The Structural Basis of the Farnesylated and Methylated KRas4B Interaction with Calmodulin

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Tunneling nanotubes: a new route for the exchange of components between animal cells

Recently, highly sensitive nanotubular structures mediating membrane continuity between mammalian cells have been discovered. With respect to their peculiar architecture, these membrane channels were termed tunneling nanotubes (TNTs). TNTs could form de novo between animal cells leading to the generation of complex cellular networks. They have been shown to facilitate the intercellular transfer of organelles as well as, on a limited scale, of membrane components and cytoplasmic molecules. It has been proposed that TNTs represent a novel and general biological principle of cell-to-cell communication and it becomes increasingly apparent that they fulfill important functions in the physiological processes of multicellular organisms.     

ASG2 is a farnesylated DWD protein that acts as ABA negative regulator in Arabidopsis

The tagging‐via‐substrate approach designed for the capture of mammal prenylated proteins was adapted to Arabidopsis cell culture. In this way, proteins are in vivo tagged with an azide‐modified farnesyl moiety and captured thanks to biotin alkyne Click‐iT® chemistry with further streptavidin‐affinity chromatography. Mass spectrometry analyses identified four small GTPases and ASG2 (ALTERED SEED GERMINATION 2), a protein previously associated to the seed germination gene network. ASG2 is a conserved protein in plants and displays a unique feature that associates WD40 domains and tetratricopeptide repeats. Additionally, we show that ASG2 has a C‐terminal CaaX‐box that is farnesylated in vitro. Protoplast transfections using CaaX prenyltransferase mutants show that farnesylation provokes ASG2 nucleus exclusion. Moreover, ASG2 interacts with DDB1 (DAMAGE DNA BINDING protein 1), and the subcellular localization of this complex depends on ASG2 farnesylation status. Finally, germination and root elongation experiments reveal that asg2 and the farnesyltransferase mutant era1 (ENHANCED RESPONSE TO ABSCISIC ACID (ABA) 1) behave in similar manners when exposed to ABA or salt stress. To our knowledge, ASG2 is the first farnesylated DWD (DDB1 binding WD40) protein related to ABA response in Arabidopsis that may be linked to era1 phenotypes.     

Increasing the length of progerin's isoprenyl anchor does not worsen bone disease or survival in mice with Hutchinson-Gilford progeria syndrome

Hutchinson-Gilford progeria syndrome (HGPS) is caused by the synthesis of a truncated prelamin A, commonly called progerin, that contains a carboxyl-terminal farnesyl lipid anchor. The farnesyl lipid anchor helps to target progerin to membrane surfaces at the nuclear rim, where it disrupts the integrity of the nuclear lamina and causes misshapen nuclei. Several lines of evidence have suggested that progerin's farnesyl lipid anchor is crucial for the emergence of disease phenotypes. Because a geranylgeranyl lipid is approximately 45-fold more potent than a farnesyl lipid in anchoring proteins to lipid membranes, we hypothesized that a geranylgeranylated version of progerin might be more potent in eliciting disease phenotypes. To test this hypothesis, we used gene targeting to create mice expressing geranylgeranylated progerin (Lmna(ggHG/+)). We then compared Lmna(ggHG/+) mice, side-by-side, with otherwise identical mice expressing farnesylated progerin (Lmna(HG/+)). Geranylgeranylation of progerin in Lmna(ggHG/+) cells and farnesylation of progerin in Lmna(HG/+) cells was confirmed by metabolic labeling. Contrary to our expectations, Lmna(ggHG/+) mice survived longer than Lmna(HG/+) mice. The Lmna(ggHG/+) mice also exhibited milder bone disease. The steady-state levels of progerin, relative to lamin C, were lower in Lmna(ggHG/+) mice than in Lmna(HG/+) mice, providing a potential explanation for the milder disease in Lmna(ggHG/+) mice.     

A novel bisphosphonate inhibitor of squalene synthase combined with a statin or a nitrogenous bisphosphonate in vitro

Statins and nitrogenous bisphosphonates (NBP) inhibit 3-hydroxy-3-methylglutaryl-coenzyme-A reductase (HMGCR) and farnesyl diphosphate synthase (FDPS), respectively, leading to depletion of farnesyl diphosphate (FPP) and disruption of protein prenylation. Squalene synthase (SQS) utilizes FPP in the first committed step from the mevalonate pathway toward cholesterol biosynthesis. Herein, we have identified novel bisphosphonates as potent and specific inhibitors of SQS, including the tetrasodium salt of 9-biphenyl-4,8-dimethyl-nona-3,7-dienyl-1,1-bisphosphonic acid (compound 5). Compound 5 reduced cholesterol biosynthesis and lead to a substantial intracellular accumulation of FPP without reducing cell viability in HepG2 cells. At high concentrations, lovastatin and zoledronate impaired protein prenylation and decreased cell viability, which limits their potential use for cholesterol depletion. When combined with lovastatin, compound 5 prevented lovastatin-induced FPP depletion and impairment of protein farnesylation. Compound 5 in combination with the NBP zoledronate completely prevented zoledronate-induced impairment of both protein farnesylation and geranylgeranylation. Cotreatment of cells with compound 5 and either lovastatin or zoledronate was able to significantly prevent the reduction of cell viability caused by lovastatin or zoledronate alone. The combination of an SQS inhibitor with an HMGCR or FDPS inhibitor provides a rational approach for reducing cholesterol synthesis while preventing nonsterol isoprenoid depletion.     

Optimal functional levels of activation-induced deaminase specifically require the Hsp40 DnaJa1

The enzyme activation-induced deaminase (AID) deaminates deoxycytidine at the immunoglobulin genes, thereby initiating antibody affinity maturation and isotype class switching during immune responses. In contrast, off-target DNA damage caused by AID is oncogenic. Central to balancing immunity and cancer is AID regulation, including the mechanisms determining AID protein levels. We describe a specific functional interaction between AID and the Hsp40 DnaJa1, which provides insight into the function of both proteins. Although both major cytoplasmic type I Hsp40s, DnaJa1 and DnaJa2, are induced upon B-cell activation and interact with AID in vitro, only DnaJa1 overexpression increases AID levels and biological activity in cell lines. Conversely, DnaJa1, but not DnaJa2, depletion reduces AID levels, stability and isotype switching. In vivo, DnaJa1-deficient mice display compromised response to immunization, AID protein and isotype switching levels being reduced by half. Moreover, DnaJa1 farnesylation is required to maintain, and farnesyltransferase inhibition reduces, AID protein levels in B cells. Thus, DnaJa1 is a limiting factor that plays a non-redundant role in the functional stabilization of AID.     

Shoot-Specific Down-Regulation of Protein Farnesyltransferase （α-Subunit） for Yield Protection against Drought in Canola

Canola （Brassica napus L.） is one of the most important oilseed crops in the world and its seed yield and quality are significantly affected by drought stress. As an innate and adaptive response to water deficit, land plants avoid potential damage by rapid biosynthesis of the phytohormone abscisic acid （ABA）, which triggers stomatal closure to reduce transpirational water loss. The ABA-mediated stomatal response is a dosage-dependent process; thus, one genetic engineering approach for achieving drought avoidance could be to sensitize the guard cell＇s responsiveness to this hormone. Recent genetic studies have pinpointed protein farnesyltransferase as a key negative regulator controlling ABA sensitivity in the guard cells. We have previously shown that down-regulation of the gene encoding Arabidopsis β-subunit of farnesyltransferase （ERA1） enhances the plant＇s sensitivity to ABA and drought tolerance. Although the β-subunit of famesyltransferase （AtFTA） is also implicated in ABA sensing, the effectiveness of using such a gene target for improving drought tolerance in a crop plant has not been validated. Here, we report the identification and characterization of the promoter of Arabidopsis hydroxypyruvate reductase （AtHPR1）, which expresses specifically in the shoot and not in non-photosynthetic tissues such as root. The promoter region of AtHPR1 contains the core motif of the well characterized dehydration-responsive cis-acting element and we have confirmed thatAtHPR1 expression is inducible by drought stress. Conditional and specific down-regulation of FTA in canola using the AtHPR1 promoter driving an RNAi construct resulted in yield protection against drought stress in the field. Using this molecular strategy, we have made significant progress in engineering drought tolerance in this important crop species.     

Farnesylation is involved in meristem organization in Arabidopsis

 Although studies in plant and animal cell culture systems indicate farnesylation is required for normal cell cycle progression, how this lipid modification of select proteins translates into whole-organism developmental decisions involving cell proliferation or differentiation is largely unknown. The era1 mutant of the higher plant Arabidopsis thaliana (L.) Heynh. offers a unique opportunity to understand the role farnesylation may play in regulating various processes during the development of a multicellular organism. Loss of farnesylation affects many aspects of Arabidopsis growth and development. In particular, apical and axillary meristem development is altered and these phenotypes are contingent on the growth conditions. Received: 25 October 1999 / Accepted: 22 December 1999     

Mouse models of laminopathies

The A‐ and B‐type lamins are nuclear intermediate filament proteins in eukaryotic cells with a broad range of functions, including the organization of nuclear architecture and interaction with proteins in many cellular functions. Over 180 disease‐causing mutations, termed ‘laminopathies,’ have been mapped throughout LMNA, the gene for A‐type lamins in humans. Laminopathies can range from muscular dystrophies, cardiomyopathy, to Hutchinson–Gilford progeria syndrome. A number of mouse lines carrying some of the same mutations as those resulting in human diseases have been established. These LMNA‐related mouse models have provided valuable insights into the functions of lamin A biogenesis and the roles of individual A‐type lamins during tissue development. This review groups these LMNA‐related mouse models into three categories: null mutants, point mutants, and progeroid mutants. We compare their phenotypes and discuss their potential implications in laminopathies and aging.     

Thematic review series: lipid posttranslational modifications. CAAX modification and membrane targeting of Ras

Proteins that terminate with a consensus sequence known as CAAX undergo a series of posttranslational modifications that include polyisoprenylation, endoproteolysis, and carboxyl methylation. These modifications render otherwise hydrophilic proteins hydrophobic at their C termini such that they associate with membranes. Whereas prenylation occurs in the cytosol, postprenylation processing is accomplished on the cytoplasmic surface of the endoplasmic reticulum and Golgi apparatus. Among the numerous CAAX proteins encoded in mammalian genomes are many signaling molecules such as monomeric GTPases, including the Ras proteins that play an important role in cancer. In the course of their processing, nascent Ras proteins traffic from their site of synthesis in the cytosol to the endomembrane and then out to the plasma membrane (PM) by at least two pathways. Recently, retrograde pathways have been discovered that deliver mature Ras from the PM back to the Golgi. The Golgi has been identified as a platform upon which Ras can signal. Thus, the subcellular trafficking of Ras proteins has the potential to increase the complexity of Ras signaling by adding a spatial dimension. The complexity of Ras trafficking also affords a wider array of potential targets for the discovery of drugs that might inhibit tumors by interfering with Ras trafficking.