Allosteric activation of yeast enzyme neutral trehalase by calcium and 14-3-3 protein.

Neutral trehalase 1 (Nth1) from Saccharomyces cerevisiae catalyzes disaccharide trehalose hydrolysis and helps yeast to survive adverse conditions, such as heat shock, starvation or oxidative stress. 14-3-3 proteins, master regulators of hundreds of partner proteins, participate in many key cellular processes. Nth1 is activated by phosphorylation followed by 14-3-3 protein (Bmh) binding. The activation mechanism is also potentiated by Ca(2+) binding within the EF-hand-like motif. This review summarizes the current knowledge about trehalases and the molecular and structural basis of Nth1 activation. The crystal structure of fully active Nth1 bound to 14-3-3 protein provided the first high-resolution view of a trehalase from a eukaryotic organism and showed 14-3-3 proteins as structural modulators and allosteric effectors of multi-domain binding partners.

Thirty-four novel mutations detected in factor VIII gene by multiplex CSGE: modeling of 13 novel amino acid substitutions.

Detection of causal mutations is required for genetic counseling. Molecular modeling combined with patients' phenotype provides significant insight into structure-function relationship of factor (F)VIII molecule. Our objective was to identify defects in the gene of FVIII by a sensitive and simple scanning technique with high throughput in order to study molecular mechanisms by which novel amino acid substitutions may lead to hemophilia A. A cohort of 81 families with mild, moderate and severe hemophilia A negative in intron 22 inversion was studied. For detection of mutations in the FVIII gene a conformation sensitive gel electrophoresis (CSGE) was modified by multiplexing. Thirteen novel amino acid substitutions were studied by molecular modeling and a correlation with the cross-reactive material (CRM) phenotype was performed. In 74 families, 59 different mutations were detected. Six different mutations were recurrent in 21 unrelated families. Thirty-four novel mutations included 19 point mutations, four small insertions, nine small deletions and two complex mutations. Thirteen novel amino acid substitutions occurred at residues conserved in FVIII orthologs. Five of them were associated with a discrepancy between FVIII activity and antigen; another five with CRM reduced phenotype and one with undetectable FVIII antigen. Multiplexing of the CSGE significantly increased its throughput without substantial loss of sensitivity. Molecular modeling suggested mechanisms by which substitutions at residues 382 and 569, located outside the proposed FIXa-binding region, may influence FVIII/FIXa interaction. His2155 was predicted to participate in FVIII/VFW binding.

Impact of splice-site mutations of the human MDR1 cDNA on its stability and expression following retroviral gene transfer.

The multidrug resistance 1 (MDR1) gene transfer to hematopoietic cells for protection against cytotoxic drugs has received considerable attention in gene therapy. However, ectopic expression of MDR1 from retroviral vectors has been hampered by its genetic instability resulting from cryptic splice sites within the cDNA. We have evaluated the efficiency of retroviral MDR1 vectors with introduced mutations of the MDR1 cryptic splice donor (cSD) located at nucleotide +339 and of the cryptic splice acceptor (cSA) at nucleotide +2319 of the cDNA. Sequence alterations of the cSD reduced the expression of MDR1 P-glycoprotein (P-gp), even when generated as silent mutations. A silent mutation of the cSA reduced the splicing activity shifting the splice acceptor site one base downstream; however, it significantly improved the expression of P-gp. The incidence of wild-type MDR1 pregenome splicing was markedly reduced when vectors were produced in human 293 packaging cells as opposed to murine PG13 and GP+envAm12. We conclude that complete splice correction of MDR1 in retroviral vectors may only be achieved with extensive alterations of the cDNA or neighboring vector sequences and that the splicing is significantly influenced by the choice of the packaging cells.