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PubMed1999

Tapasin enhances assembly of transporters associated with antigen processing-dependent and -independent peptides with HLA-A2 and HLA-B27 expressed in insect cells.

Assembly of HLA class I-peptide complexes is assisted by multiple proteins that associate with HLA molecules in loading complexes. These include the housekeeping chaperones calnexin and calreticulin and two essential proteins, the transporters associated with antigen processing (TAP) for peptide supply, and the protein tapasin which is thought to act as a specialized chaperone. We dissected functional effects of processing cofactors by co-expressing in insect cells various combinations of the human proteins HLA-A2, HLA-B27, beta(2)-microglobulin, TAP, calnexin, calreticulin, and tapasin. Stability at 37 degrees C and surface expression of class I dimers correlated closely in baculovirus-infected Sf9 cells, suggesting that these cells retain empty dimers in the endoplasmic reticulum. Both HLA molecules form substantial quantities of stable complexes with insect cell-produced peptide pools. These pools are TAP-selected cytosolic peptides for HLA-B27 but endoplasmic reticulum-derived, i.e. TAP-independent peptides for HLA-A2. This discrepancy may be due to peptide selection by human TAP which is much better adapted to the HLA-B27 than to the HLA-A2 ligand preferences. HLA class I assembly with peptides from TAP-dependent and -independent pools was enhanced strongly by tapasin. Thus, tapasin acts as a chaperone and/or peptide editor that facilitates assembly of peptides with HLA class I molecules independently of mediating their interaction with TAP and/or retention in the endoplasmic reticulum.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1996

Processing of N3, a mammalian proteasome beta-type subunit.

Proteasome subunits are encoded by members of the same gene family and can be divided into two groups based on their similarity to the alpha and beta subunits of the simpler proteasome isolated from Thermoplasma acidophilum. RN3 is the beta-type subunit, N3, of rat proteasomes which has been implicated in the peptidylglutamyl-peptide hydrolase activity of the proteinase complex. We have expressed recombinant RN3 protein in Escherichia coli in order to raise subunit-specific polyclonal antibodies. Identification of the position of RN3 on two-dimensional PAGE gels of purified rat liver proteasomes showed a single protein spot of molecular mass 24 kDa and of pI value of about 5. This protein has a free N-terminus, having undergone post-translational processing. After immunoprecipitation from [35S]methionine-labelled human embryo lung L-132 cells using anti-RN3 antibodies, two radiolabelled spots were observed on two-dimensional PAGE gels, one corresponding to the mature N3, the other of molecular mass 28.5 kDa and pI value around 5, which was probably the unprocessed form of N3. However, the latter protein had a higher molecular mass (31 kDa) than was predicted from the sequence of previously cloned cDNA. Therefore rapid amplification of cDNA ends ("RACE') was carried out to determine the full sequence. The lack of detectable RN3 precursor in purified rat liver proteasomes suggests that the processing probably accompanies assembly of the complex. The half-life of the processing was determined to be 31 min in growing L-132 cells. The unprocessed form of N3 was not observed after immunoprecipitation of 35S-labelled complexes with anti-proteasome antibodies. There was no evidence to suggest that unprocessed N3 is found in precursor complexes which have been implicated in the assembly of some other unprocessed beta-type subunits. Interestingly also, the site of cleavage of N3 (ITR decreases TQN) differs significantly from those of other processed animal beta-type proteasome subunits [(H/T)G decreases TT(T/L)], many of which resemble more closely the cleavage site of the Thermoplasma acidophilum beta subunit.

Amino Acid Sequence↗
PubMed1991

Amino acids encoded downstream of gag are not required by Rous sarcoma virus protease during gag-mediated assembly.

Rous sarcoma virus (RSV) and its relatives are unique in that they appear to encode their viral protease in the gag reading frame. As a result, this 124-amino-acid sequence is found at the carboxy terminus of each Gag precursor molecule and, upon ribosome frameshifting, embedded within each Gag-Pol molecule. However, rigorous proof has never been obtained for the activity of this 124-amino-acid Gag domain during virion assembly in vivo. If the active protease actually included amino acids encoded downstream in the pol reading frame, then the sequence organization would be more in line with those of other retroviruses. To examine this issue, mutations that disrupt the addition of amino acids by ribosome frameshifting were analyzed for their effects on particle assembly and Gag processing in a mammalian expression system (J. W. Wills, R. C. Craven, and J. A. Achacoso, J. Virol. 63:4331-4343, 1989). A 2-base substitution which created a nonsense mutation in the pol reading frame and was predicted to disrupt the hairpin structure of the ribosome frameshift signal had no effect on particle assembly or Gag processing, definitively showing that downstream amino acids are unnecessary. Mutations that fused the gag and pol reading frames to place 85 amino acids at the carboxy terminus of Gag hindered particle assembly and totally abolished the activity of the protease. A smaller fusion protein containing only the seven-amino-acid spacer peptide that links Gag and reverse transcriptase allowed particle formation but slowed processing. The reduced rate of processing exhibited by this mutant also revealed a previously unnoticed series of late maturation steps associated with the RSV capsid (CA) protein. Another mutant containing two substituted amino acids plus one additional amino acid at the carboxy terminus of protease nearly abolished processing. Together, these results demonstrate the importance of the carboxy terminus for proteolytic activity and suggest that this end must be unrestrained for optimal activity. If this hypothesis is correct, then the RSV protease may be encoded at the end of gag simply to ensure the production of a free carboxy terminus by translational termination.

Amino Acid Sequence↗
PubMed1998

The plurifunctional nucleolus.

The nucleolus of eukaryotic cells was first described in the early 19th century and was discovered in the 1960s to be the seat of ribosome synthesis. Although rRNA transcription, rRNA processing and ribosome assembly have been clearly established as major functions of the nucleolus, recent studies suggest that the nucleolus participates in many other aspects of gene expression as well. Thus, the nucleolus has been implicated in the processing or nuclear export of certain mRNAs. In addition, new results indicate that biosyntheses of signal recognition particle RNA and telomerase RNA involve a nucleolar stage and that the nucleolus is also involved in processing of U6 RNA, one of the spliceosomal small nuclear RNAs. Interestingly, these three nucleolus-associated small nuclear RNAs (signal recognition particle RNA, telomerase RNA and U6 RNA) are components of catalytic ribonucleoprotein machines. Finally, recent work has also suggested that some transfer RNA precursors are processed in the nucleolus. The nucleolus may have evolutionarily descended from a proto-eukaryotic minimal genome that was spatially linked to vicinal RNA processing and ribonucleoprotein assembly events involved in gene read-out. The nucleolus of today's eukaryotes, now surrounded by the chromatin of over 2 billion years of genome expansion, may still perform these ancient functions, in addition to ribosome biosynthesis. The plurifunctional nucleolus concept has a strong footing in contemporary data and adds a new perspective to our current picture of the spatial-functional design of the cell nucleus.

Cell Nucleolus↗
PubMed2001

Mutations in the gene encoding immunoglobulin mu-binding protein 2 cause spinal muscular atrophy with respiratory distress type 1.

Classic spinal muscular atrophy (SMA) is caused by mutations in the telomeric copy of SMN1. Its product is involved in various cellular processes, including cytoplasmic assembly of spliceosomal small nuclear ribonucleoproteins, pre-mRNA processing and activation of transcription. Spinal muscular atrophy with respiratory distress (SMARD) is clinically and genetically distinct from SMA. Here we demonstrate that SMARD type 1 (SMARD1) results from mutations in the gene encoding immunoglobulin micro-binding protein 2 (IGHMBP2; on chromosome 11q13.2-q13.4). In six SMARD1 families, we detected three recessive missense mutations (exons 5, 11 and 12), two nonsense mutations (exons 2 and 5), one frameshift deletion (exon 5) and one splice donor-site mutation (intron 13). Mutations in mouse Ighmbp2 (ref. 14) have been shown to be responsible for spinal muscular atrophy in the neuromuscular degeneration (nmd) mouse, whose phenotype resembles the SMARD1 phenotype. Like the SMN1 product, IGHMBP2 colocalizes with the RNA-processing machinery in both the cytoplasm and the nucleus. Our results show that IGHMBP2 is the second gene found to be defective in spinal muscular atrophy, and indicate that IGHMBP2 and SMN share common functions important for motor neuron maintenance and integrity in mammals.

Amino Acid Sequence↗
PubMed1994

The Sendai virus matrix protein appears to be recruited in the cytoplasm by the viral nucleocapsid to function in viral assembly and budding.

The matrix (M) protein is viewed as the regulator of paramyxovirus particle assembly and budding. Accordingly it was observed to be mutated, and/or decreased in amount, in cases where virus particle production was significantly reduced. Here, a non-productive [non-defective and defective interfering (DI)] Sendai virus infection of COS cells is presented where virus particle production is abolished in the presence of a normal amount of intracellular M protein. In this infection the haemagglutinin-neuraminidase envelope glycoprotein is shown to be dispensable for virion production, and the fusion (F0) envelope glycoprotein behaves as in a productive infection. The M protein is shown to accumulate in perinuclear patches within the cytoplasm. In contrast, localization in the plasma membrane is observed in productive infections. However in both productive and non-productive infections a significant fraction of M protein is found in association with cellular membranes. The M protein-membrane association is shown to take place in the absence of any other viral component, and the M protein-membrane complex exhibits properties similar to those observed for the integral membrane protein F0. However these properties are distinct from those of the phosphoprotein, which is thought to associate with membranes in a non-specific manner. Concomitant with the cytoplasmic accumulation of M protein and the reduction of virus particle production in this non-productive infection, DI nucleocapsids are shown not to associate with cellular membrane fractions. This is a property which coincides with their poor envelopment in virus particles. Taken together, these data indicate the need for M protein to be recruited at the perinuclear membranes by the nucleocapsids to participate in viral assembly and budding. This view is consistent with a process of viral assembly taking place on internal cytoplasmic membranes rather than at the plasma membrane.

Animals↗
PubMed1988

Dissociation and re-assembly of the endoplasmic reticulum in live cells.

The endoplasmic reticulum (ER) of a typical interphase 3T3 fibroblast consists of a compact perinuclear arrangement of cisternae and lamellae which can be observed by immunofluorescence with anti-endoplasmin. During mitosis the reticulum dissociates into small fragments from which it appears to re-assemble in the daughter cells. When interphase 3T3 cells are exposed to calcium ionophores, but not other ionophores, there is a similar dissociation of the ER into small uniform fragments, which are dispersed throughout the cytoplasm. Electron microscopy shows that the fragments consist of small vesicular structures and that essentially all of the rough ER except the nuclear envelope is dissociated. The dissociation of the ER by calcium ionophore is a relatively specific process since other organelles and supramolecular assemblies remain unaffected. When cells with dissociated ER are returned to normal medium, there is a rapid reassembly of the fragments into the continuous reticulum. In a proportion of the cells it is possible to observe linear arrays of the fragments, which probably represent intermediates in the re-assembly process. These observations demonstrate that the ER in interphase 3T3 cells can be dissociated into, and re-assembled from, small fragments. Re-assembly of the ER from the fragments is dependent on the presence of millimolar levels of calcium in the external medium. In the presence of calcium, re-assembly is inhibited by the calcium channel blocker, verapamil. Thus calcium ions appear to play an important role in ER structure and assembly.

Animals↗
PubMed1998

Very-long-chain acyl-CoA dehydrogenase subunit assembles to the dimer form on mitochondrial inner membrane.

This paper describes the process of dimer assembly of mitochondrial very-long-chain acyl-CoA dehydrogenase (VLCAD) subunit. Mature VLCAD is a homodimer of a 70-kDa protein associated with the mitochondrial membrane. Newly synthesized VLCAD was present as a monomer and the major fraction was associated with the mitochondrial inner membrane. The association of VLCAD subunit with the mitochondrial membrane was observed early during dimer formation. In contrast, a VLCAD monomeric mutant S583W, a novel mutation identified from a patient with VLCAD deficiency, did not associate with the mitochondrial membrane after import and the major fraction remained in the mitochondrial matrix. These results suggest that association of VLCAD protein with mitochondrial inner membrane is necessary for dimer assembly and formation of mature VLCAD.

Acyl-CoA Dehydrogenase, Long-Chain↗
PubMed2003

Functional cysteine-less subunits of the transporter associated with antigen processing (TAP1 and TAP2) by de novo gene assembly.

Within the adaptive immune system the transporter associated with antigen processing (TAP) plays a pivotal role in loading of peptides onto major histocompatibility (MHC) class I molecules. As a central tool to investigate the structure and function of the TAP complex, we created cysteine-less human TAP subunits by de novo gene synthesis, replacing all 19 cysteines in TAP1 and TAP2. After expression in TAP-deficient human fibroblasts, cysteine-less TAP1 and TAP2 are functional with respect to adenosine triphosphate (ATP)-dependent peptide transport and inhibition by ICP47 from herpes simplex virus. Cysteine-less TAP1 and TAP2 restore maturation and intracellular trafficking of MHC class I molecules to the cell surface.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1994

Purification of active herpes simplex virus-1 protease expressed in Escherichia coli.

Assembly of viral capsids for replication of herpes simplex virus requires the proteolytic processing of the assembly protein ICP35. The protease responsible for this process is encoded within the 635-amino acid open reading frame of the UL26 gene of the virus. A simple purification scheme is given in this report for the native, mature form of the protease expressed in Escherichia coli. The scheme allows the preparation of milligram quantities of purified enzyme for elucidation of kinetic mechanism as well as for structural studies. Utilizing a 13-residue peptide substrate representing the natural cleavage site that releases the protease, kcat and Km values of the purified native enzyme are 2.0 min-1 and 0.88 mM, respectively. Thus, peptide cleavage is less efficient than reported for other viral proteases. The possibility exists that viral or cellular factors are involved in vivo for activation of the protease for herpes capsid maturation.

Amino Acid Sequence↗
PubMed2005

Mechanisms of amyloid fibril self-assembly and inhibition. Model short peptides as a key research tool.

The formation of amyloid fibrils is associated with various human medical disorders of unrelated origin. Recent research indicates that self-assembled amyloid fibrils are also involved in physiological processes in several micro-organisms. Yet, the molecular basis for the recognition and self-assembly processes mediating the formation of such structures from their soluble protein precursors is not fully understood. Short peptide models have provided novel insight into the mechanistic issues of amyloid formation, revealing that very short peptides (as short as a tetrapeptide) contain all the necessary molecular information for forming typical amyloid fibrils. A careful analysis of short peptides has not only facilitated the identification of molecular recognition modules that promote the interaction and self-assembly of fibrils but also revealed that aromatic interactions are important in many cases of amyloid formation. The realization of the role of aromatic moieties in fibril formation is currently being used to develop novel inhibitors that can serve as therapeutic agents to treat amyloid-associated disorders.

Amino Acid Sequence↗
PubMed2004

The last C-terminal residue of VP3, glutamic acid 257, controls capsid assembly of infectious bursal disease virus.

Infectious bursal disease virus (IBDV) is a nonenveloped virus with an icosahedral capsid composed of two proteins, VP2 and VP3, that derive from the processing of the polyprotein NH(2)-pVP2-VP4-VP3-COOH. The virion contains VP1, the viral polymerase, which is both free and covalently linked to the two double-stranded RNA (dsRNA) genomic segments. In this study, the virus assembly process was studied further with the baculovirus expression system. While expression of the wild-type polyprotein was not found to be self-sufficient to give rise to virus-like particles (VLPs), deletion or replacement of the five C-terminal residues of VP3 was observed to promote capsid assembly. Indeed, the single deletion of the C-terminal glutamic acid was sufficient to induce VLP formation. Moreover, fusion of various peptides or small proteins (a green fluorescent protein or a truncated form of ovalbumin) at the C terminus of VP3 also promoted capsid assembly, suggesting that assembly required screening of the negative charges at the C terminus of VP3. The fused polypeptides mimicked the effect of VP1, which interacts with VP3 to promote VLP assembly. The C-terminal segment of VP3 was found to contain two functional domains. While the very last five residues of VP3 mainly controlled both assembly and capsid architecture, the five preceding residues constituted the VP1 (and possibly the pVP2/VP2) binding domain. Finally, we showed that capsid formation is associated with VP2 maturation, demonstrating that the protease VP4 is involved in the virus assembly process.

Amino Acid Sequence↗
PubMed2006

Micromanipulation studies of chromatin fibers in Xenopus egg extracts reveal ATP-dependent chromatin assembly dynamics.

We have studied assembly of chromatin using Xenopus egg extracts and single DNA molecules held at constant tension by using magnetic tweezers. In the absence of ATP, interphase extracts were able to assemble chromatin against DNA tensions of up to 3.5 piconewtons (pN). We observed force-induced disassembly and opening-closing fluctuations, indicating our experiments were in mechanochemical equilibrium. Roughly 50-nm (150-base pair) lengthening events dominated force-driven disassembly, suggesting that the assembled fibers are chiefly composed of nucleosomes. The ATP-depleted reaction was able to do mechanical work of 27 kcal/mol per 50 nm step, which provides an estimate of the free energy difference between core histone octamers on and off DNA. Addition of ATP led to highly dynamic behavior with time courses exhibiting processive runs of assembly and disassembly not observed in the ATP-depleted case. With ATP present, application of forces of 2 pN led to nearly complete fiber disassembly. Our study suggests that ATP hydrolysis plays a major role in nucleosome rearrangement and removal and that chromatin in vivo may be subject to highly dynamic assembly and disassembly processes that are modulated by DNA tension.

Adenosine Triphosphate↗
PubMed1998

Tenascin-C hexabrachion assembly is a sequential two-step process initiated by coiled-coil alpha-helices.

We have investigated the oligomerization process of tenascin-C using a variety of recombinant wild-type and mutant polypeptide chain fragments produced by heterologous gene expression in Escherichia coli. Biochemical and biophysical analyses of the structures and assemblies of these fragments indicated a sequential two-step oligomerization mechanism of tenascin-C involving the concerted interaction of two distinct domains and cysteines 64, 111, and 113. First, the sequence between alanine 114 and glutamine 139 initiates hexabrachion formation via a parallel three-stranded coiled coil. Subsequently, the tenascin assembly domain, which is unique to the tenascins, is responsible for the connection of two triplets to a hexamer. The oligomerization of the tenascin assembly domains by the three-stranded coiled coil increases their homophilic binding affinity and is an important prerequisite for tenascin-C hexamerization. Although formation of the characteristic hexabrachion structure involves the covalent linkage of the six subunits by cysteine residues, mutational analysis indicates that hexamer formation is not dependent on intermolecular disulfide bonds. Most interestingly, substitution of glutamate 130 within the coiled-coil domain by leucine or alanine resulted in the formation of parallel four-stranded helix structures, which further associated to dodecamers. Aside from supporting a sequential process of tenascin-C assembly, this finding provides experimental evidence that non-core residues can have profound effects on the oligomerization states of coiled coils.

Amino Acid Sequence↗
PubMed1975

Assembly of tobacco mosaic virus in vitro. Improved model for the elongation process by protein subunits.

The in vitro assembly reaction of tobacco mosaic virus (TMV), especially the elongation process of partially reconstituted RNA (PRR) by protein subunits, was observed by electron microscopy. After addition of TMV-protein subunits, the PRR appeared as rods with a clump at one end, believed to be a complex between added protein subunits and the RNA tail protruding from PRR. The subunits entrapped on the RNA tails in the forms of clumps were progressively incorporated into the growing rods on incubation, ending with the formation of completely reconstituted rods. The clumps were also observed after addition of cucumber green mottle mosaic virus (CGMMV) protein subunits to rods partially reconstituted from RNA and TMV-protein. In this case, the protein subunits, seen as clumps, did not become incorporated to form elongating rods. An improved model for the elongation of TMV rods is proposed. The elongation process is composed of two steps, with the first step being the interaction of protein subunits with the RNA tail protruding from the growing rod. Any protein having a specific binding site for TMV-rna, not limited to TMV-protein, will react in the first step. The second step is the incorporation of the protein on the RNA tail into a rod-shaped structure, with consequent elongation of the growing rod. It appears that only protein homologous with that in the partially reconstituted rods can partake in the second step.

Microscopy, Electron↗
PubMed2007

Measles virus nucleocapsid transport to the plasma membrane requires stable expression and surface accumulation of the viral matrix protein.

In measles virus (MV)-infected cells the matrix (M) protein plays a key role in virus assembly and budding processes at the plasma membrane because it mediates the contact between the viral surface glycoproteins and the nucleocapsids. By exchanging valine 101, a highly conserved residue among all paramyxoviral M proteins, we generated a recombinant MV (rMV) from cloned cDNA encoding for a M protein with an increased intracellular turnover. The mutant rMV was barely released from the infected cells. This assembly defect was not due to a defective M binding to other matrix- or nucleoproteins, but could rather be assigned to a reduced ability to associate with cellular membranes, and more importantly, to a defective accumulation at the plasma membrane which was accompanied by the deficient transport of nucleocapsids to the cell surface. Thus, we show for the first time that M stability and accumulation at intracellular membranes is a prerequisite for M and nucleocapsid co-transport to the plasma membrane and for subsequent virus assembly and budding processes.

Amino Acid Sequence↗
PubMed2001

Functional implications of temporal structure in primate cortical information processing.

Processing of information in the cerebral cortex of primates is characterized by distributed representations and processing in neuronal assemblies rather than by detector neurons, cardinal cells or command neurons. Responses of individual neurons in sensory cortical areas contain limited and ambiguous information on common features of the natural environment which is disambiguated by comparison with the responses of other, related neurons. Distributed representations are also capable to represent the enormous complexity and variability of the natural environment by the large number of possible combinations of neurons that can engage in the representation of a stimulus or other content. A critical problem of distributed representation and processing is the superposition of several assemblies activated at the same time since interpretation and processing of a population code requires that the responses related to a single representation can be identified and distinguished from other, related activity. A possible mechanism which tags related responses is the synchronization of neuronal responses of the same assembly with a precision in the millisecond range. This mechanism also supports the separate processing of distributed activity and dynamic assembly formation. Experimental evidence from electrophysiological investigations of non-human primates and human subjects shows that synchronous activity can be found in visual, auditory and motor areas of the cortex. Simultaneous recordings of neurons in the visual cortex indicate that individual neurons synchronize their activity with each other, if they respond to the same stimulus but not if they are part of different assemblies representing different contents. Furthermore, evidence for synchronous activity related to perception, expectation, memory, and attention has been observed.

Journal Article↗
PubMed2005

Formation and dissolution processes of the 6-thioguanine (6TG) self-assembled monolayer. A kinetic study.

This is a report on the kinetics of the destruction and formation processes of the 6-thioguanine self-assembled monolayer (6TG SAM) on a mercury electrode from acid solutions by chronoamperometry. The destruction of the 6TG SAM that has been previously formed under open circuit potential conditions is carried out by stepping the potential from an initial value where the chemisorbed layer is stable up to potentials where the molecules are no longer chemisorbed. The destruction of the SAM has been described by a model that involves three types of contributions: (i) a Langmuir-type adsorption process, (ii) a 2D nucleation mechanism followed by a growth controlled by surface diffusion, and (iii) a 2D nucleation mechanism followed by a growth at a constant rate. The nonlinear fit of the experimental transients by using this procedure allows the quantitative determination of the individual contributions to the overall process. The kinetics of the formation process is studied under electrochemical conditions. The chronoamperometric experiment allows us to monitor the early stages of 6TG SAM formation. The implications of the physisorbed state at low potentials in the type of monolayer formation and destruction processes as well as the influence of temperature are also discussed.

Electrochemistry↗