Prayer. Thanksgiving.
January 9, 2012
Plasma anchors inducing budding
January 9, 2012
J Biol Chem. 2011 Apr 22;286(16):14383-95. Epub 2011 Feb 7.
Protein targeting to exosomes/microvesicles by plasma membrane anchors.
Shen B, Wu N, Yang JM, Gould SJ.
Source
Department of Biological Chemistry, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA.
Abstract
Animal cells secrete small vesicles, otherwise known as exosomes and microvesicles (EMVs).
A short, N-terminal acylation tag can target a highly oligomeric cytoplasmic protein, TyA, into secreted vesicles (Fang, Y., Wu, N., Gan, X., Yan, W., Morell, J. C., and Gould, S. J. (2007) PLoS Biol. 5, 1267-1283).
However, it is not clear whether this is true for other membrane anchors or other highly oligomeric, cytoplasmic proteins.
We show here that a variety of plasma membrane anchors can target TyA-GFP to sites of vesicle budding and into EMVs, including:
- (i) a myristoylation tag;
- (ii) a phosphatidylinositol-(4,5)-bisphosphate (PIP(2))-binding domain;
- (iii), a phosphatidylinositol-(3,4,5)-trisphosphate-binding domain;
- (iv) a prenylation/palmitoylation tag,
- and (v) a type-1 plasma membrane protein, CD43.
However, the relative budding efficiency induced by these plasma membrane anchors varied over a 10-fold range, from 100% of control (AcylTyA-GFP) for the myristoylation tag and PIP(2)-binding domain, to one-third or less for the others, respectively.
Targeting TyA-GFP to endosome membranes by fusion to a phosphatidylinositol 3-phosphate-binding domain induced only a slight budding of TyA-GFP, ∼2% of control, and no budding was observed when TyA-GFP was targeted to Golgi membranes via a phosphatidylinositol 4-phosphate-binding domain.
We also found that a plasma membrane anchor can target two other highly oligomeric, cytoplasmic proteins to EMVs.
These observations support the hypothesis that plasma membrane anchors can target highly oligomeric, cytoplasmic proteins to EMVs.
Our data also provide additional parallels between EMV biogenesis and retrovirus budding, as the anchors that induced the greatest budding of TyA-GFP are the same as those that mediate retrovirus budding.
PMID:
21300796
[PubMed - indexed for MEDLINE]
PMCID: PMC3077638
[Available on 2012/4/22]
ISOPRENYLATION inhibition
January 9, 2012
Acc Chem Res. 2010 Sep 21;43(9):1216-26.
Targeting isoprenoid biosynthesis for drug discovery: bench to bedside.
Oldfield E.
Source
Department of Chemistry, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801, USA.
Abstract
The isoprenoid biosynthesis pathways produce the largest class of small molecules in Nature: isoprenoids (also called terpenoids).
Not surprisingly then, isoprenoid biosynthesis is a target for drug discovery, and many drugs–such as Lipitor (used to lower cholesterol), Fosamax (used to treat osteoporosis), and many anti-infectives--target isoprenoid biosynthesis.
However, drug resistance in malaria, tuberculosis, and staph infections is rising, cheap and effective drugs for the neglected tropical diseases are lacking, and progress in the development of anticancer drugs is relatively slow.
Isoprenoid biosynthesis is thus an attractive target, and in this Account, I describe developments in four areas, using in each case knowledge derived from one area of chemistry to guide the development of inhibitors (or drug leads) in another, seemingly unrelated, area.
- First, I describe mechanistic studies of the enzyme IspH, which is present in malaria parasites and most pathogenic bacteria, but not in humans. IspH is a 4Fe-4S protein and produces the five-carbon (C5) isoprenoids IPP (isopentenyl diphosphate) and DMAPP (dimethylallyl diphosphate) from HMBPP (E-1-hydroxy-2-methyl-but-2-enyl-4-diphosphate) via a 2H(+)/2e(-) reduction (of an allyl alcohol to an alkene). The mechanism is unusual in that it involves organometallic species: “metallacycles” (η(2)-alkenes) and η(1)/η(3)-allyls. These observations lead to novel alkyne inhibitors, which also form metallacycle.
- Second, I describe structure-function-inhibition studies of FPP synthase , the macromolecule that condenses IPP and DMAPP to the sesquiterpene farnesyl diphosphate (FPP) in a “head-to-tail” manner. This enzyme uses a carbocation mechanism and is potently inhibited by bone resorption drugs (bisphosphonates), which I show are also antiparasitic agents that block sterol biosynthesis in protozoa.
- Moreover, “lipophilic” bisphosphonates inhibit protein prenylation and invasiveness in tumor cells, in addition to activating γδ T-cells to kill tumor cells, and are important new leads in oncology.
- Third, I describe structural and inhibition studies of a “head-to-head” triterpene synthase, dehydrosqualene synthase (CrtM), from Staphylococcus aureus. CrtM catalyzes the first committed step in biosynthesis of the carotenoid virulence factor staphyloxanthin: the condensation of two FPP molecules to produce a cyclopropane (presqualene diphosphate). The structure of CrtM is similar to that of human squalene synthase (SQS), and some SQS inhibitors (originally developed as cholesterol-lowering drugs) block staphyloxanthin biosynthesis. Treated bacteria are white and nonvirulent (because they lack the carotenoid shield that protects them from reactive oxygen species produced by neutrophils), rendering them susceptible to innate immune system clearance–a new therapeutic approach.
- And finally, I show that the heart drug amiodarone, also known to have antifungal activity, blocks ergosterol biosynthesis at the level of oxidosqualene cyclase in Trypanosoma cruzi, work that has led to its use in the clinic as a novel antiparasitic agent.
- In each of these four examples, I use information from one area (organometallic chemistry, bone resorption drugs, cholesterol-lowering agents, heart disease) to develop drug leads in an unrelated area: a “knowledge-based” approach that represents an important advance in the search for new drugs.
PMID:
20560544
[PubMed - indexed for MEDLINE]
PMCID: PMC2943567
Free PMC Article
