Prion endosome recycling and FTIR strain differentiation
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Copper stimulates endocytosis of the prions
Copper chaperones: no known homology to prion protein
Cycling in early endosomes
Strain-dependent differences in beta-sheet conformations of rogue prion
Rapid acquisition of beta-sheet structure in the prion protein prior to multimer formation.
No prion disease in 33 UK haemophilic patients by 1995
Medical Progress: CJD and Related TSEs ... review by Gibbs
Sensitivity of the Western blot: detection of PrPres in natural sheep scrapie
New E200K cluster in Japan
Genetic factors affecting BSE and scrapie susceptibility

Copper Stimulates Endocytosis of the Prion Protein

J Biol Chem, Vol. 273, Issue 50, 33107-33110, December 11, 1998
Peter C. Pauly and David A. Harris
The authors report that copper rapidly and reversibly stimulates endocytosis of PrPC from the cell surface, an effect that may be physiologically relevant and suggest PrPC could serve as a recycling receptor for uptake of copper ions from the extracellular milieu.

They review newish data suggesting that PrPC may play a role in the metabolism of copper:

--The N-terminal half of PrPC contains a series of histidine- and glycine-containing peptide repeats that are capable of binding copper ions. Synthetic peptides or recombinant PrP fragments encompassing the repeats bind copper with a Kd of 5-10 M, and binding of the metal induces conformational alterations in the polypeptide chain. Copper also facilitates restoration of protease resistance and infectivity during refolding of guanidine-denatured PrPSc.

-- The total content of copper but not of several other metals is only 20% of normal in crude membranes, synaptosomes, and endosomes derived from the brains of Prn-p0/0 mice that contain a disrupted PrP gene . This result suggests that PrPC may be a major copper-binding protein in brain.

-- Neuronal Cu-Zn superoxide dismutase (SOD) from Prn-p0/0 mice is less enzymatically active and incorporates less radioactive copper than the enzyme from normal mice; the opposite is true for SOD from transgenic mice that over-express PrPC. Neurons cultured from Prn-p0/0 mice are also more sensitive to oxidative insult, perhaps because of reduced SOD activity. These observations raise the possibility that PrPC could be involved in delivery of copper ions to SOD and perhaps other cuproenzymes.

-- PC12 cells selected for resistance to copper toxicity have increased expression of PrPC, suggesting a role for the protein in removal or detoxification of copper.

Though the normal function of the prion protein remains a mystery, they note that:

-- bound copper may serve as an essential cofactor for an as yet undetermined enzymatic activity of PrPC, as it does in cuproproteins such as SOD and cytochrome c oxidase.

-- by virtue of its presence on the cell surface, PrPC could also function as a sink for chelation of extracellular copper ions or as a carrier protein for uptake and delivery of copper ions to intracellular targets.

-- PrPC is constitutively endocytosed from the plasma membrane via clathrin-coated pits and transits an early endosomal compartment before being recycled back to the cell surface with copper having a dramatic effect on this cycle.

They found, for stably transfected N2a mouse neuroblastoma cells, at concentrations of 200ݵM and above, CuSO4 significantly reduced the amounts of both mouse and chicken on the cell surface, an effect not due to a generalized increased in endocytosis of surface protein. The effect of copper was rapidly reversible: within 15min of removal, PrP began to redistribute from the inside to the outside of the cell, as assayed by accessibility to PIPLC Deleting eight hexapeptide repeats of chicken more or less abolished the effect of copper; deleting the first or last 4 attenuated the effect.

One hypothesis offered to explain "how binding of copper could stimulate endocytosis of PrPC is that the metal, by altering the conformation of PrPC, increases the affinity of the protein for a putative endocytic receptor that localizes PrPC in clathrin-coated pits. Because PrPC is attached to the cell surface by a glycosyl-phosphatidylinositol anchor, it lacks a cytoplasmic domain that could bind directly to the intracellular components of coated pits. We have therefore postulated the existence of a transmembrane receptor whose extracellular domain binds PrPC via its N-terminal region and whose cytoplasmic domain contains signals for interacting with adapter molecules and clathrin."

The copper concentrations giving rise to the effect are unnaturally high, given a micromolar binding constant, though actual copper concentrations in complex media are probably lower. One unattractive hypothesis is that PrPC serves as a receptor for uptake of copper ions analogously to transferrin or as a copper chaperone: cells already have a plethora of high affinity copper uptake systems, identified non-homologous cytoplasmic copper chaperones, and expected binding partners are missing from yeast tw0-hybrid screens. Still, there could be unknown uptake or chaperone systems, or the prion protein could be specialized, say, to remove toxic copper from the immediate vicinity of synapses. These possibilities were explored here earlier from a structure/function analysis of repeat regions. Whatever the explanation, it needs to be consistent with the data presented here and elsewhere concerning copper.

Japanese Creutzfeldt-Jakob disease patients exhibiting high incidence of the E200K PRNP mutation and located in the basin of a river.

Neurol Res 1998 Dec;20(8):684-8 
Miyakawa T, Inoue K, Iseki E, Kawanishi C, Sugiyama N, Onishi H, Yamada Y,
Suzuki K, Iwabuchi K, Kosaka K
Seven cases with Creutzfeldt-Jakob disease (CJD) located in the basin of the Fuji river (Fuji area) in Japan were examined genetically and clinicopathologically. The onset of the disease was between 1989 and 1995. All cases were from different families, although 3 cases were family members of previously reported CJD patients. They had clinical and/or neuropathological features, corresponding to subacute spongiform encephalopathy. Five of the 7 cases, including the 3 familial cases, had the E200K mutation in the gene encoding prion protein (PRNP).

It is suggested that there is a small cluster of CJD patients with a founder effect of the E200K mutation in the Fuji area, because the incidence of CJD with the E200K mutation appears to be much higher in this area than other areas in Japan. The disease penetrance of the 5 cases with the E200K mutation seems to be low, and they may have an age-related incidence in the Fuji area. These findings support the hypothesis that the phenotypes of CJD patients with the PRNP mutations are linked to the position of the mutation, but not related to ethnic or environmental factors.

Strain-dependent differences in beta-sheet conformations of abnormal prion protein

J Biol Chem, Vol. 273, Issue 48, 32230-32235, November 27, 1998
Byron Caughey, Gregory J. Raymond, and Richard A. Bessen
The abstract notes "infection of hamsters with hyper (HY), drowsy (DY), and 263K TSE strains resulted in the formation of PrP-res with different conformations using limited proteinase K (PK) digestion and infrared spectroscopy. PrP-res isolated from the brains of hamsters yielded similar SDS gel profiles prior to PK treatment. However, after limited digestion with PK, the PrP-res from the DY strain exhibited a distinct fragmentation pattern."

"Infrared spectra of HY and 263K PrP-res each had major absorption bands in the amide I region at 1626and 1636 [cm-1 wavenumbers] both prior to and after digestion with PK. These bands were not evident in the DY PrP-res spectra, which had a unique band at 1629-1630 and stronger band intensity at both 1616and 1694-1695 . Because absorbances from 1616to 1636 of protein infrared spectra are attributed primarily to beta-sheet structures, these findings indicate that the conformations of HY and 263K PrP-res differ from DY PrP-res at least in structural regions with beta-sheet secondary structure. These results support the hypothesis that strain-specific PrP-res conformers can self-propagate by converting the normal prion protein to the abnormal conformers that induce phenotypically distinct TSE diseases."

Evidence for the existence of multiple conformations of PrP-res: PrP-res associated with different strains of TSEs are cleaved at different N-terminal sites by proteinase K (PK). This was first documented with the hyper (HY) and drowsy (DY) strains of transmissible mink encephalopathy (TME) passaged in Syrian golden hamsters; the 2-kDa larger molecular mass than PrP-res from DY-infected hamsters in SDS-PAGE analysis was not due to glycosylation because it was observed in both unglycosylated and glycosylated PrP-res and supported by cell-free conversion reaction studies.

Curiously, a 7k band was observed in DY PrP-res samples and to a lesser extent in 263K and HY PrP-res; its relationship to a similar band characteristic of GSS was not determined, though 143-156 and109-112 antibody did not recognize it.

Most of the paper is technical details of IR spectroscopy of protein amides as related to secondary structure. This technique is rarely used in protein chemistry because most spectral details are not interpretable. (Synchrotron fiber diffraction is far more informative method of actual structure determination in cross-beta congophilic disorders; it has not tried for strain differentiation.)

Although the authors fairly say, "these spectral differences provide physical evidence that DY PrP-res has a distinct conformation from HY and 263K PrP-res even though they are derived from PrP-sen molecules with identical primary structures" it is important to remember that these strains fail to have the same covalent structure or even a unique structure by the time they are studied, due to proteolytic clipping and ragged ends, variable glycosylation, and possibly arginine modifications.

Thus, it has not been shown that these or any other strains differ solely by conformation. Proteins with different covalent structures cannot possibly have identical conformations even where they overlap due to properties of the Schroedinger equation (though the change might be difficult to detect, say if the change is an unordered peptide in a weakly coupled second domain).

In fact, "no significant differences in secondary structure composition were observed among the PrP-res preparations from the different TSE strains. DY PrP-res differs from HY and 263K PrP-res in the character rather than the proportion of beta-sheet structures." Unfortunately, the difference in "character" could not be translated to an differential structural description. The authors note absorption of beta-sheet carbonyls can be modulated by strength/length of hydrogen-bonding, distortions of sheet structure, solvent exposure, and local dielectric constant. ie a big change in spectra does not necessarily imply significant changes in atomic positions. Note also that beta-sheet did not come at the expense of alpha helix; the latter also increased. [Table 1 shows all 3 strains at 51% beta, 13% alpha untreated; 60% beta 17% alpha after PK treatment.]

The authors conclude that "distinguishing physical and biochemical properties of DY PrP-res could encode the distinct phenotypic properties of DY TME, which is characterized by incubation periods of 6months, a progressive lethargy, and prominent PrP-res deposits along white matter tracts in the brain" ... and that similarities between HY and 263K strains were consistent with the biological properties of these shorter incubation TSE pathogens that cause hyperexcitability, ataxia, and a widespread distribution of PrP-res in the brain gray matter ."

They propose that HY TME is an independent re-isolation of the 263K scrapie strain. Safar et al. similarly had difficulty initially in discriminating HYL and SC237 [Nat Med. 1998 Oct;4(10):1157-65 or see commentary. Safar studied HY, DY, 139H, Sc237, Me7, Me7-H, Mt-c5, and RML. The only identified strains in Collinge et al Neurosci Lett. 1998 Oct 23;255(3):159-62. were CH1641 and SSBP/1]. FTIR may thus serve as a rapid method for TSE strain typing but might have better strain separation if coupled with Safar's methods.

This paper concludes reasonably enough with the observation that "uncertainty remains as to whether PrP-res is, in and of itself, the infectious agent and whether conformational variation of PrP-res is a cause or an effect of TSE strain characteristics in vivo."

Retrospective neuropathological review of prion disease in UK haemophilic patients.

Thromb Haemost 1998 Dec;80(6):909-11 
Lee CA, Ironside JW, Bell JE, Giangrande P, Ludlam C, Esiri MM, McLaughlin JE
In 1996, the CJD surveillance unit in Edinburgh, UK described nvCJD which was thought to be the human equivalent of bovine spongiform encephalopathy (BSE). The identification of prion protein in the tonsil of an affected individual has raised the question of transmission of nvCJD via blood products. This study examines the post mortem brains of 33 patients who were treated with clotting factor concentrate of predominately UK donor source during the years 1962-1995.

The brains were examined by conventional histological methods and also for the prion protein using monoclonal antibodies KG9 and 3F4. No evidence of spongiform encephalopathy was found and the immunocytochemistry was negative for PrP in all cases. It is concluded that, at present, there is no evidence for the transmission of nvCJD via clotting factor concentrate to patients with haemophilia.

Medical Progress: Creutzfeldt-Jakob Disease and Related TSEs

Richard T. Johnson, Clarence J. Gibbs
New England Journal of Medicine -- December 31, 1998 -- Volume 339, Number 27 
This has not yet appeared on Medline. NEJM now allows fax or snailmail copies of single articles to be ordered online for $10 but does not allow instant access to fulltext online for a fee.

Rapid acquisition of beta-sheet structure in the prion protein prior to multimer formation.

Biol Chem 1998 Nov;379(11):1307-17 
Post K, Pitschke M, Schafer O, Wille H, Appel TR, Kirsch D, Mehlhorn I,
Serban H, Prusiner SB, Riesner D
The N-terminally truncated form of the prion protein, PrP 27-30, and the corresponding recombinant protein, rPrP, were solubilized in 0.2% SDS, and the transitions induced by changing the conditions from 0.2% SDS to physiological conditions, i.e. removing SDS, were characterized with respect to solubility, resistance to proteolysis, secondary structure and multimerization. Circular dichroism, electron microscopy and fluorescence correlation spectroscopy were used to study the structural transitions of PrP. Within one minute the alpha-helical structure of PrP was transformed into one that was enriched in beta-sheets and consisted mainly of dimers. Larger oligomers were found after 20 minutes and larger multimers exhibiting resistance to proteolysis were found after several hours. It was concluded that the monomeric alpha-helical conformation was stable in SDS or when attached to the membrane; however, the state of lowest free energy in aqueous solution at neutral pH seems to be the multimeric, beta-sheet enriched conformation.

Sensitivity of the Western blot detection of prion protein PrPres in natural sheep scrapie.

J Virol Methods 1998 Nov;75(2):169-77
Madec JY, Groschup MH, Buschmann A, Belli P, Calavas D, Baron T
The sensitivity of Western blot detection of PrPres using two different extraction procedures on brain material of 30 scrapie-affected sheep was compared. Whereas PrPres could be detected in all sheep after extraction with the first method, 30% did not give any signal after extraction with the second method. However, the second method, when positive, permitted the detection of PrPres from smaller amounts of infected brain tissue. When used with the ruminant specific monoclonal antibody p4, the second method gave positive signals corresponding to less than 12.5 microg of scrapie-infected brain, that, up to now, is the highest sensitivity described for PrPres detection from naturally infected ruminant brains. The overall results showed highly variable levels of PrPres between sheep and are presented in relation to breed, survival time and animal genotype data. Further progress can thus be expected for PrPres detection in prion diseases, if more efficient extraction procedures and more sensitive immunological reagents are used. Such technical improvements could contribute to more accurate diagnosis in animals affected naturally.

Genetic factors affecting BSE and scrapie susceptibility

4 Jan 99 Roslin Institute John Williams' group 
A set of decent lab web pages describes EU and MAFF-funded projects on genetic factors affecting BSE and scrapie susceptibility. They seem to work a whole lot harder than what we are used to in prions, they looked at 18,859 animals from 37 breeds in a recent polymorphism study [Anim Genet 1998 Aug;29(4):273-82 ]. Note the lab also provides a listing of recently submitted papers.

"In cattle, however, while three polymorphisms have been found in the coding region of the PrP gene (5 or 6 copies of the octapeptide repeat, and two silent base substitutions, one in the repeat and the other close by), studies on the effect of the PrP genotype on BSE susceptibility have proved inconclusive. Results from the maternal transmission cohort study show a higher incidence of BSE in the maternal BSE group. This could be explained by maternal transmission. or equally by the genetic control of susceptibility as it is likely that the maternal BSE-affected group of calves would have inherited susceptibility allele(s), if they exist, at higher frequency than the maternal BSE-unaffected group. "

"EU funded - Analysis of molecular factors affecting variability in BSE and Scrapie susceptibility

MAFF funded - Molecular analysis of putative genetic factors affecting BSE susceptibility "

Aims: Identification of mutations/polymorphisms

"If PrP is involved in the development of either BSE or scrapie, and the susceptibility or progression of the disease is dependent on the PrP allele present, the effect of a mutation/polymorphism can be tested directly at the population level by observing presence of variants in affected vs. unaffected individuals. Unlike testing linked markers, families are not required. A detailed study of the PrP gene, including non-coding sequence containing promoter and regulatory regions, will be performed. "

"In order to investigate whether there are loci other than PrP in the genome that play a role in infection or progression of BSE, large paternal half-sib families, which include affected and unaffected progeny, will be analysed. In the first instance 200 microsatellite loci spanning the whole bovine genome will be utilised in a linkage study. Also, DNA from affected and unaffected animals will be pooled both within and between families and used in a search for differences between affected and unaffected individuals using Amplification Fragment Length polymorphism (AFLP) analysis."

Other collaborators and contact information is also provided.

Early endosome cycling

3 Jan 98 webmaster: Medline highlights for 'early AND endosom* AND cycl*'

Urokinase-Type Plasminogen Activator Receptor Is Internalized by Different Mechanisms in Polarized and Nonpolarized Madin-Darby Canine Kidney Epithelial Cells

Molecular Biology of the Cell Vol. 10, Issue 1, 179-195, January 1999
Frederik Vilhardt,* Morten Nielsen, Kirsten Sandvig, and Bo van Deurs* 
'Prion' occurs in the full-text of this article:
Accumulated data indicate that endocytosis of the glycosylphosphatidyl-inositol-anchored protein urokinase plasminogen activator receptor (uPAR) depends on binding of the ligand uPA:plasminogen activator inhibitor-1 (PAI-1) and subsequent interaction with internalization receptors of the low-density lipoprotein receptor family, which are internalized through clathrin-coated pits. This interaction is inhibited by receptor-associated protein (RAP).

We show that uPAR with bound uPA:PAI-1 is capable of entering cells in a clathrin-independent process. First, HeLaK44A cells expressing mutant dynamin efficiently internalized uPA:PAI-1 under conditions in which transferrin endocytosis was blocked.

Second, in polarized Madin-Darby canine kidney (MDCK) cells, which expressed human uPAR apically, the low basal rate of uPAR ligand endocytosis, which could not be inhibited by RAP, was increased by forskolin or phorbol ester (phorbol 12-myristate 13-acetate), which selectively up-regulate clathrin-independent endocytosis from the apical domain of epithelial cells.

Third, in subconfluent nonpolarized MDCK cells, endocytosis of uPA:PAI-1 was only decreased marginally by RAP. At the ultrastructural level uPAR was largely excluded from clathrin-coated pits in these cells and localized in invaginated caveolae only in the presence of cross-linking antibodies. Interestingly, a larger fraction of uPAR in nonpolarized relative to polarized MDCK cells was insoluble in Triton X-100 at 0C, and by surface labeling with biotin we also show that internalized uPAR was mainly detergent insoluble, suggesting a correlation between association with detergent-resistant membrane microdomains and higher degree of clathrin-independent endocytosis.

Furthermore, by cryoimmunogold labeling we show that 5-10% of internalized uPAR in nonpolarized, but not polarized, MDCK cells is targeted to lysosomes by a mechanism that is regulated by ligand occupancy.

J Neurosci 1997 Aug 15;17(16):6142-51
The early endosome is the first vacuolar compartment along the endocytic pathway. It is the site of internalization and initial processing of amyloid precursor protein (APP) and apolipoprotein E (ApoE), two proteins of etiological importance in Alzheimer's disease, and a putative site of beta-amyloid peptide (Abeta) formation. Here, we identify early endosomes in human pyramidal neurons, using specific compartmental markers and morphometry, and show that in Alzheimer's disease individual endosomes display up to 32-fold larger volumes than the normal average. Endosomal enlargement contributed to an average 2.5-fold larger total endosomal volume per neuron, implying a marked increase in endocytic activity.

Endosomal alterations were evident in most pyramidal neurons in Alzheimer brain, detectable at early stages of the disease but absent in several other neurodegenerative disorders examined. In addition, mature and proenzyme forms of the proteases cathepsin B and cathepsin D, a candidate APP secretase, were identified in most early endosomes in Alzheimer brains but were detectable in only a minor proportion of endosomes in normal brain. Expression of the cation-dependent 46 kDa mannose 6-phosphate receptor was elevated in pyramidal neurons of Alzheimer brains, which could be a possible basis for the altered cathepsin trafficking pattern. Enhanced endocytic activity, coupled with increased trafficking to endosomes of proteases, which may have the ability under pathological conditions to generate Abeta, constitutes a potential mechanism by which beta-amyloidogenesis may become accelerated in sporadic AD and also be subject to influences by ApoE.

Int J Biochem 1987;19(2):179-86
Transferrin bound to K 562 cells at 4 degrees C was internalized quickly on temperature shift to 37 degrees C. Endosomes were isolated according to two different procedures. The endosome fraction was shown to be heterogeneous and consisted of two vesicle populations, differing in density properties and iron content. Iron was partially released from endosomes to the supernatant after 3 and 5 min endocytosis. Isolated endosomes, still capable of internal acidification, did not release iron on incubation with ATP. However, endosomes did release iron on incubation with the iron chelator pyridoxal-isonicotinoyl hydrazone. Gel-filtration of solubilized endosomes demonstrated the presence of the transferrin-transferrin receptor complexes, free transferrin and free low molecular weight iron.

J Physiol (Paris) 1991;85(2):90-6
Synaptic vesicles participate in a cycle of fusion with the plasma membrane and reformation by endocytosis. Endocytosis of membrane proteins by the well studied clathrin-coated vesicle pathway has been shown to involve specific sequences within the cytoplasmic tail domain. Proteins taken up by clathrin-coated vesicles are directed to early endosomes from which they may return to plasma membrane. Recent evidence suggests that the synaptic vesicle protein synaptophysin is targeted to early endosomes in transfected fibroblasts and in neuroendocrine cells, ... a model in which the COOH-terminal tail is required for coated-pit localization and hence targeting of synaptophysin to early endosomes.

J Biol Chem 1994 Apr 22;269(16):12159-66
The mannose 6-phosphate receptor cycles between the trans Golgi network and late endosomes, and between the plasma membrane and early endosomes, to deliver lysosomal enzymes to prelysosomes,... findings suggesting a role for extracellular and/or transmembrane domains in mannose 6-phosphate receptor trafficking;... the presence of the mannose 6-phosphate receptor extracellular domain may interfere with the rapid recycling of receptors from early endosomes to the cell surface and detain receptors within endosomes

J Exp Med 1998 Nov 16;188(10):1769-74
Each member of the rab guanosine triphosphatase protein family assists in the regulation of a specific step within the biosynthetic or endocytic pathways. We have found that the early endosome-associated rab4 protein controls a step critical for receptor-mediated antigen processing in a murine A20 B cell line.

J Cell Sci 1998 Nov 12;111(Pt 23):3451-3458
Whilst the rat type I integral membrane protein TGN38 (ratTGN38) is predominantly localised to the trans-Golgi network this protein does reach the cell surface from where it is internalised and delivered back to the trans-Golgi network. This protein thus provides a suitable tool for the investigation of trafficking pathways between the trans-Golgi network and the cell surface and back again. The human orthologue of ratTGN38, humTGN46, behaves in a similar fashion. These proteins are internalised from the cell surface via clathrin mediated endocytosis, a process which is dependent upon the GTPase activity of dynamin. We thus reasoned that humTGN46 would accumulate at the surface of cells rendered defective in clathrin mediated endocytosis by virtue of the fact that they express a GTPase defective mutant of dynamin I. It did not. In fact, expression of a dominant negative GTPase defective mutant of dynamin I had no detectable effect on the steady state distribution of humTGN46. Tthe major recycling pathway for humTGN46 is in fact between the trans-Golgi network and the early endosome.

Immunology 1998 May;94(1):48-55
The high-affinity receptor for immunoglobulin G (Fc gamma RI) plays a central role in the clearance of immune complexes by mediating their internalization and delivery to lysosomes. In monocytic U937 cells, receptor internalization is independent of tyrosine kinase activity. However, the tyrosine kinase inhibitor, genistein, prevents further progress of the receptor to lysosomes and traps it in a sub-plasma membrane early endosome. Similarly, Fc gamma RI expressed in COS cells is able to internalize immune complexes but is unable to translocate to lysosomes. This suggests that Fc gamma RI, whose cytoplasmic tail is devoid of known signalling motifs, must recruit tyrosine kinases via its gamma-chain to achieve lysosomal delivery. We show that a chimera of the extracellular domain of Fc gamma RI and the cytoplasmic tail of the gamma-chain is both internalized and efficiently trafficked to lysosomes. Our study suggests that a key function of the gamma-chain is recruitment of tyrosine kinases to initiate the intracellular signalling pathways required to target Fc gamma RI following immune complex aggregation to lysosomes and not to initiate endocytosis per se.

Curr Biol 1998 Jul 16;8(15):881-4
In mammalian cells, fusion between early endocytic vesicles has been shown to require the ubiquitous intracellular fusion factors N-ethylmaleimide-sensitive factor (NSF) and alpha-SNAP, as well as a factor specific for early endosomes, the small GTPase Rab5 [1-3]. We have previously demonstrated an additional requirement for phosphatidylinositol 3-kinase (PI 3-kinase) activity.

Nature 1998 Jul 30;394(6692):494-8
GTPases and lipid kinases regulate membrane traffic along the endocytic pathway by mechanisms that are not completely understood. Fusion between early endosomes requires phosphatidylinositol-3-OH kinase (PI(3)K) activity as well as the small GTPase Rab5. Excess Rab5-GTP complex restores endosome fusion when PI(3)K is inhibited. Here we identify the early-endosomal autoantigen EEA1 which binds the PI(3)K product phosphatidylinositol-3-phosphate, as a new Rab5 effector that is required for endosome fusion... The identification of EEA1 as a direct Rab5 effector provides a molecular link between PI(3)K and Rab5, and its restricted distribution to early endosomes indicates that EEA1 may confer directionality to Rab5-dependent endocytic transport.

Mol Biol Cell 1998 Aug;9(8):2125-44
TGN38 is one of the few known resident integral membrane proteins of the trans-Golgi network (TGN). Since it cycles constitutively between the TGN and the plasma membrane, TGN38 is ideally suited as a model protein for the identification of post-Golgi trafficking motifs. Several studies, employing chimeric constructs to detect such motifs within the cytosolic domain of TGN38, have identified the sequence 333YQRL336 as an autonomous signal capable of localizing reporter proteins to the TGN.

Mol Biol Cell 1998 Jun;9(6):1549-63
Synaptobrevins/vesicle-associated membrane proteins (VAMPs) together with syntaxins and a synaptosome-associated protein of 25 kDa (SNAP-25) are the main components of a protein complex involved in the docking and/or fusion of synaptic vesicles with the presynaptic membrane. The amino acid sequence of endobrevin has 32, 33, and 31% identity to those of synaptobrevin/VAMP-1, synaptobrevin/VAMP-2, and cellubrevin, respectively. Membrane fractionation studies demonstrate that endobrevin is enriched in membrane fractions that are also enriched in the asialoglycoprotein receptor. Indirect immunofluorescence microscopy establishes that endobrevin is primarily associated with the perinuclear vesicular structures of the early endocytic compartment.

EMBO J 1998 Apr 1;17(7):1941-51
Rabaptin-5 functions as an effector for the small GTPase Rab5, a regulator of endocytosis and early endosome fusion. We have searched for structural determinants that confer functional specificity on Rabaptin-5. Here we report that native cytosolic Rabaptin-5 is present in a homodimeric state and dimerization depends upon the presence of its coiled-coil predicted sequences. A 73 residue C-terminal region of Rabaptin-5 is necessary and sufficient both for the interaction with Rab5 and for Rab5-dependent recruitment of the protein on early endosomes. Surprisingly, we uncovered the presence of an additional Rab-binding domain at the N-terminus of Rabaptin-5. This domain mediates the direct interaction with the GTP-bound form of Rab4, a small GTPase that has been implicated in recycling from early endosomes to the cell surface. Based on these results, we propose that Rabaptin-5 functions as a molecular linker between two sequentially acting GTPases to coordinate endocytic and recycling traffic.

J Cell Sci 1998 Mar;111 ( Pt 6):681-9
Two storage compartments in cultured noradrenergic neurons derived from the superior cervical ganglion from fetal pig have been defined using sucrose density gradient centrifugation and electron microscopy: (1) large dense-cored vesicles (LDV) contain noradrenaline and dopamine-beta-hydroxylase (DbetaH); (2) small electron-lucent vesicles contain acetylcholine and p38 and represent the noradrenergic small synaptic vesicles (SSV); no small dense-cored vesicles (SDV) could be detected. Our results demonstrate that internalized LDV membrane constituents are retrieved into early endosomes. Recycling of the SSV membranes via an endosomal intermediate is also confirmed in noradrenergic neurons. Finally, colocalization of retrieved DbetaH and retrieved p38 in stimulated neurons indicates that the two sets of constituents intermix. These data provide the first experimental evidence for a common early endosome in which SSV and LDV membrane constituents are internalized after exocytosis and imply that endosomal sorting is an important process for the generation of different secretory vesicles in the noradrenergic nerve terminal.

J Biol Chem 1998 Jan 2;273(1):375-80
Syntaxin 7, a novel syntaxin member associated with the early endosomal compartment is 35, 34, 34, 34, 25, and 19% identical to syntaxins 1, 2, 3, 4, 5, and 6. Our results highlight the general importance of members of the syntaxin family in protein trafficking and provide new avenues for future functional and mechanistic studies of this first endosomal syntaxin as well as the endocytotic pathway.

Cytometry 1997 Sep 1;29(1):41-9
Early endosomes are very dynamic intracellular membrane organelles that undergo multiple fusion and fission events. In this study, we developed a novel assay based on multiparametric flow cytometric analyses and early endosome sorting to characterize better the mechanisms of early endosome membrane dynamics in vitro. In particular, we have investigated the role of rab4 and rab5, two small GTPases known to regulate distinct steps of membrane traffic in the endocytic pathway. We show that early endosomes undergo homotypic fusion reactions, which lead to the formation of fusion intermediates with increased size. Fusion is efficiently stimulated by recombinant rab5 but not by recombinant rab4.

Mol Biol Cell 1997 Mar;8(3):533-45
Annexin II is an abundant protein which is present in the cytosol and on the cytoplasmic face of plasma membrane and early endosomes. It is generally believed that this association occurs via Ca(2+)-dependent binding to lipids, a mechanism typical for the annexin protein family. Annexin II is involved in early endosome dynamics and organizationn. In this study, we found that approximately 50% of the total cellular annexin was associated with membranes in a Ca(2+)-independent manner. This binding was extremely tight, since it resisted high salt and, to some extent, high pH treatments. We found, however, that membrane-associated annexin II could be quantitatively released by low concentrations of the cholesterol-sequestering agents filipin and digitonin. Both treatments released an identical and limited set of proteins but had no effects on other membrane-associated proteins. Among the released proteins, we identified, in addition to annexin II itself, the cortical cytoskeletal proteins alpha-actinin, ezrin and moesin, and membrane-associated actin.

Copper chaperones: no known homology to prion protein

2 Jan 98 webmaster review of Medline abstracts containing 'copper and chaperone'
FEBS Lett 1998 Nov 27;440(1-2):141-6
Copper chaperone protein, CUC-1 from Caenorhabditis elegans had the characteristic motifs of MTCXXC and KKTGK, and showed 49.3 and 39.1% sequence identity with yeast Atx1p and human HAH1, expressed together with cua-1, the copper transporting ATPase.

J Biol Chem 1998 Jan 16;273(3):1749-54
HAH1 is a 68-amino acid protein originally identified as a human homologue of Atx1p, a multi-copy suppressor of oxidative injury in sod1 delta yeast. Molecular modeling of HAH1 predicts a protein structure of two alpha-helices overlaying a four-stranded antiparallel beta-sheet with a potential metal binding site involving two conserved cysteine residues...These data support the concept of a unique copper coordination environment in HAH1 that permits this protein to function as an intracellular copper chaperone mediating distinct biological processes in eucaryotic cells.

       61 TVSYLGLE - human

       61 AVSYLGPK - mouse

       61 KKTGKEVRSG KQL - yeast ATX1
Science 1997 Oct 31;278(5339):853-6
Reactive and potentially toxic cofactors such as copper ions are imported into eukaryotic cells and incorporated into target proteins by unknown mechanisms. Atx1, a prototypical copper chaperone protein from yeast, has now been shown to act as a soluble cytoplasmic copper(I) receptor that can adopt either a two- or three-coordinate metal center in the active site. Atx1 also associated directly with the Atx1-like cytosolic domains of Ccc2, a vesicular protein defined in genetic studies as a member of the copper-trafficking pathway. The unusual structure and dynamics of Atx1 suggest a copper exchange function for this protein and related domains in the Menkes and Wilson disease proteins.

Proc Natl Acad Sci U S A 1998 May 26;95(11):6361-6
Superoxide dismutase mutants also acquire catalytic copper in vivo through the action of CCS, a specific copper chaperone for SOD1, which in turn suggests that a search for inhibitors of this SOD1 copper chaperone may represent a therapeutic avenue.

J Biol Chem 1997 Sep 19;272(38):23469-72
Copper is distributed to distinct localizations in the cell through diverse pathways, to copper/zinc superoxide dismutase (SOD1) by a soluble factor identified as Saccharomyces cerevisiae LYS7 or human CCS (copper chaperone for SOD, GenBank 2431868). This factor is specific for SOD1 and does not deliver copper to proteins in the mitochondria, nucleus, or secretory pathway. Yeast cells containing a lys7Delta null mutation have normal levels of SOD1 protein, but fail to incorporate copper into SOD1, which is therefore devoid of superoxide scavenging activity.

J Biol Chem 1998 Sep 11;273(37):23625-8
The copper chaperone CCS directly interacts with copper/zinc superoxide dismutase. Dominantly inherited mutations in the gene encoding copper/zinc superoxide dismutase (SOD1) result in the fatal motor neuron disease familial amyotrophic lateral sclerosis (FALS). These mutations confer a gain-of-function to SOD1 with neuronal degeneration resulting from enhanced free radical generating activity of the copper present in the mutant enzyme. The delivery of copper to SOD1 is mediated through a soluble factor identified as the copper chaperone for SOD1 (CCS). Amino acid sequence alignment of SOD1 and CCS reveals a striking homology with conservation of the amino acids essential for mediating SOD1 homodimerization.

J Biol Chem 1998 Jul 24;273(30):19243-50
The expression of the melanin operon (melC) of Streptomyces antibioticus requires the chaperone-like protein MelC1 for the incorporation of two copper ions (designated as CuA and CuB) and the secretion of the apotyrosinase (MelC2) via a transient binary complex formation between these two proteins.

J Biol Chem 1993 Sep 5;268(25):18710-6
Streptomyces tyrosinase trans-activator MelC1, likely a chaperone for apotyrosinase. The melanin operon (melC) of Streptomyces antibioticus contains two genes, melC1 and melC2 (apotyrosinase). Our previous studies indicated that MelC1 forms a transient binary complex with the downstream apotyrosinase MelC2 to facilitate the incorporation of copper ion and the secretion of tyrosinase....MelC1 is indispensable in the incorporation of copper ion into MelC2 apotyrosinase via a transient, competent binary complex formation, during which a conformational transition of MelC2 has occurred. This strongly suggests that MelC1 is a chaperone for the apotyrosinase MelC2.

Biochemistry 1998 May 19;37(20):7572-7
Assembly of functional cytochrome oxidase in yeast requires the copper chaperone Cox17, which has been postulated to deliver copper ions to the mitochondrion for insertion into the enzyme. This role for Cox17 is supported by the observation that it binds copper as a binuclear cuprous-thiolate cluster.

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