Protein misfolding underlies many neurodegenerative diseases, including the Transmissible Spongiform Encephalopathies (prion diseases). While cells typically recognize and process misfolded proteins, prion proteins evade protective measures by forming stable, self-replicating aggregates. However, co-expression of dominant-negative prion mutants can overcome aggregate accumulation and disease progression through currently unknown pathways. Here, we determine the mechanisms by which two mutants of the Saccharomyces cerevisiae Sup35 protein cure the [PSI+] prion. We show that both mutants incorporate into wildtype aggregates and alter their physical properties in different ways, diminishing either their assembly rate or their thermodynamic stability. While wildtype aggregates are recalcitrant to cellular intervention, mixed aggregates are disassembled by the molecular chaperone Hsp104. Thus, rather than simply blocking misfolding, dominant-negative prion mutants target multiple events in aggregate biogenesis to enhance their susceptibility to endogenous quality control pathways.

Fructose-1,6-(bis)phosphate aldolase is a ubiquitous enzyme that catalyzes the reversible aldol cleavage of fructose-1,6-(bis)phosphate and fructose 1-phosphate to dihydroxyacetone phosphate and either glyceraldehyde-3-phosphate or glyceraldehyde, respectively. Vertebrate aldolases exist as three isozymes with different tissue distributions and kinetics: aldolase A (muscle and red blood cell), aldolase B (liver, kidney, and small intestine), and aldolase C (brain and neuronal tissue). The structures of human aldolases A and B are known and herein we report the first structure of the human aldolase C, solved by X-ray crystallography at 3.0 Å resolution. Structural differences between the isozymes were expected to account for isozyme-specific activity. However, the structures of isozymes A, B, and C are the same in their overall fold and active site structure. The subtle changes observed in active site residues Arg42, Lys146, and Arg303 are insufficient to completely account for the tissue-specific isozymic differences. Consequently, the structural analysis has been extended to the isozyme-specific residues (ISRs), those residues conserved among paralogs. A complete analysis of the ISRs in the context of this structure demonstrates that in several cases an amino acid residue that is conserved among aldolase C orthologs prevents an interaction that occurs in paralogs. In addition, the structure confirms the clustering of ISRs into discrete patches on the surface and reveals the existence in aldolase C of a patch of electronegative residues localized near the C terminus. Together, these structural changes highlight the differences required for the tissue and kinetic specificity among aldolase isozymes.Keywords: isozyme specificity; structural enzymology; protein-protein interactions; isozyme-specific residues; structure/function Fructose-1,6-(bis)phosphate aldolases are ubiquitous enzymes that catalyze the reversible cleavage of fructose-1,6-(bis)phosphate (Fru-1,6-P 2 ) and fructose 1-phosphate (Fru-1-P) to dihydroxy-acetone phosphate (DHAP) and either glyceraldehyde-3-phosphate (G3P) or glyceraldehyde, respectively. The mechanisms of these aldolases occur by two distinct chemical paths (Rutter 1964

Loss of heterozygosity (LOH) on 10q is associated with late‐stage events in urothelial neoplastic progression. The tumor suppressor gene PTEN, which is mutated or homozygously deleted in numerous cancers, maps to a region of 10q within the reported region of minimal loss in bladder tumors. In two recent studies alterations in the PTEN gene occur at a low frequency in bladder tumors displaying 10q LOH. We have screened 35 late‐stage bladder tumors for mutations in PTEN and MXI1, both genes mapping to chromosome 10q. Using single‐strand conformation polymorphism analysis, we identified 6 tumors harboring mutations in PTEN and 2 additional tumors displaying homozygous deletion at this locus. No MXI1 mutations were identified within the same tumor panel. Of 16 bladder tumor cell lines analyzed, 2 showed homozygous deletion of PTEN and 3 harbored point mutations resulting in an amino acid change. Two cell lines harbored missense mutations in MXI1. We report a significantly higher frequency of PTEN alterations in bladder carcinoma (23%) than was previously recorded, with no accompanying mutations in the MXI1 gene. Int. J. Cancer 88:620–625, 2000. © 2000 Wiley‐Liss, Inc.

As the range and duration of human ventures into space increase, it becomes imperative that we understand the effects of the cosmic environment on astronaut health. Molecular technologies now widely used in research and medicine will need to become available in space to ensure appropriate care of astronauts. The polymerase chain reaction (PCR) is the gold standard for DNA analysis, yet its potential for use on-orbit remains under-explored. We describe DNA amplification aboard the International Space Station (ISS) through the use of a miniaturized miniPCR system. Target sequences in plasmid, zebrafish genomic DNA, and bisulfite-treated DNA were successfully amplified under a variety of conditions. Methylation-specific primers differentially amplified bisulfite-treated samples as would be expected under standard laboratory conditions. Our findings establish proof of concept for targeted detection of DNA sequences during spaceflight and lay a foundation for future uses ranging from environmental monitoring to on-orbit diagnostics.

Vertebrate fructose-1,6-bisphosphate aldolase exists as three isozymes (A, B, and C) that demonstrate kinetic properties that are consistent with their physiological role and tissue-specific expression. The isozymes demonstrate specific substrate cleavage efficiencies along with differences in the ability to interact with other proteins; however, it is unknown how these differences are conferred. An alignment of 21 known vertebrate aldolase sequences was used to identify all of the amino acids that are specific to each isozyme, or isozyme-specific residues (ISRs). The location of ISRs on the tertiary and quaternary structures of aldolase reveals that ISRs are found largely on the surface (24 out of 27) and are all outside of hydrogen bonding distance to any active site residue. Moreover, ISRs cluster into two patches on the surface of aldolase with one of these patches, the terminal surface patch, overlapping with the actin-binding site of aldolase A and overlapping an area of higher than average temperature factors derived from the x-ray crystal structures of the isozymes. The other patch, the distal surface patch, comprises an area with a different electrostatic surface potential when comparing isozymes. Despite their location distal to the active site, swapping ISRs between aldolase A and B by multiple site mutagenesis on recombinant expression plasmids is sufficient to convert the kinetic properties of aldolase A to those of aldolase B. This implies that ISRs influence catalysis via changes that alter the structure of the active site from a distance or via changes that alter the interaction of the mobile C-terminal portion with the active site. The methods used in the identification and analysis of ISRs discussed here can be applied to other protein families to reveal functionally relevant residue clusters not accessible by conventional primary sequence alignment methods.Vertebrate fructose-1,6-bisphosphate aldolase is a ubiquitous tetrameric enzyme that catalyzes reactions in the glycolytic, gluconeogenic, and fructose metabolic pathways (1). The enzyme catalyzes the reversible aldol cleavage of Fru 1,6-P 2 1 into two trioses, glyceraldehyde 3-phosphate and dihydroxyacetone phosphate. It also cleaves the structurally related sugar Fru 1-P into glyceraldehyde and dihydroxyacetone phosphate (2, 3).Vertebrate aldolases exist as three or more isozymes with different tissue distributions: aldolase A (expressed predominantly in muscle), aldolase B (expressed predominantly in liver), and aldolase C (expressed predominantly in brain) (1). The sequences of the mammalian isozymes are highly conserved, exhibiting 81% sequence identity between aldolases A and C (4). Aldolase B is slightly more divergent with ϳ70% sequence identity to both aldolases A and C (5). Consistent with this sequence similarity, isozymes A and C exhibit comparable kinetic properties. Aldolases A and C have evolved to perform the glycolytic reaction, Fru 1,6-P 2 cleavage, more efficiently than aldolase B as demonstrated by a 20 -30-fold higher k ...

The self-assembly of alternative conformations of normal proteins into amyloid aggregates has been implicated in both the acquisition of new functions and in the appearance and progression of disease. However, while these amyloidogenic pathways are linked to the emergence of new phenotypes, numerous studies have uncoupled the accumulation of aggregates from their biological consequences, revealing currently underappreciated complexity in the determination of these traits. Here, to explore the molecular basis of protein-only phenotypes, we focused on the S. cerevisiae Sup35/[PSI+] prion, which confers a translation termination defect and expression level-dependent toxicity in its amyloid form. Our studies reveal that aggregated Sup35 retains its normal function as a translation release factor. However, fluctuations in the composition and size of these complexes specifically alter the level of this aggregate-associated activity and thereby the severity and toxicity of the amyloid state. Thus, amyloid heterogeneity is a crucial contributor to protein-only phenotypes.

Conformational flexibility is emerging as a central theme in enzyme catalysis. Thus, identifying and characterizing enzyme dynamics is critical for understanding catalytic mechanisms. Herein, coupling analysis, which uses thermodynamic analysis to assess cooperativity/coupling between distal regions on an enzyme, is used to interrogate substrate specificity among fructose-1,6-(bis)phosphate aldolase (aldolase) isozymes. Aldolase exists as three isozymes, A, B, and C distinguishable by their unique substrate preferences despite the fact that the structures of the active sites of the three isozymes are nearly identical. While conformational flexibility has been observed in aldolase A, its function in the catalytic reaction of aldolase has not been demonstrated. To explore the role of conformational dynamics in substrate specificity, those residues associated with isozyme specificity (ISRs) were swapped and the resulting chimeras were subjected to steady-state kinetics. Thermodynamic analyses suggest cooperativity between a terminal surface patch (TSP) and a distal surface patch (DSP) of ISRs that are separated by >8.9Å. Notably, the coupling energy (ΔG I ) is anti-correlated with respect to the two substrates, fructose 1,6-bisphosphate and fructose 1-phosphate. The difference in coupling energy with respect to these two substrates accounts for about 70% of the energy difference for the ratio of k cat /K m for the two substrates between aldolase A and aldolase B. These non-additive mutational effects between the TSP and DSP provide functional evidence that coupling interactions arising from conformational flexibility during catalysis are a major determinant of substrate specificity.Recent evidence shows dynamic fluctuations of structure are essential components of enzyme catalysis (1). While it is likely that most enzymes exhibit flexibility as part of the catalytic process, the role of this movement is enzyme specific. For example, in the case of dihydrofolate reductase (DHFR), backbone and side-chain motions are essential for cofactor binding, † This work was supported by Grants GM60616 (to D.R.T. and K.N.A.), DK065089 (to D.R.T), and Training Grant HL07291 (to J.A.P.) from the National Institutes of Health.*To whom correspondence may be addressed: Dean R. Tolan, Biology Department, 5 Cummington St., Boston MA 02215; (617) 353-5310 (tel), (617) 358-0338 (fax), email: tolan@bu.edu., Karen N. Allen, Department of Physiology and Biophysics, 715 Albany St., Boston University School of Medicine, Boston MA 02118; (617) 638-4398 (tel), (617) 638-4273 (fax), allen@med-xtal.bu.edu. ⊥ Present address: Department of Molecular Biology, Cell Biology and Biochemistry, Brown University, 185 Meeting Street, Box G-L2, Providence RI 02912 1 ISR, isozyme-specific residue; Fru 1,6-P 2 , fructose 1,6-bisphosphate; Fru 1-P, fructose 1-phosphate; CTR, C-terminal region; TSP, terminal surface patch; DSP, distal surface patch; DTNB, 5,5′dithiobis(2-nitrobenzoic acid). Recently, the importance of conformational flexibility has ...

Protein-only (prion) epigenetic elements confer unique phenotypes by adopting alternate conformations that specify new traits. Given the conformational flexibility of prion proteins, protein-only inheritance requires efficient self-replication of the underlying conformation. To explore the cellular regulation of conformational self-replication and its phenotypic effects, we analyzed genetic interactions between [PSI ؉ ], a prion form of the S. cerevisiae Sup35 protein (Sup35 [PSI ؉ ] ), and the three N ␣ -acetyltransferases, NatA, NatB, and NatC, which collectively modify ϳ50% of yeast proteins. Although prion propagation proceeds normally in the absence of NatB or NatC, the [PSI ؉ ] phenotype is reversed in strains lacking NatA. Despite this change in phenotype, [PSI ؉ ] NatA mutants continue to propagate heritable Sup35 [PSI ؉ ] . This uncoupling of protein state and phenotype does not arise through a decrease in the number or activity of prion templates (propagons) or through an increase in soluble Sup35. Rather, NatA null strains are specifically impaired in establishing the translation termination defect that normally accompanies Sup35 incorporation into prion complexes. The NatA effect cannot be explained by the modification of known components of the [PSI ؉ ] prion cycle including Sup35; thus, novel acetylated cellular factors must act to establish and maintain the tight link between Sup35 [PSI ؉ ] complexes and their phenotypic effects. INTRODUCTIONThe transmission of phenotypes from one individual to another is a fundamental process in biology. Much of our understanding of these events arises from decades of study on nucleic acid metabolism, but new traits may also be passed between individuals without changes in nucleic acid content through a number of epigenetic mechanisms. One particularly intriguing example of such a process is the prion phenomenon, in which the activity of a protein is altered in a heritable way to transmit a new phenotype. How is such a feat accomplished? In 1967, Griffith proposed that some proteins, now known as prions (Prusiner, 1982), can adopt more than one stable form in vivo (Griffith, 1967). Since a protein's structure determines its function, two cells containing the same protein but in diffrent physical states will have distinct phenotypes. This protein-based process has been linked to a number of previously inexplicable events, including the development and spread of the transmissible spongiform encephalopathies in mammals (Prusiner, 1982) and the non-Mendelian inheritance of some traits in fungi (Wickner, 1994).Protein-based traits can only become transmissible, however, if the inherent structural flexibility of prion proteins can be constrained by regulatory mechanisms to create an epigenetic element. For example, if each newly synthesized prion polypeptide chain independently folded to a unique form, all cells would display the same phenotype, which would reflect the average of the accessible states. The appearance of distinct protein-based phenotypes suggests that ...