Effect of mutations of murine lens alphaB crystallin on transfected neural cell viability and cellular translocation in response to stress.

We examined the influence of over-expressed native and mutant murine lens alphaB crystallin on the response of a murine neural cell line to heat and ionic strength shock. Native and mutant (F27R and KK174/175LL) murine alphaB crystallin amplicons were subcloned into a Lac-Switch IPTG-inducible RSV promoter eukaryotic vector, and transfected into N1E-115 cells using lipofectin. Expression was induced maximally 8 h after addition of IPTG (optimal final concentration 1 mM) to the medium. Cells grew normally after transfection with native and mutant murine alphaB crystallin. We demonstrated expression of the protein using specific anti-alpha crystallin antibodies. Viability under severe heat and ionic strength stress increased when cells had been transfected with native alphaB crystallin, but not with mutants F27R or KK174/175LL. Heat shock caused translocation of both native and mutant alphaB crystallins into the central region of the cells. These results show that mutations in alphaB crystallin that effect its chaperone-like activity may also influence viability of N1E-115 neural cells under stress, while not influencing the distribution of the protein within the cell.

Translocation of beta crystallin in neural cells in response to stress.

While extralenticular expression of proteins in the alpha crystallin (small heat shock protein) family is well documented, that for proteins in the beta/gamma-superfamily is less well established. Here we show, using SDS-PAGE, Western blotting and confocal microscopy, that there is a constitutive level of beta crystallin expression in mouse N1E-115 neural cells. Furthermore, upon heat shock at 43 degrees C or 55 degrees C, or cold shock at 30 degrees C, beta crystallin immunoreactivity translocated predominantly from the nuclear region into the cytoplasmic region of the cells. In conditions of stress, it may be important for beta crystallin to be recruited into the cytoplasm to stabilise other proteins via its high beta-sheet content, and/or to ensure that storage levels of cytoplasmic Ca2+ are maintained.

Polyketide synthesis in vitro on a modular polyketide synthase.

BACKGROUND: The 6-deoxyerythronolide B synthase (DEBS) of Saccharopolyspora erythraea, which synthesizes the aglycone core of the antibiotic erythromycin A, contains some 30 active sites distributed between three multienzyme polypeptides (designated DEBS1-3). This complexity has hitherto frustrated mechanistic analysis of such enzymes. We previously produced a mutant strain of S. erythraea in which the chain-terminating cyclase domain (TE) is fused to the carboxyl-terminus of DEBS1, the multienzyme that catalyzes the first two rounds of polyketide chain extension in S. erythraea. This mutant strain produces triketide lactone in vivo. We set out to purify the chimaeric enzyme and to determine its activity in vitro. RESULTS: The purified DEBS1-TE multienzyme catalyzes synthesis of triketide lactones in vitro. The synthase specifically uses the (2S)-isomer of methylmalonyl-CoA, as previously proposed, but has a more relaxed specificity for the starter unit than in vivo. CONCLUSIONS: We have obtained a purified polyketide synthase system, derived from DEBS, which retains catalytic activity. This approach opens the way for mechanistic and structural analyses of active multienzymes derived from any modular polyketide synthase.

Repositioning of a domain in a modular polyketide synthase to promote specific chain cleavage.

Macrocyclic polyketides exhibit an impressive range of medically useful activities, and there is great interest in manipulating the genes that govern their synthesis. The 6-deoxyerythronolide B synthase (DEBS) of Saccharopolyspora erythraea, which synthesizes the aglycone core of the antibiotic erythromycin A, has been modified by repositioning of a chain-terminating cyclase domain to the carboxyl-terminus of DEBS1, the multienzyme that catalyzes the first two rounds of polyketide chain extension. The resulting mutant markedly accelerates formation of the predicted triketide lactone, compared to a control in which the repositioned domain is inactive. Repositioning of the cyclase should be generally useful for redirecting polyketide synthesis to obtain polyketides of specified chain lengths.