The purpose of this study was to identify the receptor responsible for endocytosis of denatured collagen from blood. The major site of clearance of this material (at least 0.5 g/day in humans) is a receptor on liver sinusoidal endothelial cells (LSECs). We have now identified an 180-kDa endocytic receptor on LSECs, peptide mass fingerprinting of which revealed it to be the mannose receptor. Challenge of mannose-receptor knockout mice and their cultured LSECs revealed significantly reduced blood clearance and a complete absence of LSEC endocytosis of denatured collagen. Organ analysis of wild-type versus knockout mice after injection of denatured collagen revealed significantly reduced liver uptake in the knockout mice. Clearance/endocytosis of ligands for other receptors in these animals was as that for wild-type mice, and denatured collagen uptake in wild-type mice was not affected by other ligands of the mannose receptor, namely mannose and mannan. Furthermore, unlike that of mannose and mannan, endocytosis of denatured collagen by the mannose receptor is calcium independent. This suggests that the binding site for denatured collagen is distinct from that for mannose/mannan. Mannose receptors on LSECs appear to have less affinity for circulating triple helical type I collagen. Conclusion: The mannose receptor is the main candidate for being the endocytic denatured collagen receptor on LSECs. (HEPATOLOGY 2007;45:1454-1461

Background: The fish pathogen Aliivibrio salmonicida is the causative agent of cold-water vibriosis in marine aquaculture. The Gram-negative bacterium causes tissue degradation, hemolysis and sepsis in vivo.

The roles of 2-oxoglutarate (2OG)-dependent prolyl-hydroxylases in eukaryotes include collagen stabilization, hypoxia sensing, and translational regulation. The hypoxia-inducible factor (HIF) sensing system is conserved in animals, but not in other organisms. However, bioinformatics imply that 2OG-dependent prolyl-hydroxylases (PHDs) homologous to those acting as sensing components for the HIF system in animals occur in prokaryotes. We report cellular, biochemical, and crystallographic analyses revealing that Pseudomonas prolyl-hydroxylase domain containing protein (PPHD) contain a 2OG oxygenase related in structure and function to the animal PHDs. A Pseudomonas aeruginosa PPHD knockout mutant displays impaired growth in the presence of iron chelators and increased production of the virulence factor pyocyanin. We identify elongation factor Tu (EF-Tu) as a PPHD substrate, which undergoes prolyl-4-hydroxylation on its switch I loop. A crystal structure of PPHD reveals striking similarity to human PHD2 and a Chlamydomonas reinhardtii prolyl-4-hydroxylase. A crystal structure of PPHD complexed with intact EF-Tu reveals that major conformational changes occur in both PPHD and EF-Tu, including a >20-Å movement of the EF-Tu switch I loop. Comparison of the PPHD structures with those of HIF and collagen PHDs reveals conservation in substrate recognition despite diverse biological roles and origins. The observed changes will be useful in designing new types of 2OG oxygenase inhibitors based on various conformational states, rather than active site iron chelators, which make up most reported 2OG oxygenase inhibitors. Structurally informed phylogenetic analyses suggest that the role of prolylhydroxylation in human hypoxia sensing has ancient origins.T he hypoxia-inducible transcription factor (HIF) is a major regulator of the response to limited oxygen availability in humans and other animals (1-3). A hypoxia-sensing component of the HIF system is provided by 2-oxoglutarate (2OG)-dependent and Fe(II)-dependent oxygenases, which catalyze prolyl-4-hydroxylation of HIF-α subunits, a posttranslational modification that enhances binding of HIF-α to the von Hippel-Lindau protein (pVHL), so targeting HIF-α for proteasomal degradation. The HIF prolyl-hydroxylases (PHDs) belong to a subfamily of 2OG oxygenases that catalyze prolyl-hydroxylation, which also includes the collagen prolyl-3-hydroxylases (CP3Hs) and prolyl-4-hydroxylases (CP4Hs) (4). Subsequently identified prolyl-hydroxylases include the ribosomal prolyl-hydroxylases (OGFOD1 and Tpa1), which catalyze ribosomal protein 23 prolyl-3-hydroxylation in many eukaryotes, and slime-mold enzymes, which catalyze prolyl-4-hydroxylation of Skp1, a ubiquitin ligase subunit (5-9). The HIF-PHD-VHL triad is likely present in all animals, but probably not in other organisms (3). However, structurally informed bioinformatic analyses imply the presence of PHD homologs in bacteria (10, 11), including in Pseudomonas spp, suggesting PHDs may have ancient origins. ResultsPseudomonas spp. Cont...

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It is shown from molecular statistical considerations that a demixing instability exists in the moment space of a microbial protein expression profile. Although avoidance of demixing is generally requisite for biological function, a comparison with proteomic and genomic data suggests that many microbes lie close to the onset of this instability. Over evolutionary time scales, straying too close or into the immiscible domain may be associated with intracellular compartmentalization. DOI: 10.1103/PhysRevLett.94.178105 PACS numbers: 87.16.-b Molecular statistical approaches to demixing thermodynamics have long focused on industrially important contexts such as polymer blends, colloids and crude oil [1]. Similar avenues might also present useful insights into the intracellular thermodynamics of microbial organisms. Odijk [2] for example, proposes an equilibrium thermodynamic view of the bacterial nucleoid according to which, under conditions of excess salt, DNA tends to reversibly collapse and demix from the cytosol proteome.The proteome itself features only as a secondary focus in Odijk's particular analysis, but it is also of interest to examine how from a statistical mechanical perspective microbes apparently manage to avoid a similar intraproteomic demixing effect [3]. Expressed proteins of course do not disperse in perfectly miscible souplike fashion, but we can reasonably suppose that they must remain essentially miscible in respect of their macroscopic phase behavior. It is known for molecular mixtures in general that miscibility is sensitive to low moments of the size distribution [4], so we might anticipate that microbial intracellular stability depends analogously on moments of the proteomic expression level profile with respect to sequence length. Our principal objective here is to demonstrate this more explicitly within a model framework.Consider a crude molecular statistical formulation of the Helmholtz free energy F U ÿ TS describing the expressed protein ensemble, where U and S denote, respectively, internal energy and entropy at temperature T. We assume a continuous distribution l over length l in amino acid residues. With the total protein number density, l dl is the concentration in the cytosol having length between l and l dl. Assuming proteins with the same l can be considered indistinguishable with respect to their mutual interactions, we can then write for the entropy density over volume V of the cytosolwhere k B is Boltzmann's constant.Next we assume that the dominant contribution to the internal energy U comes from nonspecific adhesive interaction between proteins. For a system of monodisperse adhesive well particles U=V ' ÿ 2 2 ad 2 , where d is the particle diameter, a is the well width, and is its depth. In this spirit, we writewhere h. . .i denotes the distribution-averaged moment.Here we have identified a with the amino acid length scale, and set d al 1=3 to represent a compact protein comprising l residues. To look for a miscibility gap in the parameter space of this description, we...