In this article we describe the molecular cloning of Pirin, a novel highly conserved 32-kDa protein consisting of 290 amino acids. Pirin was isolated by a yeast two-hybrid screen as an interactor of nuclear factor I/CCAAT box transcription factor (NFI/CTF1), which is known to stimulate adenovirus DNA replication and RNA polymerase II-driven transcription.

Intestinal cholesterol absorption is an important regulator of serum cholesterol levels. Ezetimibe is a specific inhibitor of intestinal cholesterol absorption recently introduced into medical practice; its mechanism of action, however, is still unknown. Ezetimibe neither influences the release of cholesterol from mixed micelles in the gut lumen nor the transfer of cholesterol to the enterocyte brush border membrane.

Prions mediate the pathogenesis of certain neurodegenerative diseases, including bovine spongiform encephalopathy in cattle and Creutzfeldt-Jakob disease in humans. The prion particle consists mainly, if not entirely, of PrP Sc , a posttranslationally modified isoform of the cellular host-encoded prion protein (PrP c). It has been suggested that additional cellular factors might be involved in the physiological function of PrP c and in the propagation of PrP Sc. Here we employ a Saccharomyces cerevisiae two-hybrid screen to search for proteins which interact specifically with the Syrian golden hamster prion protein. Screening of a HeLa cDNA library identified heat shock protein 60 (Hsp60), a cellular chaperone as a major interactor for PrP c. The specificity of the interaction was confirmed in vitro for the recombinant proteins PrP c 23-231 and rPrP27-30 fused to glutathione S-transferase with recombinant human Hsp60 as well as the bacterial GroEL. The interaction site for recombinant Hsp60 and GroEL proteins was mapped between amino acids 180 and 210 of the prion protein by screening with a set of recombinant PrP c fragments. The binding of Hsp60 and GroEL occurs within a region which contains parts of the putative ␣-helical domains H3 and H4 of the prion protein.

Members of the CCAAT-binding transcription factor (CTF) family of proteins stimulate the initiation of adenovirus DNA replication and act as transcriptional activators. To investigate the mechanisms underlying CTF-mediated transactivation patterns, we expressed several natural CTF variants in Saccharomyces cerevisiae and determined their transactivating activities in enzymatic assays. CTF7, which lacks the entire proline-rich region previously thought to mediate transcriptional activation by CTF proteins, enhances transcription to a greater degree than full-length CTF1, which contains the putative activation domain. CTF2, which contains a partially deleted proline-rich activation region, does not stimulate transcription at all. These findings indicate that the proline-rich region of CTF proteins is not essential for transcriptional activation in yeast. Our studies also suggest a bipartite two-domain structure of CTF-tpe transcriptional activation domains.Transcriptional activation by many DNA sequence-specific regulatory factors is thought to be mediated via protein domains rich in either acidic side chains, glutamine residues, or proline residues (1). Recent data suggest that these domains are recognized by a class of polypeptides, termed TATA box binding protein-associated factors, or TAFs, which are part ofthe multisubunit protein complex, TFIID (2, 3), that plays a pivotal role in controlling RNA polymerase II-dependent transcription.

To identify protein components of the intestinal cholesterol transporter, rabbit small intestinal brush border membrane vesicles were submitted to photoaffinity labeling using photoreactive derivatives of 2-azetidinone cholesterol absorption inhibitors. An integral membrane protein of M r 145.3 þ 7.5 kDa was specifically labeled in brush border membrane vesicles from rabbit jejunum and ileum. Its labeling was concentrationdependently inhibited by the presence of cholesterol absorption inhibitors whereas bile acids, D-glucose, fatty acids or cephalexin had no effect. The inhibitory potency of 2-azetidinones to inhibit photolabeling of the 145 kDa protein correlated with their in vivo activity to inhibit intestinal cholesterol absorption. These results suggest that an integral membrane protein of M r 145 kDa is (a component of) the cholesterol absorption system in the brush border membrane of small intestinal enterocytes. ß