A comparison of the phosphorylated and unphosphorylated forms of isocitrate dehydrogenase from Escherichia coli ML308

AbstractNADP+ can protect active isocitrate dehydrogenase against attack by several proteases. Inactive phosphorylated isocitrate dehydrogenase is much less susceptible to proteolysis than the active enzyme, and it is not protected by NADP+. The results suggest that binding of NADP+ to, or phosphorylation of, active isocitrate dehydrogenase induces similar conformational states. Fluorescence titration experiments show that NADPH can bind to active but not to inactive isocitrate dehydrogenase. It is suggested that the phosphorylation of isocitrate dehydrogenase may occur close to its coenzyme binding site

An Atypical Form of αB-crystallin Is Present in High Concentration in Some Human Cataractous Lenses IDENTIFICATION AND CHARACTERIZATION OF ABERRANT N- AND C-TERMINAL PROCESSING

Two unique polypeptides, 22.4 and 16.4 kDa, were prominent in some human cataracts. Both proteins were identified as modified forms of the small heat shock protein, αB-crystallin. The concentration of total αB-crystallin in most of these cataracts was significantly increased. The 22.4-kDa protein was subsequently designated as αBg. Mass spectrometric analyses of tryptic and Asp-N digests showed αBg is αB-crystallin minus the C-terminal lysine. αBg constituted 10–90% of the total αB-crystallin in these cataracts and was preferentially phosphorylated over the typical form of αB-crystallin. Human αBg and αB-crystallin were cloned and expressed inEscherichia coli. The differences in electrophoretic mobility and the large difference in native pI values suggest some structural differences exist. The chaperone-like activity of recombinant human αBg was comparable to that of recombinant human αB-crystallin in preventing the aggregation of lactalbumin induced by dithiothreitol. The mechanism involved in generating αBg is not known, but a premature termination of the αB-crystallin gene was ruled out by sequencing the polymerase chain reaction products of the last exon for the αB-crystallin gene from lenses containing αBg. The 16.4-kDa protein was an N-terminally truncated fragment of αBg. The high concentration of αB-crystallin in these cataracts is the first observation of this kind in human lenses

Late-onset retinal degeneration pathology de to mutations in CTRP5 is mediated through HTRA1

Late-onset retinal degeneration (L-ORD) is an autosomal dominant macular degeneration characterized by the formation of sub-retinal pigment epithelium (RPE) deposits and neuroretinal atrophy. L-ORD results from mutations in the C1q-tumor necrosis factor-5 protein (CTRP5), encoded by the CTRP5/C1QTNF5 gene. To understand the mechanism underlying L-ORD pathology, we used a human cDNA library yeast two-hybrid screen to identify interacting partners of CTRP5. Additionally, we analyzed the Bruch's membrane/choroid (BM-Ch) from wild-type (Wt), heterozygous S163R Ctrp5 mutation knock-in (Ctrp5S163R/wt ), and homozygous knock-in (Ctrp5S163R/S163R ) mice using mass spectrometry. Both approaches showed an association between CTRP5 and HTRA1 via its C-terminal PDZ-binding motif, stimulation of the HTRA1 protease activity by CTRP5, and CTRP5 serving as an HTRA1 substrate. The S163R-CTRP5 protein also binds to HTRA1 but is resistant to HTRA1-mediated cleavage. Immunohistochemistry and proteomic analysis showed significant accumulation of CTRP5 and HTRA1 in BM-Ch of Ctrp5S163R/S163R and Ctrp5S163R/wt mice compared with Wt. Additional extracellular matrix (ECM) components that are HTRA1 substrates also accumulated in these mice. These results implicate HTRA1 and its interaction with CTRP5 in L-ORD pathology

Late-onset retinal degeneration pathology due to mutations in CTRP5 is mediated through HTRA1.

Late-onset retinal degeneration (L-ORD) is an autosomal dominant macular degeneration characterized by the formation of sub-retinal pigment epithelium (RPE) deposits and neuroretinal atrophy. L-ORD results from mutations in the C1q-tumor necrosis factor-5 protein (CTRP5), encoded by the CTRP5/C1QTNF5 gene. To understand the mechanism underlying L-ORD pathology, we used a human cDNA library yeast two-hybrid screen to identify interacting partners of CTRP5. Additionally, we analyzed the Bruchs membrane/choroid (BM-Ch) from wild-type (Wt), heterozygous S163R Ctrp5 mutation knock-in (Ctrp5S163R/wt ), and homozygous knock-in (Ctrp5S163R/S163R ) mice using mass spectrometry. Both approaches showed an association between CTRP5 and HTRA1 via its C-terminal PDZ-binding motif, stimulation of the HTRA1 protease activity by CTRP5, and CTRP5 serving as an HTRA1 substrate. The S163R-CTRP5 protein also binds to HTRA1 but is resistant to HTRA1-mediated cleavage. Immunohistochemistry and proteomic analysis showed significant accumulation of CTRP5 and HTRA1 in BM-Ch of Ctrp5S163R/S163R and Ctrp5S163R/wt mice compared with Wt. Additional extracellular matrix (ECM) components that are HTRA1 substrates also accumulated in these mice. These results implicate HTRA1 and its interaction with CTRP5 in L-ORD pathology