Identification of hPin1 inhibitors that induce apoptosis in a mammalian Ras transformed cell line.

The authors have developed a class of potent inhibitors against the phosphate specific prolyl isomerase hPin1, which induced apoptosis in transformed cell lines.

The factors governing the thermal stability of frataxin orthologues: how to increase a protein's stability.

Understanding the factors governing the thermal stability of proteins and correlating them to the sequence and structure is a complex and multiple problem that can nevertheless provide important information on the molecular forces involved in protein folding. Here, we have carried out a comparative genomic study to analyze the effects that different intrinsic and environmental factors have on the thermal stability of frataxins, a family of small mitochondrial iron-binding proteins found in organisms ranging from bacteria to humans. Low expression of frataxin in humans causes Friedreich's ataxia, an autosomal recessive neurodegenerative disease. The human, yeast, and bacterial orthologues were selected as representatives of different evolutionary steps. Although sharing high sequence homology and the same three-dimensional fold, the three proteins have a large variability in their thermal stabilities. Whereas bacterial and human frataxins are thermally stable, well-behaved proteins, under the same conditions yeast frataxin exists in solution as an unstable species with apprechable tracts in a conformational exchange. By designing suitable mutants, we show and justify structurally that the length of the C-terminus is an important intrinsic factor that directly correlates with the thermal stabilities of the three proteins. Thermal stability is also gained by the addition of Fe(2+). This effect, however, is not uniform for the three orthologues nor highly specific for iron: a similar albeit weaker stabilization is observed with other mono- and divalent cations. We discuss the implications that our findings have for the role of frataxins as iron-binding proteins.

Structural analysis of the mitotic regulator hPin1 in solution: insights into domain architecture and substrate binding.

The peptidyl-prolyl cis/trans isomerase hPin1 is a phosphorylation-dependent regulatory enzyme whose substrates are proteins involved in regulation of cell cycle, transcription, Alzheimer's disease, and cancer pathogenesis. We have determined the solution structure of the two domain protein hPin1-(1-163) and its separately expressed PPIase domain (50-163) (hPin1PPIase) with an root mean square deviation of <0.5 A over backbone atoms using NMR. Domain organization of hPin1 differs from that observed in structures solved by x-ray crystallography. Whereas PPIase and WW domain are tightly packed onto each other and share a common binding interface in crystals, our NMR-based data revealed only weak interaction of both domains at their interface in solution. Interaction between the two domains of full-length hPin1 is absent when the protein is dissected into the catalytic and the WW domain. It indicates that the flexible linker, connecting both domains, promotes binding. By evaluation of NOESY spectra we can show that the alpha1/beta1 loop, which was proposed to undergo a large conformational rearrangement in the absence of sulfate and an Ala-Pro peptide, remained in the closed conformation under these conditions. Dissociation constants of 0.4 and 2.0 mm for sulfate and phosphate ions were measured at 12 degrees C by fluorescence spectroscopy. Binding of sulfate prevents hPin1 aggregation and changes surface charges across the active center and around the reactive and catalytically essential Cys113. In the absence of sulfate and/or reducing agent this residue seems to promote aggregation, as observed in hPin1 solutions in vitro.

The N-terminal basic domain of human parvulin hPar14 is responsible for the entry to the nucleus and high-affinity DNA-binding.

We have studied the cellular localization and the DNA-binding capability of human peptidyl-prolyl cis/trans isomerase hPar14. The cellular expression pattern shows an uneven distribution of the protein between cytoplasm and nucleus. To determine the nuclear localization of hPar14 in vivo the molecule was fused to green fluorescent protein and expressed in human HeLa cells. Deletion mutants of hPar14 were used to restrict a sequence, necessary for nuclear targeting, to Ser7-Lys14 of the N terminus of the protein. DNA-cellulose affinity experiments were performed to demonstrate that hPar14, which is present in the nuclear fraction, could bind to double-stranded native DNA in vitro. On the basis of homologies and similarities of hPar14 to members of the high-mobility group proteins, double-stranded DNA constructs were developed and tested for their hPar14 binding affinity in fluorescence titration assays. The protein binds preferentially to bent A-tract sequences. The binding interface of the protein was determined by 1-D and 2-D NMR studies of the complex of unlabeled DNA and uniformly 15N-labeled hPar14((1-131)). Experiments with a truncated hPar14((25-131)) showed that the unstructured N-terminal 25 amino acid residues are necessary for high-affinity binding to DNA. These findings in connection with sequence and structural homologies of hPar14 to members of the HMGB/HMGN protein family suggest a function of hPar14 in cell-cycle regulation or gene transcription.