Protein engineered variants of hepatocyte growth factor/scatter factor promote proliferation of primary human hepatocytes and in rodent liver.

BACKGROUND & AIMS: Hepatocyte growth factor/scatter factor (HGF/SF) stimulates hepatocyte DNA synthesis and protects against apoptosis; in vivo it promotes liver regeneration and reduces fibrosis. However, its therapeutic value is limited by its complex domain structure, high cost of production, instability, and poor tissue penetration due to sequestration by heparin sulfate proteoglycans (HSPGs). METHODS: Using protein engineering techniques, we created a full-length form of HGF/SF (called HP21) and a form of the small, naturally occurring HGF/SF fragment, NK1 (called 1K1), which have reduced affinity for HSPG. We characterized the stability and proliferative and anti-apoptotic effects of these variants in primary human hepatocytes and in rodents. RESULTS: Analytical ultracentrifugation showed that 1K1 and NK1 were more stable than the native, full-length protein. All 4 forms of HGF/SF induced similar levels of DNA synthesis in human hepatocytes; 1K1 and NK1 required heparin, an HSPG analogue, for full agonistic activity. All the proteins reduced levels of Fas ligand-mediated apoptosis, reducing the activity of caspase-3/7 and cleavage of poly(adenosine diphosphate-ribose) polymerase. 1K1 was more active than NK1 in rodents; in healthy mice, 1K1 significantly increased hepatocyte DNA synthesis, and in mice receiving carbon tetrachloride, it reduced fibrosis. In rats, after 70% partial hepatectomy, daily administration of 1K1 for 5 days significantly increased liver mass and the bromodeoxyuridine labeling index compared with mice given NK1. CONCLUSIONS: 1K1, an engineered form of the small, naturally occurring HGF/SF fragment NK1, has reduced affinity for HSPG and exerts proliferative and antiapoptotic effects in cultured hepatocytes. In rodents, 1K1 has antifibrotic effects and promotes liver regeneration. The protein has better stability and is easier to produce than HGF/SF and might be developed as a therapeutic for acute and chronic liver disease.

A new autocatalytic activation mechanism for cysteine proteases revealed by Prevotella intermedia interpain A.

Prevotella intermedia is a major periodontopathogen contributing to human gingivitis and periodontitis. Such pathogens release proteases as virulence factors that cause deterrence of host defenses and tissue destruction. A new cysteine protease from the cysteine-histidine-dyad class, interpain A, was studied in its zymogenic and self-processed mature forms. The latter consists of a bivalved moiety made up by two subdomains. In the structure of a catalytic cysteine-to-alanine zymogen variant, the right subdomain interacts with an unusual prodomain, thus contributing to latency. Unlike the catalytic cysteine residue, already in its competent conformation in the zymogen, the catalytic histidine is swung out from its active conformation and trapped in a cage shaped by a backing helix, a zymogenic hairpin, and a latency flap in the zymogen. Dramatic rearrangement of up to 20A of these elements triggered by a tryptophan switch occurs during activation and accounts for a new activation mechanism for proteolytic enzymes. These findings can be extrapolated to related potentially pathogenic cysteine proteases such as Streprococcus pyogenes SpeB and Porphyromonas gingivalis periodontain.

Papaya glutamine cyclotransferase shows a singular five-fold beta-propeller architecture that suggests a novel reaction mechanism.

Cyclisation of N-terminal glutamine and/or glutamate to yield pyroglutamate is an essential posttranslational event affecting a plethora of bioactive peptides and proteins. It is directly linked with pathologies ranging from neurodegenerative diseases to inflammation and several types of cancers. The reaction is catalysed by ubiquitous glutaminyl cyclotransferases (QCs), which present two distinct prototypes. Mammalian QCs are zinc-dependent enzymes with an alpha/beta-hydrolase fold. Here we present the 1.6-A-resolution structure of the other prototype, the plant analogue from Carica papaya (PQC). The hatbox-shaped molecule consists of an unusual five-fold beta-propeller traversed by a central channel, a topology that has hitherto been described only for some sugar-binding proteins and an extracellular nucleotidase. The high resistance of the enzyme to denaturation and proteolytic degradation is explained by its architecture, which is uniquely stabilised by a series of tethering elements that confer rigidity. Strikingly, the N-terminus of PQC specifically interacts with residues around the entrance to the central channel of a symmetry-related molecule, suggesting that this location is the putative active site. Cyclisation would follow a novel general-acid/base working mechanism, pivoting around a strictly conserved glutamate. This study provides a lead structure not only for plant QC orthologues, but also for bacteria, including potential human pathogens causing diphtheria, plague and malaria.