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Pro-inflammatory and amyloidogenic S100A9 protein is central to the amyloid-neuroinflammatory cascade in neurodegenerative diseases. Polyoxometalates (POMs) constitute a diverse group of nanomaterials, which showed potency in amyloid inhibition. Here, we have demonstrated that two selected nanosized niobium POMs, Nb10 and TiNb9, can act as potent inhibitors of S100A9 amyloid assembly. Kinetics analysis based on ThT fluorescence experiments showed that addition of either Nb10 or TiNb9 reduces the S100A9 amyloid formation rate and amyloid quantity. Atomic force microscopy imaging demonstrated the complete absence of long S100A9 amyloid fibrils at increasing concentrations of either POM and the presence of only round-shaped and slightly elongated aggregates. Molecular dynamics simulation revealed that both Nb10 and TiNb9 bind to native S100A9 homo-dimer by forming ionic interactions with the positively charged Lys residue-rich patches on the protein surface. The acrylamide quenching of intrinsic fluorescence showed that POM binding does not perturb the Trp 88 environment. The far and near UV circular dichroism revealed no large-scale perturbation of S100A9 secondary and tertiary structures upon POM binding. These indicate that POM binding involves only local conformational changes in the binding sites. By using intrinsic and 8-anilino-1-naphthalene sulfonate fluorescence titration experiments, we found that POMs bind to S100A9 with a Kd of ca. 2.5 μM. We suggest that the region, including Lys 50 to Lys 54 and characterized by high amyloid propensity, could be the key sequences involved in S1009 amyloid self-assembly. The inhibition and complete hindering of S100A9 amyloid pathways may be used in the therapeutic applications targeting the amyloid-neuroinflammatory cascade in neurodegenerative diseases.

The amyloid cascade is central for the neurodegeneration disease pathology, including Alzheimer’s and Parkinson’s, and remains the focus of much current research. S100A9 protein drives the amyloid-neuroinflammatory cascade in these diseases. DOPA and cyclen-based compounds were used as amyloid modifiers and inhibitors previously, and DOPA is also used as a precursor of dopamine in Parkinson’s treatment. Here, by using fluorescence titration experiments we showed that five selected ligands: DOPA-D-H-DOPA, DOPA-H-H-DOPA, DOPA-D-H, DOPA-cyclen, and H-E-cyclen, bind to S100A9 with apparent Kd in the sub-micromolar range. Ligand docking and molecular dynamic simulation showed that all compounds bind to S100A9 in more than one binding site and with different ligand mobility and H-bonds involved in each site, which all together is consistent with the apparent binding determined in fluorescence experiments. By using amyloid kinetic analysis, monitored by thioflavin-T fluorescence, and AFM imaging, we found that S100A9 co-aggregation with these compounds does not hinder amyloid formation but leads to morphological changes in the amyloid fibrils, manifested in fibril thickening. Thicker fibrils were not observed upon fibrillation of S100A9 alone and may influence the amyloid tissue propagation and modulate S100A9 amyloid assembly as part of the amyloid-neuroinflammatory cascade in neurodegenerative diseases.

Mismatch repair (MMR) is a major DNA repair pathway in cells from all branches of life that removes replication errors in a strand-specific manner, such that mismatched nucleotides are preferentially removed from the newly replicated strand of DNA. Here we demonstrate a role for MMR in helping create new phenotypes in nondividing cells. We show that mispairs in yeast that escape MMR during replication can later be subject to MMR activity in a replication strand-independent manner in nondividing cells, resulting in either fully wild-type or mutant DNA sequence. In one case, this activity is responsible for what appears to be adaptive mutation. This replication strand-independent MMR activity could contribute to the formation of tumors arising in nondividing cells and could also contribute to mutagenesis observed during somatic hypermutation of Ig genes.

High-ordered aggregates (amyloids) may disrupt cell functions, cause toxicity at certain conditions and provide a basis for self-perpetuated, protein-based infectious heritable agents (prions). Heat shock proteins acting as molecular chaperones counteract protein aggregation and influence amyloid propagation. The yeast Hsp104/Hsp70/Hsp40 chaperone complex plays a crucial role in interactions with both ordered and unordered aggregates. The main focus of this review will be on the Hsp104 chaperone, a molecular "disaggregase".

Prions are self-perpetuating aggregated fibrous proteins that associated with fatal transmissible spongiform encephalopathies (TSEs) in mammals (including humans ), and manifest themselves as non-Mendelian heritable elements in yeast and other fungi. The infectious agent responsible for TSEs is the prion, an abnormally folded and aggregated protein that propagates itself by imposing its conformation onto the cellular prion protein (PrPC) of the host. PrPC is necessary for prion replication and for prion-induced neurodegeneration, yet causes of neuronal injury and death are still poorly understood. Here we view of the prion concept, models describing of the replication and transport of prions particles, structural features and functions of the cellular PrP, the prion strain phenomenon, current developments in diagnostics of prion and potential antiprion therapies. Finally, we discuss how prion-like mechanisms may apply to other protein aggregation diseases.

Human urokinase-type plasminogen activator (uPA) is poorly secreted by yeast cells. Here, we have selected Hansenula polymorpha mutants with increased productivity of active extracellular uPA. Several of the obtained mutants also demonstrated a defect of sorting of carboxypeptidase Y to the vacuole and the mutant loci have been identified in six of them. All these mutations damaged genes involved in protein traffic between the Golgi apparatus and the vacuole, namely PEP3, VPS8, VPS10, VPS17, and VPS35. We have shown that inactivation of the VPS10 gene encoding the vacuolar protein sorting receptor does not increase uPA secretion but stimulates its proteolytic processing.

Human urokinase-type plasminogen activator (uPA) is poorly secreted and aggregates in the endoplasmic reticulum of yeast cells due to inefficient folding. A screen for Hansenula polymorpha mutants with improved uPA secretion revealed a gene encoding a homologue of the Saccharomyces cerevisiae protein-O-mannosyltransferase Pmt1p. Expression of the H. polymorpha PMT1 gene (HpPMT1) abolished temperature sensitivity of the S. cerevisiae pmt1 pmt2 double mutant. As in S. cerevisiae, inactivation of the HpPMT1 gene affected electrophoretic mobility of the O-glycosylated protein, extracellular chitinase. In contrast to S. cerevisiae, disruption of HpPMT1 alone caused temperature sensitivity. Inactivation of the HpPMT1 gene decreased intracellular aggregation of uPA, suggesting that enhanced secretion of uPA was due to improvement of its folding in the endoplasmic reticulum. Unlike most of the endoplasmic reticulum membrane proteins, HpPmt1p possesses the C-terminal KDEL retention signal. The GenBank Accession No. for the H. polymorpha PMT1 sequence is AY701415. Copyright © 2005 John Wiley & Sons, Ltd.

DNA mismatch repair greatly increases genome fidelity by recognizing and removing replication errors. In order to understand how this fidelity is maintained, it is important to uncover the relative specificities of the different components of mismatch repair. There are two major mispair recognition complexes in eukaryotes that are homologues of bacterial MutS proteins, MutSα and MutSβ, with MutSα recognizing base-base mismatches and small loop mispairs and MutSβ recognizing larger loop mispairs. Upon recognition of a mispair, the MutS complexes then interact with homologues of the bacterial MutL protein. Loops formed on the primer strand during replication lead to insertion mutations, whereas loops on the template strand lead to deletions. We show here in yeast, using oligonucleotide transformation, that MutSα has a strong bias toward repair of insertion loops, while MutSβ has an even stronger bias toward repair of deletion loops. Our results suggest that this bias in repair is due to the different interactions of the MutS complexes with the MutL complexes. Two mutants of MutLα, pms1-G882E and pms1-H888R, repair deletion mispairs but not insertion mispairs. Moreover, we find that a different MutL complex, MutLγ, is extremely important, but not sufficient, for deletion repair in the presence of either MutLα mutation. MutSβ is present in many eukaryotic organisms, but not in prokaryotes. We suggest that the biased repair of deletion mispairs may reflect a critical eukaryotic function of MutSβ in mismatch repair., Author Summary DNA mismatch repair is a major pathway that prevents both base substitution and insertion or deletion errors during replication. Most eukaryotes have two recognition complexes, MutSα and MutSβ, homologues of prokaryotic MutS and differing in their affinity for mismatches, with MutSα recognizing base-base mismatches and small insertion/deletion loops and MutSβ recognizing larger loops. We show that repair mediated by these complexes has opposite biases for insertion versus deletion mispairs with MutSα-directed repair favoring insertion loops and MutSβ-directed repair favoring deletion loops. This bias is mediated by differing interactions with downstream MutL complexes. We suggest that MutSα represents a prokaryotic MutS biased for repair of insertion loops and that MutSβ represents a new eukaryotic activity biased for repair of deletion loops.

Polyglutamine expansion causes diseases in humans and other mammals. One example is Huntington's disease. Fragments of human huntingtin protein having an expanded polyglutamine stretch form aggregates and cause cytotoxicity in yeast cells bearing endogenous QN-rich proteins in the aggregated (prion) form. Attachment of the proline(P)-rich region targets polyglutamines to the large perinuclear deposit (aggresome). Aggresome formation ameliorates polyglutamine cytotoxicity in cells containing only the prion form of Rnq1 protein. Here we show that expanded polyglutamines both with (poly-QP) or without (poly-Q) a P-rich stretch remain toxic in the presence of the prion form of translation termination (release) factor Sup35 (eRF3). A Sup35 derivative that lacks the QN-rich domain and is unable to be incorporated into aggregates counteracts cytotoxicity, suggesting that toxicity is due to Sup35 sequestration. Increase in the levels of another release factor, Sup45 (eRF1), due to either disomy by chromosome II containing the SUP45 gene or to introduction of the SUP45-bearing plasmid counteracts poly-Q or poly-QP toxicity in the presence of the Sup35 prion. Protein analysis confirms that polyglutamines alter aggregation patterns of Sup35 and promote aggregation of Sup45, while excess Sup45 counteracts these effects. Our data show that one and the same mode of polyglutamine aggregation could be cytoprotective or cytotoxic, depending on the composition of other aggregates in a eukaryotic cell, and demonstrate that other aggregates expand the range of proteins that are susceptible to sequestration by polyglutamines., Author Summary Polyglutamine diseases, including Huntington disease, are associated with expansions of polyglutamine tracts, resulting in aggregation of respective proteins. The severity of Huntington disease is controlled by both DNA and non–DNA factors. Mechanisms of such a control are poorly understood. Polyglutamine may sequester other cellular proteins; however, different experimental models have pointed to different sequestered proteins. By using a yeast model, we demonstrate that the mechanism of polyglutamine toxicity is driven by the composition of other (endogenous) aggregates (for example, yeast prions) present in a eukaryotic cell. Although these aggregates do not necessarily cause significant toxicity on their own, they serve as mediators in protein sequestration and therefore determine which specific proteins are to be sequestered by polyglutamines. We also show that polyglutamine deposition into an aggresome, a perinuclear compartment thought to be cytoprotective, fails to ameliorate cytotoxicity in cells with certain compositions of pre-existing aggregates. Finally, we demonstrate that an increase in the dosage of a sequestered protein due to aneuploidy by a chromosome carrying a respective gene may rescue cytotoxicity. Our data shed light on genetic and epigenetic mechanisms modulating polyglutamine cytotoxicity and establish a new approach for identifying potential therapeutic targets through characterization of the endogenous aggregated proteins.

We have developed a set of cloning vectors possessing a modified Tn903 kanamycin resistance gene that enables the selection of both kanamycin-resistant transformants in Escherichia coli and G418-resistant transformants in the yeasts Saccharomyces cerevisiae, Hansenula polymorpha and Pichia pastoris. Expression of this gene in yeast is controlled by the H. polymorpha glyceraldehyde-3-phosphate dehydrogenase promoter, while expression in E. coli is governed by an upstream E. coli lacZ promoter. Applicability of the vectors for gene disruption in H. polymorpha and S. cerevisiae was demonstrated by inactivation of the HpMAL1 and URA3 genes, respectively. One of the vectors possesses a H. polymorpha ARS allowing plasmid maintenance in an episomal state. The small size of the vectors (2-2.5 kb) makes them convenient for routine DNA cloning. In addition, we report a novel approach for construction of gene disruption cassettes.

In mammalian cells, abnormal proteins that escape proteasome-dependent degradation form small aggregates that can be transported into a centrosome-associated structure, called an aggresome. Here we demonstrate that in yeast a single aggregate formed by the huntingtin exon 1 with an expanded polyglutamine domain (103QP) represents a bona fide aggresome that colocalizes with the spindle pole body (the yeast centrosome) in a microtubule-dependent fashion. Since a polypeptide lacking the proline-rich region (P-region) of huntingtin (103Q) cannot form aggresomes, this domain serves as an aggresome-targeting signal. Coexpression of 103Q with 25QP, a soluble polypeptide that also carries the P-region, led to the recruitment of 103Q to the aggresome via formation of hetero-oligomers, indicating the aggresome targeting in trans. To identify additional factors involved in aggresome formation and targeting, we purified 103QP aggresomes and 103Q aggregates and identified the associated proteins using mass spectrometry. Among the aggresome-associated proteins we identified, Cdc48 (VCP/p97) and its cofactors, Ufd1 and Nlp4, were shown genetically to be essential for aggresome formation. The 14-3-3 protein, Bmh1, was also found to be critical for aggresome targeting. Its interaction with the huntingtin fragment and its role in aggresome formation required the huntingtin N-terminal N17 domain, adjacent to the polyQ domain. Accordingly, the huntingtin N17 domain, along with the P-region, plays a role in aggresome targeting. We also present direct genetic evidence for the protective role of aggresomes by demonstrating genetically that aggresome targeting of polyglutamine polypeptides relieves their toxicity.—Wang, Y., Meriin, A. B., Zaarur, N., Romanova, N. V., Chernoff, Y. O., Costello, C. E., Sherman, M. Y. Abnormal proteins can form aggresome in yeast: aggresome-targeting signals and components of the machinery.

In yeast, functions of the endoplasmic reticulum (ER) depend on the Golgi apparatus Ca2+ pool, which is replenished by the medial-Golgi ion pump Pmr1p. Here, to dissect the role of the Golgi Ca2+ pool in protein folding and elimination of unfolded proteins in the ER, the manifestations of the pmr1 mutation in yeast Hansenula polymorpha were studied. The PMR1 gene was disrupted in a H. polymorpha diploid strain. Haploid segregants of this diploid bearing the disruption allele were viable, though they showed a severe growth defect on synthetic medium and rapidly died during storage at low temperature. Disruption of H. polymorpha PMR1 led to defects of the Golgi-hosted protein glycosylation and vacuolar protein sorting. This mutation increased the survival rate of H. polymorpha cells upon treatment with the proapoptotic drug amiodarone. Unlike Saccharomyces cerevisiae , the H. polymorpha pmr1 mutant was not hypersensitive to chemicals that induce the accumulation of unfolded proteins in the ER, indicating that the elimination of unfolded proteins from the ER was not essentially affected. At the same time, the pmr1 mutation improved the secretion of human urokinase and decreased its intracellular aggregation, indicating an influence of the mutation on the protein folding in the ER.

In eukaryotic cells, COPI vesicles retrieve resident proteins to the endoplasmic reticulum and mediate intra-Golgi transport. Here, we studied the Hansenula polymorpha homologue of the Saccharomyces cerevisiae RET1 gene, encoding -COP, a subunit of the COPI protein complex. H. polymorpha ret1 mutants, which expressed truncated -COP lacking more than 300 C-terminal amino acids, manifested an enhanced ability to secrete human urokinase-type plasminogen activator (uPA) and an inability to grow with a shortage of Ca 2 ions, whereas a lack of -COP expression was lethal. The -COP defect also caused alteration of intracellular transport of the glycosylphosphatidylinositol-anchored protein Gas1p, secretion of abnormal uPA forms, and reductions in the levels of Pmr1p, a Golgi Ca 2 -ATPase. Overexpression of Pmr1p suppressed some ret1 mutant phenotypes, namely, Ca 2 dependence and enhanced uPA secretion. The role of COPI-dependent vesicular transport in cellular Ca 2 homeostasis is discussed. In eukaryotic cells, secretory proteins synthesized at the endoplasmic reticulum (ER) pass through multiple distinct membrane-bound organelles comprising the secretory pathway. Transport of proteins and lipids between membrane compartments of the early secretory system is mediated by COPIand COPII-coated vesicles that capture cargo, bud from the donor membrane, and then target, dock, and fuse with an appropriate acceptor compartment (40). Both the COPI and COPII protein coat complexes employ a GTP switch mechanism for coating and uncoating. The COPII complex mediates selective protein export from the ER, while COPI-coated vesicles retrieve resident proteins to the ER and mediate intraGolgi transport (8, 38).

Exposure to high temperature or other stresses induces a synthesis of heat shock proteins. Many of these proteins are molecular chaperones, and some of them help cells to cope with heat induced denaturation and aggregation of other proteins. In the last decade, chaperones have received increased attention in connection with their role in maintenance and propagation of the Saccharomyces cerevisiae prions, infectious or heritable agents transmitted at the protein level. Recent data suggest that functioning of the chaperones in reactivation of heat damaged proteins and in propagation of prions is based on the same molecular mechanisms but may lead to different consequences depending on the type of aggregate. In both cases the concerted and balanced action of “chaperones’ team”, including Hsp104, Hsp70, Hsp40 and possibly other proteins, determines whether a misfolded protein is to be incorporated into an aggregate, rescued to the native state or targeted for degradation.

The aggresome is an organelle that recruits aggregated proteins for storage and degradation. We performed an siRNA screen for proteins involved in aggresome formation and identified novel mammalian AAA+ protein disaggregases RuvbL1 and RuvbL2. Depletion of RuvbL1 or RuvbL2 suppressed aggresome formation and caused buildup of multiple cytoplasmic aggregates. Similarly, downregulation of RuvbL orthologs in yeast suppressed the formation of an aggresome-like body and enhanced the aggregate toxicity. In contrast, their overproduction enhanced the resistance to proteotoxic stress independently of chaperone Hsp104. Mammalian RuvbL associated with the aggresome, and the aggresome substrate synphilin-1 interacted directly with the RuvbL1 barrel-like structure near the opening of the central channel. Importantly, polypeptides with unfolded structures and amyloid fibrils stimulated the ATPase activity of RuvbL. Finally, disassembly of protein aggregates was promoted by RuvbL. These data indicate that RuvbL complexes serve as chaperones in protein disaggregation.