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PubMed2003

The likelihood of aggregation during protein renaturation can be assessed using the second virial coefficient.

Protein aggregation is commonly observed during protein refolding. To better understand this phenomenon, the intermolecular interactions experienced by a protein during unfolding and refolding are inferred from second virial coefficient (SVC) measurements. It is accepted that a negative SVC is indicative of protein-protein interactions that are attractive, whereas a positive SVC indicates net repulsive interactions. Lysozyme denatured and reduced in guanidinium hydrochloride exhibited a decreasing SVC as the denaturant was diluted, and the SVC approached zero at approximately 3 M GdnHCl. Further dilution of denaturant to renaturation conditions (1.25 M GdnHCl) led to a negative SVC, and significant protein aggregation was observed. The inclusion of 500 mM L-arginine in the renaturation buffer shifted the SVC to positive and suppressed aggregation, thereby increasing refolding yield. The formation of mixed disulfides in the denatured state prior to refolding also increased protein solubility and suppressed aggregation, even without the use of L-arginine. Again, the suppression of aggregation was shown to be caused by a shift from attractive to repulsive intermolecular interactions as reflected in a shift from a negative to a positive SVC value. To the best of our knowledge, this is the first time that SVC data have been reported for renaturation studies. We believe this technique will aid in our understanding of how certain conditions promote renaturation and increase protein solubility, thereby suppressing aggregation. SVC measurements provide a useful link, for protein folding and aggregation, between empirical observation and thermodynamics.

Animals↗
PubMed2001

beta-Amyloid protein aggregation: its implication in the physiopathology of Alzheimer's disease.

beta-Amyloid protein (A beta), a 39-42 residue peptide resulting from the proteolytic processing of a membrane-bound beta-amyloid precursor protein (APP), is one of the major components of the fibrillar deposits observed in Alzheimer patients. A beta fibril formation is a complex process which involves changes in A beta conformation and self-association to form cross-beta pleated sheets, protofibrils, and fibrils. Since the aggregation of soluble A beta peptide into fibrils is viewed as a critical event in the physiopathology of Alzheimer's disease (AD), preventing, altering, or reversing fibril formation may thus be of therapeutic value. This review will focus on the current state of knowledge of A beta fibril formation, with special emphasis on physiological and exogenous inhibitors which may have a therapeutic potential.

Alzheimer Disease↗
PubMed2006

Molecular morphology and toxicity of cytoplasmic prion protein aggregates in neuronal and non-neuronal cells.

Recent studies have revealed that accumulation of prion protein (PrP) in the cytoplasm results in the production of aggregates that are insoluble in non-ionic detergents and partially resistant to proteinase K. Transgenic mice expressing PrP in the cytoplasm develop severe ataxia with cerebellar degeneration and gliosis, suggesting that cytoplasmic PrP may play a role in the pathogenesis of prion diseases. The mechanism of cytoplasmic PrP neurotoxicity is not known. In this report, we determined the molecular morphology of cytoplasmic PrP aggregates by immunofluorescence and electron microscopy, in neuronal and non-neuronal cells. Transient expression of cytoplasmic PrP produced juxtanuclear aggregates reminiscent of aggresomes in human embryonic kidney 293 cells, human neuroblastoma BE2-M17 cells and mouse neuroblastoma N2a cells. Time course studies revealed that discrete aggregates form first throughout the cytoplasm, and then coalesce to form an aggresome. Aggresomes containing cytoplasmic PrP were 1-5-microm inclusion bodies and were filled with electron-dense particles. Cytoplasmic PrP aggregates induced mitochondrial clustering, reorganization of intermediate filaments, prevented the secretion of wild-type PrP molecules and diverted these molecules to the cytoplasm. Cytoplasmic PrP decreased the viability of neuronal and non-neuronal cells. We conclude that any event leading to accumulation of PrP in the cytoplasm is likely to result in cell death.

Animals↗
PubMed1990

Identification of a virB10 protein aggregate in the inner membrane of Agrobacterium tumefaciens.

Products of the virB operon are proposed components of a membrane-associated T-DNA transport apparatus in Agrobacterium tumefaciens. Here we identified the virB10 gene product and raised specific antiserum to the protein. While the virB10 reading frame contains two potential ATG translation start sites located 32 codons apart, we found that only the downstream ATG was required for efficient VirB10 synthesis. Cellular localization studies and analysis of translational fusions with the Escherichia coli alkaline phosphatase gene (phoA) indicated that VirB10 was anchored in the inner membrane and contained a periplasmic domain. This work also demonstrated the utility of alkaline phosphatase as a reporter for secreted proteins in A. tumefaciens. Several high-molecular-weight forms of VirB10 were observed after treatment of A. tumefaciens whole cells or inner membranes with protein cross-linking agents, suggesting that VirB10 exists as a native oligomer or forms an aggregate with other membrane proteins. These results provide the first biochemical evidence that a VirB protein complex is membrane associated in A. tumefaciens.

Amino Acid Sequence↗
PubMed2006

Structural characterization of beta-sheeted oligomers formed on the pathway of oxidative prion protein aggregation in vitro.

The pathology of transmissible spongiform encephalopathies (TSEs) is strongly associated with the structural conversion of the cellular prion protein (PrPC) into a misfolded isoform (PrPSc) that assembles into amyloid fibrils. Since increased levels of oxidative stress have been linked to prion diseases, we investigated the metal-induced oxidation of human PrP (90-231). A novel in vitro conversion assay based on aerobic incubation of PrP in the presence of elemental copper pellets at pH 5 was established, resulting in aggregation of highly beta-sheeted prion proteins. We show for the first time that two discrete oligomeric species of elongated shape, approx. 25 mers and 100 mers, are formed on the pathway of oxidative PrP aggregation in vitro, which are well characterized regarding shape and size using small-angle X-ray scattering (SAXS), dynamic light scattering (DLS), and electron microscopy (EM). Considering that small oligomers of highly similar size have recently been reported to show the highest specific infectivity within TSE-infected brain tissues of hamsters, the novel oligomers observed in this study are interesting candidates as agent causing neurodegenerative and/or self-propagating effects. Moreover, our results significantly strengthen the theory that oxidative stress might be an influence that leads to substantial structural conversions of PrP in vivo.

Amino Acid Sequence↗
PubMed2001

Crowbars and ratchets: hsp100 chaperones as tools in reversing protein aggregation.

Molecular chaperones have the capacity to prevent inappropriate interactions between aggregation-prone folding or unfolding intermediates created in the cell during protein synthesis or in response to physical and chemical stress. What happens when surveillance by molecular chaperones is evaded or overwhelmed and aggregates accumulate? Recent progress in the elucidation of Hsp100/Clp function suggests that intracellular aggregates or stable complexes can be progressively dissolved by the action of chaperones that act as molecular crowbars or ratchets. These insights set the stage for new progress in the understanding and treatment of diseases of protein folding.

ATPases Associated with Diverse Cellular Activitie↗
PubMed1999

Unfolded protein response-induced BiP/Kar2p production protects cell growth against accumulation of misfolded protein aggregates in the yeast endoplasmic reticulum.

Overproduction of delta(pro), a mutated secretory proteinase derived from a filamentous fungus Rhizopus niveus, results in formation of gross aggregates (delta(pro) aggregates) in the yeast endoplasmic reticulum (ER) lumen, activation of the unfolded protein response (UPR) and ER membrane proliferation. To investigate the roles of the UPR against the delta(pro) aggregates, we constructed an IRE1-deleted ((delta)ire1) strain. In contrast to wild-type cells, (delta)ire1 cells ceased to grow several hours after the overproduction of (delta)pro. Two lines of evidence argued against the possibility that the growth defect was due to the inability to make extra ER membrane which accommodates the (delta)pro aggregates. First, by electron microscopy, ER membrane proliferation was observed in (delta)ire1 cells overproducing (delta)pro. Second, disruption of the OPI1 gene in the (delta)ire1 mutant, which is considered to derepress the activities of phospholipid-synthesizing enzymes, did not restore the growth upon the overproduction of (delta)pro. Instead, the growth was restored when an extra copy of the KAR2 gene, which encodes yeast BiP, was introduced, indicating that an increase in the amount of BiP is essential for cell growth when the (delta)pro aggregates accumulate in the ER. Since BiP is included in the insoluble (delta)pro aggregates, it is likely that the amount of free BiP in the ER lumen is insufficient without the UPR to fully exert its functions. Consistently, overproduction of (delta)pro impaired protein translocation and folding in (delta)ire1 cells but not in wild-type cells. The tunicamycin sensitivity of (delta)ire1 cells was also suppressed by extra expression of KAR2, suggesting that BiP plays a principal role in protecting cell growth against misfolded proteins accumulated in the ER.

Base Sequence↗
PubMed2006

A motor neuron disease-associated mutation in p150Glued perturbs dynactin function and induces protein aggregation.

The microtubule motor cytoplasmic dynein and its activator dynactin drive vesicular transport and mitotic spindle organization. Dynactin is ubiquitously expressed in eukaryotes, but a G59S mutation in the p150Glued subunit of dynactin results in the specific degeneration of motor neurons. This mutation in the conserved cytoskeleton-associated protein, glycine-rich (CAP-Gly) domain lowers the affinity of p150Glued for microtubules and EB1. Cell lines from patients are morphologically normal but show delayed recovery after nocodazole treatment, consistent with a subtle disruption of dynein/dynactin function. The G59S mutation disrupts the folding of the CAP-Gly domain, resulting in aggregation of the p150Glued protein both in vitro and in vivo, which is accompanied by an increase in cell death in a motor neuron cell line. Overexpression of the chaperone Hsp70 inhibits aggregate formation and prevents cell death. These data support a model in which a point mutation in p150Glued causes both loss of dynein/dynactin function and gain of toxic function, which together lead to motor neuron cell death.

Animals↗
PubMedDate not supplied

[The non-functioning chaperonin GroEL stimulates protein aggregation].

To clarify the role of chaperones in the development of amyloid diseases, the interaction of the chaperonin GroEL with misfolded proteins and recombinant prions has been studied. The efficiency of the chaperonin-assisted folding of denatured glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was shown to decrease in the presence of prions. Prions are capable of binding to GroEL immobilized on Sepharose, but this does not prevent the interaction between GroEL and other denatured proteins. The sizes of individual proteins (GroEL, GAPDH, and the recombinant prion), as well as aggregates formed after their mixing, were determined by the dynamic light scattering method. It was shown that at 25 degrees C the non-functioning chaperonin (equimolar mixture of GroEL and GroES in the absence of Mg-ATP) bound prion yielding large aggregates (greater than 400 nm). The addition of Mg-ATP decreased significantly the aggregate size to 70-80 nm. On the blocking of one of the chaperonin centers by oxidized denatured GAPDH, the aggregate size increased to 1200 nm, and the addition of Mg-ATP did not prevent the aggregation. These data indicate the significant role of chaperonins in the formation of amyloid structures and demonstrate the acceleration of aggregation in the presence of functionally inactive chaperonins. The suggested model can be used for the analysis of the efficiency of antiaggregants in the system containing chaperonins.

Amyloid↗
PubMed2002

Induction of protein aggregation in an early secretory compartment by elevation of expression level.

A variety of valuable therapeutic proteins are expressed in mammalian cells. Currently, rate-limiting for secretion of recombinant glycoproteins are activities in the secretory pathway of eukaryotic cells, i.e., folding and glycosylation of the naked polypeptide chain. In this paper we provide evidence that elevation of expression level alone is sufficient to cause intracellular aggregation of a structurally relatively simple glycoprotein, antithrombin III (ATIII). Elevation of expression level by selection for increased drug resistance in Chinese hamster ovary cells stably expressing ATIII resulted in formation of disulfide-bonded aggregates of ATIII. Aggregated ATIII displayed incomplete sialylation and Endo H-sensitivity and located to the endoplasmic reticulum and the cis-Golgi compartment in subcellular fractionations. To explore possible causes for aggregation of ATIII at elevated expression levels we investigated the influence of the two major energy sources of cultured mammalian cells, D-glucose and L-glutamine, on the ATIII-yield. We found that utilization of D-glucose was not limiting for synthesis of ATIII at elevated expression levels. However, the amount of ATIII-synthesized per L-glutamine consumed did not seem to increase steadily with expression level for ATIII, indicating that secretion of ATIII may be limited by the capacity of the cell to utilize L-glutamine.

Animals↗
PubMed1975

The effect of manipulation of reticuloendothelial system activity on glomerular deposition of aggregated protein and immune complexes in two different strains of mice.

Glomerular uptake of intravenously administered aggregated albumen or immune complexes in mice appears to be inversely related to the activity of the reticuloendothelial system (RES). Stimulation of RES activity diminishes the amount of material appearing in the glomerulus whereas RES blockade enhances glomerular uptake. The possible relevance of these observations to experimental models of immune complex disease is discussed.

Anaphylaxis↗
PubMedDate not supplied

Analysis of the two p97/VCP/Cdc48p proteins of Caenorhabditis elegans and their suppression of polyglutamine-induced protein aggregation.

A class of inherited neurodegenerative diseases including Huntington's disease is caused by polyglutamine (polyQ) expansion in the responsible proteins. Pathology is typically associated with polyQ expansions of greater than 40 residues, and the longer the length of the expansion, the earlier the onset of disease. It has been reported that p97/VCP/Cdc48p, a member of AAA family of proteins, can bind to longer polyQ tracts. In Caenorhabditis elegans, two p97/VCP/Cdc48p homologues, C41C4.8 and C06A1.1, have been identified. Our results indicate that these p97/VCP/Cdc48p homologues have essential but redundant functions in C. elegans. To provide a model system for investigating the molecular basis of pathogenesis, we have expressed polyQ expansions fused to green fluorescent protein in the body wall muscle cells of C. elegans. When the repeats are longer than 40, discrete cytoplasmic aggregates are formed and these appear at an early stage of embryogenesis. The formation of aggregates was partially suppressed by co-expression of either C41C4.8 or C06A1.1. These results suggest that these p97/VCP/Cdc48p homologues, AAA chaperones, may play a protective role in polyQ aggregation.

Adenosine Triphosphatases↗
PubMed1997

Accumulation of misfolded protein aggregates leads to the formation of russell body-like dilated endoplasmic reticulum in yeast.

RNAP-1, an aspartic proteinase from a filamentous fungus Rhizopus niveus, is secreted very efficiently in Saccharomyces cerevisiae. It is synthesized first as a precursor form with signal sequence and prosequence in its amino-terminus. Our previous study indicated that the prosequence of RNAP-I had important roles in its correct folding and secretion in yeast, and that a prosequence-deleted derivative of RNAP-I, delta pro, was not secreted but was retained and degraded in the yeast endoplasmic reticulum (ER). In the present study, we show that the accumulation of delta pro in the yeast ER caused elevated synthesis of ER resident chaperones, indicating that delta pro is recognized as an unfolded protein species in the ER. Our biochemical data demonstrated that delta pro formed aggregates which contained BiP, but not protein disulfide isomerase (PDI), in the ER. Immunoelectron microscopical analysis revealed that the delta pro aggregates were indeed visible as electron-dense regions in the ER and nuclear envelope. Such 'chaperone-associated misfolded protein bodies' were observed for the first time in yeast. Morphologies of the ER and nucleus were drastically altered by the accumulation of the delta pro aggregates. The ER lost its flat cisternal shape; the ER lumen extended aberrantly and the ER membrane irregularly proliferated. The misfolded delta pro proteins are probably sorted from the ordinary ER lumen to form the aggregates so that the ER function would not be grossly impaired, and the dilated ER may represent an ER subcompartment where the delta pro aggregates are degraded.

Amino Acid Sequence↗
PubMed2005

Cytoplasmic targeting of mutant poly(A)-binding protein nuclear 1 suppresses protein aggregation and toxicity in oculopharyngeal muscular dystrophy.

Oculopharyngeal muscular dystrophy (OPMD) is an adult-onset disorder characterized by progressive eyelid drooping, swallowing difficulties and proximal limb weakness. The autosomal dominant form of this disease is caused by a polyalanine expansion from 10 to 12-17 residues, located at the N-terminus of the poly(A)-binding protein nuclear 1 (PABPN1). A distinct pathological hallmark of OPMD is the presence of filamentous intranuclear aggregates in patients' skeletal muscle cells. Wildtype PABPN1 protein is expressed ubiquitously and was shown to be mostly concentrated in discrete nuclear domains called 'speckles'. Using an established cell- culture model, we show that most mutant PABPN1- positive (alanine expanded form) intranuclear aggregates are structures distinct from intranuclear speckles. In contrast, the promyelocytic leukaemia protein, a major component of nuclear bodies, strongly colocalized to intranuclear aggregates of mutant PABPN1. Wildtype PABPN1 can freely shuttle between the nucleus and cytoplasm. We determined whether the nuclear environment is necessary for mutant PABPN1 inclusion formation and cellular toxicity. This was achieved by inactivating the mutant PABPN1 nuclear localization signal and by generating full-length mutant PABPN1 fused to a strong nuclear export sequence. A green fluorescence protein tag inserted at the N-terminus of both wildtype PABPN1 (ala10) and mutant PABPN1 (ala17) proteins allowed us to visualize their subcellular localization. Targeting mutant PABPN1 to the cytoplasm resulted in a significant suppression of both intranuclear aggregates formation and cellular toxicity, two histological consequences of OPMD. Our results indicate that the nuclear localization of mutant PABPN1 is crucial to OPMD pathogenesis.

Amino Acid Sequence↗
PubMed2002

Guanidine hydrochloride inhibits the generation of prion "seeds" but not prion protein aggregation in yeast.

[PSI(+)] strains of the yeast Saccharomyces cerevisiae replicate and transmit the prion form of the Sup35p protein but can be permanently cured of this property when grown in millimolar concentrations of guanidine hydrochloride (GdnHCl). GdnHCl treatment leads to the inhibition of the replication of the [PSI(+)] seeds necessary for continued [PSI(+)] propagation. Here we demonstrate that the rate of incorporation of newly synthesized Sup35p into the high-molecular-weight aggregates, diagnostic of [PSI(+)] strains, is proportional to the number of seeds in the cell, with seed number declining (and the levels of soluble Sup35p increasing) in the presence of GdnHCl. GdnHCl does not cause breakdown of preexisting Sup35p aggregates in [PSI(+)] cells. Transfer of GdnHCl-treated cells to GdnHCl-free medium reverses GdnHCl inhibition of [PSI(+)] seed replication and allows new prion seeds to be generated exponentially in the absence of ongoing protein synthesis. Following such release the [PSI(+)] seed numbers double every 20 to 22 min. Recent evidence (P. C. Ferreira, F. Ness, S. R. Edwards, B. S. Cox, and M. F. Tuite, Mol. Microbiol. 40:1357-1369, 2001; G. Jung and D. C. Masison, Curr. Microbiol. 43:7-10, 2001), together with data presented here, suggests that curing yeast prions by GdnHCl is a consequence of GdnHCl inhibition of the activity of molecular chaperone Hsp104, which in turn is essential for [PSI(+)] propagation. The kinetics of elimination of [PSI(+)] by coexpression of a dominant, ATPase-negative allele of HSP104 were similar to those observed for GdnHCl-induced elimination. Based on these and other data, we propose a two-cycle model for "prionization" of Sup35p in [PSI(+)] cells: cycle A is the GdnHCl-sensitive (Hsp104-dependent) replication of the prion seeds, while cycle B is a GdnHCl-insensitive (Hsp104-independent) process that converts these seeds to pelletable aggregates.

Cell Division↗