Alleviation of a defect in protein folding by increasing the rate of subunit assembly.

Understanding the nature of protein grammar is critical because amino acid substitutions in some proteins cause misfolding and aggregation of the mutant protein resulting in a disease state. Amino acid substitutions in phage P22 coat protein, known as tsf (temperature-sensitive folding) mutations, cause folding defects that result in aggregation at high temperatures. We have isolated global su (suppressor) amino acid substitutions that alleviate the tsf phenotype in coat protein (Aramli, L. A., and Teschke, C. M. (1999) J. Biol. Chem. 274, 22217-22224). Unexpectedly, we found that a global su amino acid substitution in tsf coat proteins made aggregation worse and that the tsf phenotype was suppressed by increasing the rate of subunit assembly, thereby decreasing the concentration of aggregation-prone folding intermediates.

Single amino acid substitutions globally suppress the folding defects of temperature-sensitive folding mutants of phage P22 coat protein.

The amino acid sequence of a polypeptide defines both the folding pathway and the final three-dimensional structure of a protein. Eighteen amino acid substitutions have been identified in bacteriophage P22 coat protein that are defective in folding and cause their folding intermediates to be substrates for GroEL and GroES. These temperature-sensitive folding (tsf) substitutions identify amino acids that are critical for directing the folding of coat protein. Additional amino acid residues that are critical to the folding process of P22 coat protein were identified by isolating second site suppressors of the tsf coat proteins. Suppressor substitutions isolated from the phage carrying the tsf coat protein substitutions included global suppressors, which are substitutions capable of alleviating the folding defects of numerous tsf coat protein mutants. In addition, potential global and site-specific suppressors were isolated, as well as a group of same site amino acid substitutions that had a less severe phenotype than the tsf parent. The global suppressors were located at positions 163, 166, and 170 in the coat protein sequence and were 8-190 amino acid residues away from the tsf parent. Although the folding of coat proteins with tsf amino acid substitutions was improved by the global suppressor substitutions, GroEL remained necessary for folding. Therefore, we believe that the global suppressor sites identify a region that is critical to the folding of coat protein.

The direct embryotoxicity of immunoglobulin G fractions from patients with systemic lupus erythematosus.

PROBLEM: To determine if IgG fractions from sera of individuals with systemic lupus erythematosus (SLE) were toxic to cultures of whole rat embryos. METHODS: Head-fold stage rat embryos (9.5 days of gestation) were cultured on media consisting of 50% rat serum containing IgG fractions isolated from plasmapheresis plasma of six subjects with SLE and six with other autoimmune diseases. Each fraction was tested at 11 mg/ml and those toxic were also tested at 7.5 and 4 mg/ml. RESULTS: Of the six SLE IgG fractions, four were embryotoxic (embryolethal or teratogenic) while only one of the six non-SLE fractions were embryotoxic. CONCLUSION: IgG fractions from subjects with SLE can be toxic to cultures of whole rat embryos in the absence of maternal tissues or influence. Such cultures of whole embryos may be useful to identify those antibodies that represent a risk for fetal loss as well as to understand their mechanisms of embryotoxicity.

Phosphotyrosine phosphatase activity in human platelets.

Using O-phosphotyrosine as a substrate, human platelets were shown to contain a highly active phosphotyrosine phosphatase (PTPase) activity. This activity was potently inhibited by vanadate, molybdate, and HgCl2. About 80% of the PTPase activity was particulate. When Triton-solubilized PTPase activity from whole platelets was applied to a DEAE Sephacel column about 40% came through unbound. The activity that bound was eluted by a NaCl gradient as a broad, heterogeneous peak. The possibility is raised for the existence of multiple forms of phosphotyrosine phosphatases in human platelets. That one or more of these forms may be regulated by activators of platelet aggregation and secretion, such as thrombin and collagen, is discussed.