TUGAS MATA KULIAH BIOKIMIA

Disusun oleh:

Dita Angga Sukma J1004150

FAKULTAS PETERNAKAN

UNIVERSITAS PADJADJARAN

SUMEDANG

2010

LIPID

Extensive sphingolipid depletion does not affect lipid raft

integrity or lipid raft localization and efflux function of the ABC

transporter MRP1

Karin Klappe*, Anne-Jan Dijkhuis*, Ina Hummel*, Annie van Dam†,

Pavlina T. Ivanova‡, Stephen B. Milne‡, David S. Myers‡, H. Alex Brown‡,

Hjalmar Permentier† and Jan W. Kok*1

*Department of Cell Biology, Section Membrane Cell Biology, University Medical Center

Groningen, A. Deusinglaan 1, 9713 AV Groningen, The Netherlands, †Mass Spectrometry

Core Facility, University of Groningen, A. Deusinglaan 1, 9713 AV Groningen, The

Netherlands, and ‡Departments of Pharmacology and Chemistry, Vanderbilt University

School of Medicine, 23rd Avenue South at Pierce, Nashville, TN 37232, U.S.A.

We show that highly efficient depletion of sphingolipids in two different cell lines does not

abrogate the ability to isolate Lubrol-based DRMs (detergent-resistant membranes) or

detergent-free lipid rafts from these cells. Compared with control, DRM/detergent-free lipid

raft fractions contain equal amounts of protein, cholesterol and phospholipid, whereas the

classical DRM/lipid raft markers Src, caveolin-1 and flotillin display the same gradient

distribution. DRMs/detergent-free lipid rafts themselves are severely depleted of

sphingolipids. The fatty acid profile of the remaining sphingolipids as well as that of the

glycerophospholipids shows several differences compared with control, most prominently an

increase in highly saturated C16 species. The glycerophospholipid headgroup composition is

unchanged in sphingolipid-depleted cells and cell-derived detergent-free lipid rafts.

Sphingolipid depletion does not alter the localization of MRP1 (multidrug-resistance-related

protein 1) in DRMs/detergent-free lipid rafts or MRP1-mediated efflux of

carboxyfluorescein. We conclude that extensive sphingolipid depletion does not affect lipid

raft integrity in two cell lines and does not affect the function of the lipid-raft-associated

protein MRP1.

Key words: caveolin, detergent-free lipid raft, flotillin, multidrug-resistance-related protein 1

(MRP1), Neuro-2a cell, Src.

Abbreviations used: ABC, ATP-binding cassette; BHK, baby hamster kidney; Cav-1,

caveolin-1; Cer, ceramide; CFDA, 5-carboxyfluorescein diacetate; DRM, detergent-resistant

membrane; ECL, enhanced chemiluminescence; ESI, electrospray ionization; FBS, fetal

bovine serum; GlcCer, glucosylceramide; GCS, glucosylceramide synthase; HBSS, Hanks

balanced salt solution; HPTLC, high-performance TLC; LacCer, lactosylceramide; LC, liquid

chromatography; LCB, long-chain base subunit; MDR, multidrug resistance; MRP1,

multidrug-resistance-related protein 1; MS/MS, tandem MS; MTT, 3-(4,5-dimethylthiazol-2-

yl)-2,5-diphenyl-2H-tetrazolium bromide; PA, phosphatidic acid; PC, phosphatidylcholine;

PE, phosphatidylethanolamine; PG, phosphatidylglycerol; Pgp, P-glycoprotein; PI,

phosphatidylinositol; PNS, post-nuclear supernatant; PS, phosphatidylserine; RNAi, RNA

interference; siRNA, small interfering RNA; SM, sphingomyelin; SPTLC, serine

palmitoyltransferase long-chain base subunit.

1To whom correspondence should be addressed (email j.w.kok@med.umcg.nl).

Received 16 December 2009/2 July 2010; accepted 6 July 2010

Published as BJ Immediate Publication 6 July 2010, doi:10.1042/BJ20091882

© The Authors Journal compilation © 2010 Biochemical Society

VITAMIN

Regulation of renal sodium-dependent phosphate co-transporter

genes (Npt2a and Npt2c) by all-trans-retinoic acid and its

receptors

Masashi Masuda*1, Hironori Yamamoto*12, Mina Kozai*, Sarasa Tanaka*,

Mariko Ishiguro*, Yuichiro Takei*, Otoki Nakahashi*, Shoko Ikeda*,

Takashi Uebanso*, Yutaka Taketani*, Hiroko Segawa†, Ken-ichi Miyamoto† and

Eiji Takeda*

*Department of Clinical Nutrition, Institute of Health Biosciences, University of Tokushima

Graduate School, Kuramoto-Cho 3-18-15, Tokushima City, 770-8503, Japan, and

†Department of Molecular Nutrition, Institute of Health Biosciences, University of

Tokushima Graduate School, Kuramoto-Cho 3-18-15, Tokushima City, 770-8503, Japan

The type II sodium-dependent phosphate co-transporters Npt2a and Npt2c play critical roles

in the reabsorption of Pi by renal proximal tubular cells. The vitamin A metabolite ATRA

(all-trans-retinoic acid) is important for development, cell proliferation and differentiation,

and bone formation. It has been reported that ATRA increases the rate of Pi transport in renal

proximal tubular cells. However, the molecular mechanism is still unknown. In the present

study, we observed the effects of a VAD (vitamin A-deficient) diet on Pi homoeostasis and

the expression of Npt2a and Npt2c genes in rat kidney. There was no change in the plasma

levels of Pi, but VAD rats significantly increased renal Pi excretion. Renal brush-border

membrane Pi uptake activity and renal Npt2a and Npt2c expressions were significantly

decreased in VAD rats. The transcriptional activity of a luciferase reporter plasmid

containing the promoter region of human Npt2a and Npt2c genes was increased markedly by

ATRA and a RAR (retinoic acid receptor)-specific analogue TTNPB {4-[E-2-(5,6,7,8-

tetrahydro-5,5,8,8-tetra-methyl-2-naphtalenyl)-1-propenyl] benzoic acid} in renal proximal

tubular cells overexpressing RARs and RXRs (retinoid X receptors). Furthermore, we

identified RAREs (retinoic acid-response elements) in both gene promoters. Interestingly, the

half-site sequences (5′-GGTTCA-3′: −563 to −558) of 2c-RARE1 overlapped the vitamin D-

responsive element in the human Npt2c gene and were functionally important motifs for

transcriptional regulation of human Npt2c by ATRA and 1,25(OH)2D3 (1α,25-

dihydroxyvitamin D3), in both independent or additive actions. In summary, we conclude that

VAD induces hyperphosphaturia through the down-regulation of Npt2a and Npt2c gene

expression in the kidney.

Key words: all-trans retinoic acid (ATRA), gene promoter analysis, phosphate homoeostasis,

retinoic acid nuclear receptor, renal type II sodium-dependent phosphate co-transporter (Npt),

vitamin A.

Abbreviations used: ATRA, all-trans-retinoic acid; BBMV, brush-border membrane vesicle;

β-gal, β-galactosidase; Cr, creatinine; 1,25(OH)2D3, 1α,25-dihydroxyvitamin D3; DR, direct

repeat; EMSA, electrophoretic mobility-shift assay; FBS, fetal bovine serum; FEI, fractional

excretion index; FGF23, fibroblast growth factor 23; Npt, sodium-dependent phosphate co-

transporter; NF-κB, nuclear factor κB; OK cell, opossum kidney cell; PTH, parathyroid

hormone; RAR, retinoic acid receptor; RARE, retinoic acid-responsive element; RXR,

retinoid X receptor; TTNPB, 4-[E-2-(5,6,7,8-tetrahydro-5,5,8,8-tetra-methyl-2-naphtalenyl)-

1-propenyl] benzoic acid; VAD, vitamin A-deficient; VDR, vitamin D receptor; VDRE,

vitamin D-responsive element.

1These authors contributed equally to this work.

2To whom correspondence should be addressed (email yamamoto@nutr.med.tokushima-

u.ac.jp).

Received 6 April 2010/17 May 2010; accepted 27 May 2010

Published as BJ Immediate Publication 27 May 2010, doi:10.1042/BJ20100484

© The Authors Journal compilation © 2010 Biochemical Society

KARBOHIDRAT

Botulinum neurotoxin serotype D attacks neurons via two

carbohydrate-binding sites in a ganglioside-dependent manner

Jasmin Strotmeier*1, Kwangkook Lee†1, Anne K. Völker*1, Stefan Mahrhold‡,

Yinong Zong†, Johannes Zeiser*, Jie Zhou†, Andreas Pich*, Hans Bigalke*,

Thomas Binz‡, Andreas Rummel*2 and Rongsheng Jin†2

*Institut für Toxikologie, Medizinische Hochschule Hannover, Carl-Neuberg-Str. 1, 30625

Hannover, Germany, †Center for Neuroscience, Aging and Stem Cell Research, Sanford-

Burnham Medical Research Institute, 10901 North Torrey Pines Road, La Jolla, CA 92037,

U.S.A., and ‡Institut für Biochemie, Medizinische Hochschule Hannover, Carl-Neuberg-Str.

1, 30625 Hannover, Germany

The extraordinarily high toxicity of botulinum neurotoxins primarily results from their

specific binding and uptake into neurons. At motor neurons, the seven BoNT (botulinum

neurotoxin) serotypes A–G inhibit acetylcholine release leading to flaccid paralysis. Uptake

of BoNT/A, B, E, F and G requires a dual interaction with gangliosides and the synaptic

vesicle proteins synaptotagmin or SV2 (synaptic vesicle glycoprotein 2), whereas little is

known about the cell entry mechanisms of the serotypes C and D, which display the lowest

amino acid sequence identity compared with the other five serotypes. In the present study we

demonstrate that the neurotoxicity of BoNT/D depends on the presence of gangliosides by

employing phrenic nerve hemidiaphragm preparations derived from mice expressing the

gangliosides GM3, GM2, GM1 and GD1a, or only GM3 [a description of our use of

ganglioside nomenclature is given in Svennerholm (1994) Prog. Brain Res. 101, XI–XIV].

High-resolution crystal structures of the 50 kDa cell-binding domain of BoNT/D alone and in

complex with sialic acid, as well as biological analyses of single-site BoNT/D mutants

identified two carbohydrate-binding sites. One site is located at a position previously

identified in BoNT/A, B, E, F and G, but is lacking the conserved SXWY motif. The other

site, co-ordinating one molecule of sialic acid, resembles the second ganglioside-binding

pocket (the sialic-acid-binding site) of TeNT (tetanus neurotoxin).

Key words: botulinum neurotoxin D (BoNT/D), crystal structure, ganglioside-binding site,

HC fragment, sialic acid complex.

Abbreviations used: BoNT, botulinum neurotoxin; CNT, clostridial neurotoxin; GD3S, GD3

synthetase; GM3S, GM3 synthetase; HC, heavy chain; HCA, BoNT/A HC fragment; HCB,

BoNT/B HC fragment; HCD, BoNT/D HC fragment; KO, knockout; LC, light chain; MALDI,

matrix-assisted laser-desorption ionization; MPN, mice phrenic nerve; NAcGal, N-

acetylgalactosamine; NAcGalT, β-1,4-N-acetylgalactosamine transferase; NAcNeu, N-

acetylneuraminic acid (salic acid); PEG, poly(ethylene glycol); RMSD, root mean square

deviation; SV, synaptic vesicle; Syt, synaptotagmin; TeNT, tetanus neurotoxin; TOF, time-

of-flight.

1These authors contributed equally to this work.

2Correspondence may be addressed to either of these authors (email rummel.andreas@mh-

hannover.de or rjin@sanfordburnham.org).

The co-ordinates and diffraction data for the apo-BoNT/D HC fragment and BoNT/D HC

fragment–NAcNeu have been deposited in the PDB under codes 3OBR and 3OBT

respectively.

Received 12 July 2010/11 August 2010; accepted 12 August 2010

Published as BJ Immediate Publication 12 August 2010, doi:10.1042/BJ20101042

© The Authors Journal compilation © 2010 Biochemical Society

ENZIM

NAD-malic enzymes of Arabidopsis thaliana display distinct kinetic

mechanisms that support differences in physiological control

Marcos A. Tronconi*, Mariel C. Gerrard Wheeler*, Verónica G. Maurino†,

María F. Drincovich* and Carlos S. Andreo*1

*Centro de Estudios Fotosintéticos y Bioquímicos (CEFOBI), Universidad Nacional de

Rosario, Suipacha 531, Rosario, Argentina, and †Botanisches Institut, Universität zu Köln,

Zülpicher Str. 47b, 50674, Cologne, Germany.

The Arabidopsis thaliana genome contains two genes encoding NAD-MEs [NAD-dependent

malic enzymes; NAD-ME1 (TAIR accession number At4G13560) and NAD-ME2 (TAIR

accession number At4G00570)]. The encoded proteins are localized to mitochondria and

assemble as homo- and hetero- dimers in vitro and in vivo. In the present work, the kinetic

mechanisms of NAD-ME1 and -ME2 homodimers and NAD-MEH (NAD-ME heterodimer)

were studied as an approach to understand the contribution of these enzymes to plant

physiology. Product-inhibition and substrate-analogue analyses indicated that NAD-ME2

follows a sequential ordered Bi-Ter mechanism, NAD being the leading substrate followed

by L-malate. On the other hand, NAD-ME1 and NAD-MEH can bind both substrates

randomly. However, NAD-ME1 shows a preferred route that involves the addition of NAD

first. As a consequence of the kinetic mechanism, NAD-ME1 showed a partial inhibition by

L-malate at low NAD concentrations. The analysis of a protein chimaeric for NAD-ME1 and

-ME2 indicated that the first 176 amino acids are associated with the differences observed in

the kinetic mechanisms of the enzymes. Furthermore, NAD-ME1, -ME2 and -MEH catalyse

the reverse reaction (pyruvate reductive carboxylation) with very low catalytic activity,

supporting the notion that these isoforms act only in L-malate oxidation in plant

mitochondria. The different kinetic mechanism of each NAD-ME entity suggests that, for a

metabolic condition in which the mitochondrial NAD level is low and the L-malate level is

high, the activity of NAD-ME2 and/or -MEH would be preferred over that of NAD-ME1.

Key words: Arabidopsis thaliana, kinetic mechanism, malate decarboxylation, NAD-

dependent malic enzyme (NAD-ME), product inhibition, pyruvate carboxylation.

Abbreviations used: E, free enzyme; EC, Enzyme Commission; MDH, malate

dehydrogenase; E-MAL, enzyme linked to L-malate; ME, malic enzyme; NAD-ME, NAD-

dependent ME; NADP-ME, NADP-dependent ME; NAD-MEH, NAD-ME heterodimer;

OAA, oxaloacetate; TAIR, The Arabidopsis Information Resource.

1To whom correspondence should be addressed (carlosandreo@cefobi-conicet.gov.ar)

Received 1 April 2010/1 June 2010; accepted 9 June 2010

Published as BJ Immediate Publication 9 June 2010, doi:10.1042/BJ20100497

© The Authors Journal compilation © 2010 Biochemical Society

PROTEIN

The substrates and binding partners of protein kinase Cε

Philip M. Newton*1 and Robert O. Messing†

*School of Medicine, Swansea University, Grove Building, Singleton Park Campus, Swansea

SA2 8PP, Wales, U.K., and †Ernest Gallo Clinic and Research Center, Department of

Neurology, University of California San Francisco, 5858 Horton Street, Emeryville, CA

94608, U.S.A.

The ε isoform of protein kinase C (PKCε) has important roles in the function of the cardiac,

immune and nervous systems. As a result of its diverse actions, PKCε is the target of active

drug-discovery programmes. A major research focus is to identify signalling cascades that

include PKCε and the substrates that PKCε regulates. In the present review, we identify and

discuss those proteins that have been conclusively shown to be direct substrates of PKCε by

the best currently available means. We will also describe binding partners that anchor PKCε

near its substrates. We review the consequences of substrate phosphorylation and discuss

cellular mechanisms by which target specificity is achieved. We begin with a brief overview

of the biology of PKCε and methods for substrate identification, and proceed with a

discussion of substrate categories to identify common themes that emerge and how these may

be used to guide future studies.

Key words: anchoring protein, drug discovery, phosphorylation, protein kinase Cε (PKCε),

signalling.

Abbreviations used: AS, analogue-selective; cMyBPC, cardiac myosin-binding protein C;

DAG, sn-1,2-diacylglycerol; ENH1, enigma homologue protein-1; eNOS, endothelial nitric

oxide synthase; F-actin, filamentous actin; GABAA, type A γ-aminobutyric acid; GAP,

GTPase-activating protein; IFN, interferon; IQGAP1, IQ motif-containing GAP1; LPS,

lipopolysaccharide; MAPK, mitogen-activated protein kinase; mTOR, mammalian target of

rapamycin; PDK1, phospholipid-dependent kinase 1; PDLIM5, PDZ and LIM domain

protein 5; PKC, protein kinase C; aPKC, atypical PKC; cPKC, conventional PKC; nPKC,

novel PKC; PKD, protein kinase D; RACK, receptor for activated C-kinase; STAT3, signal

transducer and activator of transcription 3; TLR4, Toll-like receptor 4; TRAM, TRIF [TIR

(Toll/interleukin-1 receptor) domain-containing adaptor protein inducing IFNβ]-related

adaptor molecule; TRH, thyrotropin-releasing hormone; TRPV1, transient receptor potential

vanilloid 1; VDAC1, voltage-dependent anion channel 1.

1To whom correspondence should be addressed (email p.newton@swansea.ac.uk).

Received 20 August 2009/25 January 2010; accepted 26 January 2010

Published online 29 March 2010, doi:10.1042/BJ20091302

© The Authors Journal compilation © 2010 Biochemical Society

MINERAL

Milk minerals (including trace elements) and bone health

Kevin D. Cashman, a,

aDepartment of Food and Nutritional Sciences, and Department of Medicine, University

College, Cork, Ireland

Received 12 September 2005;

accepted 31 May 2006.

Available online 28 August 2006.

Abstract

Osteoporosis is a global health problem that will take on increasing significance as people

live longer and the world's population continues to increase in number. Thus, there is an

urgent need to develop and implement nutritional approaches and policies for the prevention

and treatment of osteoporosis. However, to develop preventative strategies, it is important to

determine which modifiable factors, especially nutritional factors, are able to improve bone

health throughout life. The present review will firstly, and very briefly, define the principal

disease of bone mass (i.e., osteoporosis) and its risk factors, and will then focus on the

importance of ‘milk minerals’ in bone health. While there are 20 essential minerals, and all of

these are present in milk at some concentration, for the purposes of this review only a

selected number of minerals (calcium, phosphorus, magnesium, sodium, potassium and zinc)

will be discussed in relation to bone health.

Keywords: Milk minerals; Trace elements; Bone