Analysis of Hepatocyte Secretion Pathways: A Case Study on Hepatic Apolipoproteins, Serum Albumin, and Hepatitis C Virus

The hepatocyte is one of the major secretory cell types in the body. It fulfills many of the liver\u27s essential functions in protein secretion, lipid storage and transport, and excretion. Some of these functions are carried out via polarized secretion of simple protein cargo, such as serum albumin, or large macromolecular lipid-protein complexes, the lipoproteins. The hepatocyte is also the site of infection of several hepatotropic viruses. Of these, hepatitis C virus (HCV) is peculiar due to its close structural and functional association with the hepatic lipoproteins. All these cargoes are transported from the endoplasmic reticulum (ER) to the cell surface by the vesicular secretory pathway, yet insufficient knowledge exists regarding the molecular regulation of their secretion by the hepatocyte. Furthermore, differential modalities of regulation may be involved in the shuttling of such a diverse set of cargoes as albumin, the lipoproteins and HCV. The work presented here head-starts a comprehensive examination of how the hepatocyte regulates the secretion of the following cargoes: serum albumin, the apolipoproteins E and B100 (ApoE and ApoB100, respectively, both lipoprotein components, and surrogate markers for these complex macromolecular particles), and HCV, a lipoprotein-associated virus. I propose to combine genetic, biochemical, virological and imaging approaches to identify which vesicular secretory pathways are utilized by each of these cargoes. These approaches include inactivation of specific vesicular transport pathways, accompanied by measurements of their effects on cargo secretion efficiencies, and establishment of functional fluorescent protein-tagged cargo markers to be used in live cell imaging experiments. I begin by describing a dominant negative (DN) Rab GTPase screen that I performed to identify Rab proteins involved in ApoE, ApoB100 or albumin secretion. The small Rab GTPases control individual steps of vesicular transport. I analyzed how expression of individual dominant negative Rab proteins affected cargo secretion compared to expression of their wild type (WT) counterparts. I identified several Rabs that caused significant changes in secretion, many of which had previously been described as regulators of various exocytic vesicular transport steps. I next present ongoing work that aims to define the involvement of the Rabs 11a, 11b, 8a, and 8b in hepatic cargo secretion. Their dominant negative mutants exhibited some of the largest secretion phenotypes in my dominant negative Rab screen. These Rabs have been implicated in various aspects of post-Golgi secretion in polarized and non-polarized cell types. I thus discuss the implications of their involvement in cargo secretion in the polarized hepatocyte and outline my ongoing efforts to define the parameters of this involvement. I also investigated the function of Rab1b in hepatic secretion. I show that inactivation of Rab1 function, by expression of a set of dominant negative mutants, or by expression of a bacterial effector which affects Rab1 function, led to impairment of albumin, ApoE, ApoB100 and HCV secretion. I implicate Rab1, for the first time to my knowledge, in the transport of these cargoes. I also document differences in the sensitivity of cargo secretion to the various means of Rab1 inactivation. ApoE secretion, in particular, was insensitive to several means of transport inactivation, consistent with existing models of differential regulation of hepatic cargo transport. Lastly, I functionally characterize an ApoE-green fluorescent protein fusion (ApoE-GFP). I show that while ApoE-GFP does not support infectious HCV release, a hallmark function of untagged ApoE, ApoE-GFP nevertheless reproduces several known behaviors of ApoE that have been associated with lipoprotein release. I thus conclude that ApoE-GFP may be a useful marker for live cell imaging of lipoprotein release. This work therefore identifies potential regulators of hepatic cargo transport, establishes molecular tools useful for the continued study of cargo secretion in hepatocytes and elsewhere, and advances the understanding of the involvement of Rabs 11, 8, and, in particular, Rab1, in the regulation of hepatic cargo transport. I propose that this work forms a solid foundation for extensive studies on how these biomedically relevant hepatic cargoes are secreted

D-amino acids govern stationary phase cell wall remodeling in bacteria

4 pages, 4 figures.-- PMID: 19762646 [PubMed].-- Supporting information available at: http://www.sciencemag.org/cgi/content/full/sci;325/5947/1552/DC1In all known organisms, amino acids are predominantly thought to be synthesized and used as their L-enantiomers. Here, we found that bacteria produce diverse D-amino acids as well, which accumulate at millimolar concentrations in supernatants of stationary phase cultures. In Vibrio cholerae, a dedicated racemase produced D-Met and D-Leu, whereas Bacillus subtilis generated D-Tyr and D-Phe. These unusual D-amino acids appear to modulate synthesis of peptidoglycan, a strong and elastic polymer that serves as the stress-bearing component of the bacterial cell wall. D-Amino acids influenced peptidoglycan composition, amount, and strength, both by means of their incorporation into the polymer and by regulating enzymes that synthesize and modify it. Thus, synthesis of D-amino acids may be a common strategy for bacteria to adapt to changing environmental conditions.This work was supported by Howard Hughes Medical Institute (HHMI); NIH AI-R37-42347 (M.K.W.) and CA24487 and GM086258 (J.C.); Ministry of Education and Science, Spain (MEC) BFU2006-04574 and Fundación Ramón Areces (M.A.P.); Jane Coffin Childs Fellowship (H.L.); MEC Fellowship (F.C.); and HHMI Exceptional Research Opportunities (EXROP) (C.N.T.).Peer reviewe

Differential Regulation of Lipoprotein and Hepatitis C Virus Secretion by Rab1b

Secretory cells produce diverse cargoes, yet how they regulate concomitant secretory traffic remains insufficiently explored. Rab GTPases control intracellular vesicular transport. To map secretion pathways, we generated a library of lentivirus-expressed dominant-negative Rab mutants and used it in a large-scale screen to identify regulators of hepatic lipoprotein secretion. We identified several candidate pathways, including those mediated by Rab11 and Rab8. Surprisingly, inhibition of Rab1b, the major regulator of transport from the endoplasmic reticulum to the Golgi, differently affected the secretion of the very-low-density lipoprotein components ApoE and ApoB100, despite their final association on mature secreted lipoprotein particles. Since hepatitis C virus (HCV) incorporates ApoE and ApoB100 into its virus particle, we also investigated infectious HCV secretion and show that its regulation by Rab1b mirrors that of ApoB100. These observations reveal differential regulation of hepatocyte secretion by Rab1b and advance our understanding of lipoprotein assembly and lipoprotein and HCV secretion

D-amino acids govern stationary phase cell wall remodeling in bacteria

4 pages, 4 figures.-- PMID: 19762646 [PubMed].-- Supporting information available at: http://www.sciencemag.org/cgi/content/full/sci;325/5947/1552/DC1In all known organisms, amino acids are predominantly thought to be synthesized and used as their L-enantiomers. Here, we found that bacteria produce diverse D-amino acids as well, which accumulate at millimolar concentrations in supernatants of stationary phase cultures. In Vibrio cholerae, a dedicated racemase produced D-Met and D-Leu, whereas Bacillus subtilis generated D-Tyr and D-Phe. These unusual D-amino acids appear to modulate synthesis of peptidoglycan, a strong and elastic polymer that serves as the stress-bearing component of the bacterial cell wall. D-Amino acids influenced peptidoglycan composition, amount, and strength, both by means of their incorporation into the polymer and by regulating enzymes that synthesize and modify it. Thus, synthesis of D-amino acids may be a common strategy for bacteria to adapt to changing environmental conditions.This work was supported by Howard Hughes Medical Institute (HHMI); NIH AI-R37-42347 (M.K.W.) and CA24487 and GM086258 (J.C.); Ministry of Education and Science, Spain (MEC) BFU2006-04574 and Fundación Ramón Areces (M.A.P.); Jane Coffin Childs Fellowship (H.L.); MEC Fellowship (F.C.); and HHMI Exceptional Research Opportunities (EXROP) (C.N.T.).Peer reviewe

MreB Drives De Novo Rod Morphogenesis in Caulobacter crescentus via Remodeling of the Cell Wall▿ †

MreB, the bacterial actin-like cytoskeleton, is required for the rod morphology of many bacterial species. Disruption of MreB function results in loss of rod morphology and cell rounding. Here, we show that the widely used MreB inhibitor A22 causes MreB-independent growth inhibition that varies with the drug concentration, culture medium conditions, and bacterial species tested. MP265, an A22 structural analog, is less toxic than A22 for growth yet equally efficient for disrupting the MreB cytoskeleton. The action of A22 and MP265 is enhanced by basic pH of the culture medium. Using this knowledge and the rapid reversibility of drug action, we examined the restoration of rod shape in lemon-shaped Caulobacter crescentus cells pretreated with MP265 or A22 under nontoxic conditions. We found that reversible restoration of MreB function after drug removal causes extensive morphological changes including a remarkable cell thinning accompanied with elongation, cell branching, and shedding of outer membrane vesicles. We also thoroughly characterized the composition of C. crescentus peptidoglycan by high-performance liquid chromatography and mass spectrometry and showed that MreB disruption and recovery of rod shape following restoration of MreB function are accompanied by considerable changes in composition. Our results provide insight into MreB function in peptidoglycan remodeling and rod shape morphogenesis and suggest that MreB promotes the transglycosylase activity of penicillin-binding proteins

Growth medium-dependent glycine incorporation into the peptidoglycan of Caulobacter crescentus.

The peptidoglycan (PG) is a macromolecular component of the bacterial cell wall that maintains the shape and integrity of the cell. The PG of Caulobacter crescentus, unlike that of many other Gram-negative bacteria, has repeatedly been shown to contain significant amounts of glycine. This compositional peculiarity has been deemed an intrinsic characteristic of this species. By performing a comprehensive qualitative and quantitative analysis of the C. crescentus PG by high-performance liquid chromatography (HPLC) and mass spectrometry (MS), we show here that glycine incorporation into the C. crescentus PG depends on the presence of exogenous glycine in the growth medium. High levels of glycine were detected at the fifth position of the peptide side chains of PG isolated from C. crescentus cells grown in the complex laboratory medium PYE or in defined medium (M2G) supplemented with casamino acids or glycine alone. In contrast, glycine incorporation was undetectable when cells were grown in M2G medium lacking glycine. Remarkably, glycine incorporation into C. crescentus peptidoglycan occurred even in the presence of low millimolar to sub-millimolar concentrations of free glycine. High glycine content in the PG had no obvious effects on growth rates, mode of PG incorporation or cell morphology. Hence, the C. crescentus PG is able to retain its physiological functions in cell growth and morphogenesis despite significant alterations in its composition, in what we deem to be unprecedented plasticity

Summarized composition of the PG of <i>C. crescentus</i> cells grown in PYE or M2G media.

<p>Summarized composition of the PG of <i>C. crescentus</i> cells grown in PYE or M2G media.</p

Cell dimensions and growth rates of <i>C. crescentus</i> in various growth media.

<p>Cell dimensions and growth rates of <i>C. crescentus</i> in various growth media.</p

Comparison between the PG composition of <i>C. crescentus</i> cells grown in unsupplemented and glycine-supplemented M2G media.

<p>(A). Overlay of HPLC profiles of muropeptides obtained from <i>C. crescentus</i> cultures grown in the indicated media. CAA, casamino acids; empty arrowheads, Penta(Gly)-containing muropeptide peaks; filled arrowheads, peak corresponding to the PentaTri muropeptide species. (B). Relative representation (molar percentage) of each muropeptide species in PG digests obtained from <i>C. crescentus</i> cultures grown in the indicated media. Red rectangles denote glycine-containing species; Tri, Glc<i>N</i>Ac-Mur<i>N</i>Ac-l-Ala-d-γ-Glu-m-Dap ; Tetra, Glc<i>N</i>Ac-Mur<i>N</i>Ac-l-Ala-d-γ-Glu-m-Dap-d-Ala; Penta, Glc<i>N</i>Ac-Mur<i>N</i>Ac-l-Ala-d-γ-Glu-m-Dap-d-Ala-d-Ala; Penta(Gly), Glc<i>N</i>Ac-Mur<i>N</i>Ac-l-Ala-d-γ-Glu-m-Dap-d-Ala-Gly; Anh, 1,6-anhydro- Mur<i>N</i>Ac; (D,D), m-Dap-D-Ala crosslink; (L,D), m-Dap-m-Dap crosslink. Bars represent averages ± standard deviation for two (M2G, M2G + 0.2 mM Gly, M2G + 2 mM Gly) or three (M2G + 0.1% CAA) samples analyzed. (C). Summary of the composition of <i>C. crescentus</i> PG digests obtained from <i>C. crescentus</i> cultures grown in the indicated media. Major PG characteristics are shown, namely the total degree of crosslinkage, the relative amounts of differentially crosslinked muropeptide classes (monomers, dimers, trimers and tetramers), side chain types (Tri, l-Ala-d-γ-Glu-m-Dap ; Tetra, l-Ala-d-γ-Glu-m-Dap-d-Ala; Penta(Ala), l-Ala-d-γ-Glu-m-Dap-d-Ala-d-Ala; Penta(Gly), l-Ala-d-γ-Glu-m-Dap-d-Ala-Gly), or chain ends (anhydro, 1,6-anhydro-Mur<i>N</i>Ac ). The red rectangle highlights the relative amounts of the Penta(Gly) side chain. Bars are as in (B).</p

Comparison between the PG composition of <i>C. crescentus</i> cells grown in PYE and M2G media.

<p>(A). Overlay of HPLC profiles of muropeptides obtained from <i>C. crescentus</i> cultures grown in PYE (black trace) and M2G (red trace) media. Black arrowheads, Penta(Gly)-containing muropeptide peaks present only in the PYE-derived sample; red arrowhead, peak corresponding to the PentaTri muropeptide species, identified in the M2G-derived sample. (B). Relative representation (molar percentage) of each muropeptide species in PG digests obtained from <i>C. crescentus</i> cultures grown in PYE (black) and M2G (grey) media. Red rectangles denote glycine-containing species. Tri, Glc<i>N</i>Ac-Mur<i>N</i>Ac-l-Ala-d-γ-Glu-m-Dap; Tetra, Glc<i>N</i>Ac-Mur<i>N</i>Ac-l-Ala-d-γ-Glu-m-Dap-d-Ala; Penta, Glc<i>N</i>Ac-Mur<i>N</i>Ac-l-Ala-d-γ-Glu-m-Dap-d-Ala-d-Ala; Penta(Gly), Glc<i>N</i>Ac-Mur<i>N</i>Ac-l-Ala-d-γ-Glu-m-Dap-d-Ala-Gly; Anh, 1,6-anhydro- Mur<i>N</i>Ac; (D,D), m-Dap-D-Ala crosslink; (L,D), m-Dap-m-Dap crosslink. Bars represent averages ± standard deviation for the three samples analyzed. (C). Summary of the composition of <i>C. crescentus</i> PG digests obtained from cultures grown in the indicated media. Major PG characteristics are shown, namely the total degree of crosslinkage, the relative amounts of differentially crosslinked muropeptide classes (monomers, dimers, trimers and tetramers), side chain types (Tri, l-Ala-d-γ-Glu-m-Dap; Tetra, l-Ala-d-γ-Glu-m-Dap-d-Ala; Penta(Ala), l-Ala-d-γ-Glu-m-Dap-d-Ala-d-Ala; Penta(Gly), l-Ala-d-γ-Glu-m-Dap-d-Ala-Gly), or chain ends (anhydro, 1,6-anhydro-Mur<i>N</i>Ac ). Bars are as in (B). The red rectangle highlights the relative representation of the Penta(Gly) side chain. (B and C).</p