Lysine 188 substitutions convert the pattern of proteasome activation by REGgamma to that of REGs alpha and beta.

11S REGs (PA28s) are multimeric rings that bind proteasomes and stimulate peptide hydrolysis. Whereas REGalpha activates proteasomal hydrolysis of peptides with hydrophobic, acidic or basic residues in the P1 position, REGgamma only activates cleavage after basic residues. We have isolated REGgamma mutants capable of activating the hydrolysis of fluorogenic peptides diagnostic for all three active proteasome beta subunits. The most robust REGgamma specificity mutants involve substitution of Glu or Asp for Lys188. REGgamma(K188E/D) variants are virtually identical to REGalpha in proteasome activation but assemble into less stable heptamers/hexamers. Based on the REGalpha crystal structure, Lys188 of REGgamma faces the aqueous channel through the heptamer, raising the possibility that REG channels function as substrate-selective gates. However, covalent modification of proteasome chymotrypsin-like subunits by 125I-YL3-VS demonstrates that REGgamma(K188E)'s activation of all three proteasome active sites is not due to relaxed gating. We propose that decreased stability of REGgamma(K188E) heptamers allows them to change conformation upon proteasome binding, thus relieving inhibition of the CT and PGPH sites normally imposed by the wild-type REGgamma molecule.

The proteasome activator 11 S REG or PA28: chimeras implicate carboxyl-terminal sequences in oligomerization and proteasome binding but not in the activation of specific proteasome catalytic subunits.

The REG homologs, alpha, beta and gamma, activate mammalian proteasomes in distinct ways. REGalpha and REGbeta activate the trypsin-like, chymotrypsin-like and peptidylglutamyl-preferring active sites, whereas REGgamma only activates the proteasome's trypsin-like subunit. The three REG homologs differ in carboxyl-terminal sequences that are located next to activation loops on their proteasome binding surface. To assess the importance of these carboxyl-terminal sequences in the activation of specific proteasome beta catalytic subunits, we characterized chimeras in which 8 or 12 residues were exchanged among the three proteins. Like the wild-type molecule, REGalpha chimeras activated all three proteasome catalytic subunits regardless of the carboxyl-terminal sequence. However, REGalpha-beta chimeras activated the proteasome at lower concentrations than wild-type REGalpha and higher levels of REGalpha-gamma chimeras were needed for maximal activation because exchanged carboxyl-terminal sequences can stabilize (REGalpha-beta) or destabilize (REGalpha-gamma) the REGalpha heptamer. REGgamma chimeras were equivalent to REGgamma in their activation properties, but they bound the proteasome less tightly than the wild-type molecule. REGbeta chimeras also bound the proteasome more weakly than wild-type REGbeta and were virtually unable to activate it. Our findings demonstrate that the carboxyl-terminal sequences of REG subunits can affect heptamer stability and proteasome affinity, but they do not determine which proteasome beta subunits become activated.

Synthesis and characterization of poly(ethylene glycol)-insulin conjugates.

Human insulin was modified by covalent attachment of short-chain (750 and 2000 Da) methoxypoly (ethylene glycol) (mPEG) to the amino groups of either residue PheB1 or LysB29, resulting in four distinct conjugates: mPEG(750)-PheB1-insulin, mPEG(2000)-PheB1-insulin, mPEG(750)-LysB29-insulin, and mPEG(2000)-LysB29-insulin. Characterization of the conjugates by MALDI-TOF mass spectrometry and N-terminal protein sequence analyses verified that only a single polymer chain (750 or 2000 Da) was attached to the selected residue of interest (PheB1 or LysB29). Equilibrium sedimentation experiments were performed using analytical ultracentrifugation to quantitatively determine the association state(s) of insulin derivatives. In the concentration range studied, all four of the conjugates and Zn-free insulin exist as stable dimers while Zn(2+)-insulin was exclusively hexameric and Lispro was monomeric. In addition, insulin (conjugate) self-association was evaluated by circular dichroism in the near-ultraviolet wavelength range (320-250 nm). This independent method qualitatively suggests that mPEG-insulin conjugates behave similarly to Zn-free insulin in the concentration range studied and complements results from ultracentrifugation studies. The physical stability/resistance to fibrillation of mPEG-insulin conjugates in aqueous solution were assessed. The data proves that mPEG(750 and 2000)-PheB1-insulin conjugates are substantially more stable than controls but the mPEG(750 and 2000)-LysB29-insulin conjugates were only slightly more stable than commercially available preparations. Circular dichroism studies done in the far ultraviolet region confirm insulin's tertiary structure in aqueous solution is essentially conserved after mPEG conjugation. In vivo pharmacodynamic assays reveal that there is no loss in biological activity after conjugation of mPEG(750) to either position on the insulin B-chain. However, attachment of mPEG(2000) decreased the bioactivity of the conjugates to about 85% of Lilly's HumulinR formulation. The characterization presented in this paper provides strong testimony to the fact that attachment of mPEG to specific amino acid residues of insulin's B-chain improves the conjugates' physical stability without appreciable perturbations to its tertiary structure, self-association behavior, or in vivo biological activity.

Spt16 and Pob3 of Saccharomyces cerevisiae form an essential, abundant heterodimer that is nuclear, chromatin-associated, and copurifies with DNA polymerase alpha.

Previously we showed that the yeast proteins Spt16 (Cdc68) and Pob3 are physically associated, and interact physically and genetically with the catalytic subunit of DNA polymerase alpha, Pol1 [Wittmeyer and Formosa (1997) Mol. Cell. Biol. 17, 4178-4190]. Here we show that purified Spt16 and Pob3 form a stable, abundant, elongated heterodimer and provide evidence that this is the functional form of these proteins. Genetic interactions between mutations in SPT16 and POB3 support the importance of the Spt16-Pob3 interaction in vivo. Spt16, Pob3, and Pol1 proteins were all found to localize to the nucleus in S. cerevisiae. A portion of the total cellular Spt16-Pob3 was found to be chromatin-associated, consistent with the proposed roles in modulating chromatin function. Some of the Spt16-Pob3 complex was found to copurify with the yeast DNA polymerase alpha/primase complex, further supporting a connection between Spt16-Pob3 and DNA replication.

Characterization of interactions between the anti-apoptotic protein BAG-1 and Hsc70 molecular chaperones.

The anti-cell death protein BAG-1 binds to 70-kDa heat shock proteins (Hsp70/Hsc70) and modulates their chaperone activity. Among other facilitory roles, BAG-1 may serve as a nucleotide exchange factor for Hsp70/Hsc70 family proteins and thus represents the first example of a eukaryotic homologue of the bacterial co-chaperone GrpE. In this study, the interactions between BAG-1 and Hsc70 are characterized and compared with the analogous GrpE-DnaK bacterial system. In contrast to GrpE, which binds DnaK as a dimer, BAG-1 binds to Hsc70 as a monomer with a 1:1 stoichiometry. Dynamic light scattering, sedimentation equilibrium, and circular dichroism measurements provided evidence that BAG-1 exists as an elongated, highly helical monomer in solution. Isothermal titration microcalorimetry was used to determine the complex stoichiometry and an equilibrium dissociation constant, KD, of 100 nM. Kinetic analysis using surface plasmon resonance yielded a KD consistent with the calorimetrically determined value. Molecular modeling permitted a comparison of structural features between the functionally homologous BAG-1 and GrpE proteins. These data were used to propose a mechanism for BAG-1 in the regulation of Hsp70/Hsc70 chaperone activity.

Interpreting kinetic rate constants from optical biosensor data recorded on a decaying surface.

A capturing assay was used to monitor a Fab-antigen interaction using a BIACORE optical biosensor. The antigen, a truncated single-site mutant (F43V) version of the CD4 receptor, was captured onto the sensor surface using an immobilized nonneutralizing monoclonal antibody. While this assay design created an oriented antigen surface, the antigen slowly dissociated during subsequent binding of the Fab, thus complicating the binding responses. In this paper, we illustrate how binding events occurring on a decaying surface can be accurately described by globally fitting the response data to a model that accounts for the background surface decay. Support for the method was obtained by showing the equilibrium dissociation constant calculated from the kinetic rate constants (Kd = 2.20 +/- 0.01 nM) was similar to the value measured in solution using titration calorimetry (Kd = 2.6 +/- 0.5 nM). The ability to interpret rate constants from decaying surfaces significantly extends the types of experimental systems that can be quantitatively studied on optical biosensors.

Beta-lactoglobulin B: a proposed standard for the study of reversible self-association reactions in the analytical ultracentrifuge?

Bovine beta-lactoglobulin B is proposed for use as a standard in the measurement of reversible self-association reactions in the analytical ultracentrifuge. The protein is well understood on a molecular level, is readily obtainable, and is stable under harsh conditions. Bovine beta-lactoglobulin B undergoes a simple monomer-dimer equilibrium which can be predictably controlled and consistently reproduced. In this investigation bovine beta-lactoglobulin B has been studied via sedimentation equilibrium experiments in the XL-A analytical ultracentrifuge at 5-30 degrees C in buffers of ionic strength 0.1-0.2 M and pH 2.0-3.0. Samples subjected to a number of different treatments and storage methods all yielded similar results. Molar equilibrium constants for the association reaction were determined by nonlinear regression fitting of a monomer-dimer model of association either to concentration versus radius data, using the programs NONLIN and ORIGIN, or to Omega versus concentration data using the program DUGOM. At 20 degrees C and pH 2.6, over the ionic strength range 0. 1-0.2 M, the equilibrium constant for the association reaction ranges between 1 x 10(4) and 5 x 10(4) M-1. The parameters of nonideal self-association behavior were found to be independent of the particular analysis strategy. Fitting to the concentration distribution, the apparent weight-average molecular weight, or the Omega function all returned identical parameters.