization in receptor-mediated G protein activation is unclear. Previous studies of the yeast alpha-factor receptor (STE2 gene product) have indicated that oligomerization promotes signaling. Here we have addressed the mechanism by which oligomerization facilitates G protein signaling by examining the ability of ligand binding- and G protein coupling-defective alpha-factor receptors to form complexes in vivo and to correct their signaling defects when co-expressed (trans complementation). Newly and previously identified receptor mutants indicated that ligand binding involves the exofacial end of transmembrane domain (TM) 4, whereas G protein coupling involves ic1, ic3, the C-terminal tail, and the intracellular ends of TM2 and TM3. Mutant receptors bearing substitutions in these domains formed homo-oligomeric or hetero-oligomeric complexes in vivo, as indicated by results of fluorescence resonance energy transfer experiments. Co-expression of ligand binding- and G protein coupling-defective mutant receptors did not significantly improve signaling. In contrast, co-expression of ic1 and ic3 mutations in trans but not in cis significantly increased signaling efficiency. Therefore, we suggest that subunits of the alpha-factor receptor: 1) are activated independently rather than cooperatively by agonist, and 2) function in a concerted fashion to promote G protein activation, possibly by contacting different subunits or regions of the G protein heterotrimer.

Cell Division↗
PubMed2004

Hetero-oligomerization between beta2- and beta3-adrenergic receptors generates a beta-adrenergic signaling unit with distinct functional properties.

The ability of the closely related beta(2)- and beta(3)-adrenergic receptors (AR) to form hetero-oligomers was assessed by bioluminescence resonance energy transfer. Quantitative bioluminescence resonance energy transfer titration curves revealed that the beta(2)AR has identical propensity to hetero-oligomerize with the beta(3)AR than to form homo-oligomers. To determine the influence of heterooligomerization, a HEK293 cell line stably expressing an excess of beta(3)AR over beta(2)AR was generated so that all beta(2)AR are engaged in hetero-oligomerization with beta(3)AR, providing a tool to study the effect of hetero-oligomerization on beta(2)AR function in the absence of any beta(2)AR homooligomer. The hetero-oligomerization had no effect on the ligand binding properties of various beta(2)AR ligands and did not affect the potency of isoproterenol to stimulate adenylyl cyclase. Despite the unaltered ligand binding properties of the beta(2/3)AR hetero-oligomer, the stable association of the beta(2)AR with the beta(3)AR completely blocked agonist-stimulated internalization of the beta(2)AR. Given that the beta(3)AR is resistant to agonist-promoted endocytosis, the results indicate that the beta(3)AR acted as a dominant negative of the beta(2)AR endocytosis process. Consistent with this notion, the beta(2/3)AR hetero-oligomer displayed a lower propensity to recruit beta-arrestin-2 than the beta(2)AR. The hetero-oligomerization also led to a change in G protein coupling selectivity. Indeed, in contrast to beta(2)AR and beta(3)AR, which regulate adenylyl cyclase and extracellular signal-regulated kinase activity through a coupling to G(s) and G(i/o), no G(i/o) coupling was observed for the beta(2/3)AR hetero-oligomer. Together, these results demonstrate that hetero-oligomerization between beta(2)AR and beta(3)AR forms a beta-adrenergic signaling unit that possesses unique functional properties.

Adenylyl Cyclases↗
PubMed2004

Functional domains of Brevibacillus thermoruber lon protease for oligomerization and DNA binding: role of N-terminal and sensor and substrate discrimination domains.

Lon protease is a multifunctional enzyme, and its functions include the degradation of damaged proteins and naturally short lived proteins, ATPase and chaperone-like activities, as well as DNA binding. A thermostable Lon protease from Brevibacillus thermoruber WR-249 (Bt-Lon) has been cloned and characterized with an N-terminal domain, a central ATPase domain that includes a sensor and substrate discrimination (SSD) domain, and a C-terminal protease domain. Here we present a detailed structure-function characterization of Bt-Lon, not only dissecting the individual roles of Bt-Lon domains in oligomerization, catalytic activities, chaperone-like activity, and DNA binding activity but also describing the nature of oligomerization. Seven truncated mutants of Bt-Lon were designed, expressed, and purified. Our results show that the N-terminal domain is essential for oligomerization. The truncation of the N-terminal domain resulted in the failure of oligomerization and led to the inactivation of proteolytic, ATPase, and chaperone-like activities but retained the DNA binding activity, suggesting that oligomerization of Bt-Lon is a prerequisite for its catalytic and chaperone-like activities. We further found that the SSD is involved in DNA binding based on gel mobility shift assays. On the other hand, the oligomerization of Bt-Lon proceeds through a dimer <--> tetramer <--> hexamer assembly model revealed by chemical cross-linking experiments. The results also showed that hydrophobic interactions may play important roles in the dimerization of Bt-Lon, and ionic interactions are mainly responsible for the assembly of hexamers.

ATP-Dependent Proteases↗
PubMed2004

Tetrameric oligomerization mediates transcriptional repression by the BRCA1-dependent Kruppel-associated box-zinc finger protein ZBRK1.

The Kruppel-associated box (KRAB)-zinc finger protein ZBRK1 has been implicated in the transcriptional regulation of DNA damage-response genes that function in cell growth control and survival. Recently, we described a novel BRCA1-dependent C-terminal transcriptional repression domain (CTRD) within ZBRK1, the mode of repression of which is functionally distinguishable from that of the N-terminal KRAB repression domain within ZBRK1. The identification of BRCA1 binding-competent but repression-defective CTRD mutants further revealed that BRCA1 binding is necessary, but not sufficient, for ZBRK1 CTRD function. During an unbiased search for possible co-regulators of the CTRD, we identified ZBRK1 itself, suggesting that ZBRK1 can oligomerize through its CTRD. Herein we explore the physical and functional requirements for ZBRK1 oligomerization in ZBRK1-directed transcriptional repression. Protein interaction analyses confirmed that ZBRK1 can homo-oligomerize both in vitro and in vivo and further mapped the ZBRK1 oligomerization domain to the CTRD C terminus. Biochemical analyses, including protein cross-linking and gel filtration chromatography, revealed that ZBRK1 homo-oligomers exist as tetramers in solution. Functionally, ZBRK1 oligomerization facilitates ZBRK1-directed transcriptional repression through ZBRK1 response elements; requirements for oligomerization-dependent repression include the ZBRK1 CTRD and KRAB repression domains but not the DNA binding activity of ZBRK1. These observations suggest that higher order oligomers of ZBRK1 may assemble on target ZBRK1 response elements through both protein-DNA and CTRD-dependent protein-protein interactions. These findings thus reveal an unanticipated dual function for ZBRK1 in both DNA binding-dependent and -independent modes of transcriptional repression and further establish the CTRD as a novel protein interaction surface responsible for directing homotypic and heterotypic interactions necessary for ZBRK1-directed transcriptional repression.

BRCA1 Protein↗
PubMed2004

Multiple signals are required for alpha2,6-sialyltransferase (ST6Gal I) oligomerization and Golgi localization.

A single amino acid difference in the catalytic domain of two isoforms of the alpha2,6-sialyltransferase (ST6Gal I) leads to differences in their trafficking, processing, and oligomerization. The STtyr isoform is transiently localized in the Golgi and is ultimately cleaved and secreted, whereas the STcys isoform is stably localized in the Golgi and is not cleaved and secreted. The stable localization of STcys is correlated with its enhanced ability to oligomerize. To test the hypothesis that multiple signals can mediate Golgi localization and further evaluate the role of oligomerization in the localization process, we evaluated the effects of individually and simultaneously altering the cytosolic tail and transmembrane region of the STcys isoform. We found that the localization, processing, and oligomerization of STcys were not substantially changed when either the core amino acids of the cytosolic tail were deleted or the sequence and length of the transmembrane region were altered. In contrast, when these changes were made simultaneously, the STcys isoform was converted into a form that was processed, secreted, and weakly oligomerized like STtyr. We propose that STcys oligomerization is a secondary event resulting from its concentration in the Golgi via mechanisms independently mediated by its cytosolic tail and transmembrane region.

Animals↗