Nucleic acid detection aboard the International Space Station by colorimetric loop-mediated isothermal amplification (LAMP).

Human spaceflight endeavors present an opportunity to expand our presence beyond Earth. To this end, it is crucial to understand and diagnose effects of long-term space travel on the human body. Developing tools for targeted, on-site detection of specific DNA sequences will allow us to establish research and diagnostics platforms that will benefit space programs. We describe a simple DNA diagnostic method that utilizes colorimetric loop-mediated isothermal amplification (LAMP) to enable detection of a repetitive telomeric DNA sequence in as little as 30 minutes. A proof of concept assay for this method was carried out using existing hardware on the International Space Station and the results were read instantly by an astronaut through a simple color change of the reaction mixture. LAMP offers a novel platform for on-orbit DNA-based diagnostics that can be deployed on the International Space Station and to the broader benefit of space programs.

Erratum: Author Correction: Successful amplification of DNA aboard the International Space Station.

[This corrects the article DOI: 10.1038/s41526-017-0033-9.].

Successful amplification of DNA aboard the International Space Station.

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.

Amyloid-associated activity contributes to the severity and toxicity of a prion phenotype.

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 Saccharomyces 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.

Dominant prion mutants induce curing through pathways that promote chaperone-mediated disaggregation.

Protein misfolding underlies many neurodegenerative diseases, including the transmissible spongiform encephalopathies (prion diseases). Although cells typically recognize and process misfolded proteins, prion proteins evade protective measures by forming stable, self-replicating aggregates. However, coexpression 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 wild-type aggregates and alter their physical properties in different ways, diminishing either their assembly rate or their thermodynamic stability. Whereas wild-type 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.

The NatA acetyltransferase couples Sup35 prion complexes to the [PSI+] phenotype.

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(alpha)-acetyltransferases, NatA, NatB, and NatC, which collectively modify approximately 50% 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.

Thermodynamic analysis shows conformational coupling and dynamics confer substrate specificity in fructose-1,6-bisphosphate aldolase.

Conformational flexibility is emerging as a central theme in enzyme catalysis. Thus, identifying and characterizing enzyme dynamics are critical for understanding catalytic mechanisms. Herein, coupling analysis, which uses thermodynamic analysis to assess cooperativity and 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, distinguished 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 >8.9 A. Notably, the coupling energy (DeltaGI) is anticorrelated 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 approximately 70% of the energy difference for the ratio of kcat/Km for the two substrates between aldolase A and aldolase B. These nonadditive 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.

Prion propagation: the role of protein dynamics.

The transfer of phenotypes from one individual to another is a fundamental aspect of biology. In addition to traditional nucleic acid-based genetic determinants, unique proteins known as prions can also act as elements of inheritance, infectivity, and disease. Nucleic acids and proteins encode genetic information in distinct ways, either in the sequence of bases in DNA or RNA or in the three dimensional structure of the polypeptide chain. Given these differences in the nature of the genetic repository, the mechanisms underlying the transmission of nucleic acid-based and protein-based phenotypes are necessarily distinct. While the appearance, persistence and transfer of nucleic acid determinants require the synthesis of new polymers, recent studies indicate that prions are propagated through dynamic transitions in the structure of existing protein.

Structure of human brain fructose 1,6-(bis)phosphate aldolase: linking isozyme structure with function.

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 glyceral-dehyde-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 A 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.

Intein-mediated purification of a recombinantly expressed peptide.

A 26 amino acid peptide has successfully been purified via recombinant expression as an intein fusion protein accompanied by cleavage without the need for any exogenous proteases or cofactors, thus offering a practical, inexpensive approach to produce isotopically labelled peptides.

Spatial clustering of isozyme-specific residues reveals unlikely determinants of isozyme specificity in fructose-1,6-bisphosphate aldolase.

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.