Cystathionine beta-synthase mutants exhibit changes in protein unfolding: conformational analysis of misfolded variants in crude cell extracts

Protein misfolding has been proposed to be a common pathogenic mechanism in many inborn errors of metabolism including cystathionine β-synthase (CBS) deficiency. In this work, we describe the structural properties of nine CBS mutants that represent a common molecular pathology in the CBS gene. Using thermolysin in two proteolytic techniques, we examined conformation of these mutants directly in crude cell extracts after expression in E. coli. Proteolysis with thermolysin under native conditions appeared to be a useful technique even for very unstable mutant proteins, whereas pulse proteolysis in a urea gradient had limited values for the study of the majority of CBS mutants due to their instability. Mutants in the active core had either slightly increased unfolding (p.A114V, p.E302K and p.G307S) or extensive unfolding with decreased stability (p.H65R, p.T191M, p.I278T and p.R369C). The extent of the unfolding inversely correlated with the previously determined degree of tetrameric assembly and with the catalytic activity. In contrast, mutants bearing aminoacid substitutions in the C-terminal regulatory domain (p.R439Q and p.D444N) had increased global stability with decreased flexibility. This study shows that proteolytic techniques can reveal conformational abnormalities even for CBS mutants that have activity and/or a degree of assembly similar to the wild-type enzyme. We present here a methodological strategy that may be used in cell lysates to evaluate properties of proteins that tend to misfold and aggregate and that may be important for conformational studies of disease-causing mutations in the field of inborn errors of metabolism

Cytomegalovirus Expresses the Chemokine Homologue vXCL1 Capable of Attracting XCR1+ CD4- Dendritic Cells

Cytomegaloviruses (CMV) have developed various strategies to escape the immune system of the host. One strategy involves the expression of virus-encoded chemokines to modulate the host chemokine network. We have identified in the English isolate of rat CMV (murid herpesvirus 8; MuHV8) an open reading frame encoding a protein homologous to the chemokine XCL1, the only known C-chemokine. Viral XCL1 (vXCL1), a glycosylated protein of 96 amino acids, can be detected 13 hours post infection in the supernatant of MuHV8-infected rat embryo fibroblasts. vXCL1 exclusively binds to CD4- rat dendritic cells (DC), a DC subset that expresses the corresponding chemokine-receptor XCR1. Like endogenous rat XCL1, vXCL1 selectively chemoattracts XCR1+ CD4- DC. Since XCR1+ DC in mice and humans have been shown to excel in antigen cross-presentation and thus in the induction of cytotoxic CD8+ T lymphocytes, the virus has apparently hijacked this gene to subvert cytotoxic immune responses. The biology of vXCL1 offers an interesting opportunity to study the role of XCL1 and XCR1+ DC in the cross-presentation of viral antigens