Cautionary Tale of Using Tris(alkyl)phosphine Reducing Agents with NAD+-Dependent Enzymes

Protein biochemistry protocols typically include disulfide bond reducing agents to guard against unwanted thiol oxidation and protein aggregation. Commonly used disulfide bond reducing agents include dithiothreitol, β-mercaptoethanol, glutathione, and the tris(alkyl)phosphine compounds tris(2-carboxyethyl)phosphine (TCEP) and tris(3-hydroxypropyl)phosphine (THPP). While studying the catalytic activity of the NAD(P)H-dependent enzyme Δ1-pyrroline-5-carboxylate reductase, we unexpectedly observed a rapid non-enzymatic chemical reaction between NAD+ and the reducing agents TCEP and THPP. The product of the reaction exhibits a maximum ultraviolet absorbance peak at 334 nm and forms with an apparent association rate constant of 231–491 M−1 s−1. The reaction is reversible, and nuclear magnetic resonance characterization (1H, 13C, and 31P) of the product revealed a covalent adduct between the phosphorus of the tris(alkyl)phosphine reducing agent and the C4 atom of the nicotinamide ring of NAD+. We also report a 1.45 Å resolution crystal structure of short-chain dehydrogenase/reductase with the NADP+–TCEP reaction product bound in the cofactor binding site, which shows that the adduct can potentially inhibit enzymes. These findings serve to caution researchers when using TCEP or THPP in experimental protocols with NAD(P)+. Because NAD(P)+-dependent oxidoreductases are widespread in nature, our results may be broadly relevant

The Crowdsourced Replication Initiative: Investigating Immigration and Social Policy Preferences. Executive Report.

In an era of mass migration, social scientists, populist parties and social movements raise concerns over the future of immigration-destination societies. What impacts does this have on policy and social solidarity? Comparative cross-national research, relying mostly on secondary data, has findings in different directions. There is a threat of selective model reporting and lack of replicability. The heterogeneity of countries obscures attempts to clearly define data-generating models. P-hacking and HARKing lurk among standard research practices in this area.This project employs crowdsourcing to address these issues. It draws on replication, deliberation, meta-analysis and harnessing the power of many minds at once. The Crowdsourced Replication Initiative carries two main goals, (a) to better investigate the linkage between immigration and social policy preferences across countries, and (b) to develop crowdsourcing as a social science method. The Executive Report provides short reviews of the area of social policy preferences and immigration, and the methods and impetus behind crowdsourcing plus a description of the entire project. Three main areas of findings will appear in three papers, that are registered as PAPs or in process

Sequence-structure relationships, expression profiles, and disease-associated mutations in the paralogs of phosphoglucomutase 1.

The key metabolic enzyme phosphoglucomutase 1 (PGM1) controls glucose homeostasis in most human cells. Four proteins related to PGM1, known as PGM2, PGM2L1, PGM3 and PGM5, and referred to herein as paralogs, are encoded in the human genome. Although all members of the same enzyme superfamily, these proteins have distinct substrate preferences and different functional roles. The recent association of PGM1 and PGM3 with inherited enzyme deficiencies prompts us to revisit sequence-structure and other relationships among the PGM1 paralogs, which are understudied despite their importance in human biology. Using currently available sequence, structure, and expression data, we investigated evolutionary relationships, tissue-specific expression profiles, and the amino acid preferences of key active site motifs. Phylogenetic analyses indicate both ancient and more recent divergence between the different enzyme sub-groups comprising the human paralogs. Tissue-specific protein and RNA expression profiles show widely varying patterns for each paralog, providing insight into function and disease pathology. Multiple sequence alignments confirm high conservation of key active site regions, but also reveal differences related to substrate specificity. In addition, we find that sequence variants of PGM2, PGM2L1, and PGM5 verified in the human population affect residues associated with disease-related mutants in PGM1 or PGM3. This suggests that inherited diseases related to dysfunction of these paralogs will likely occur in humans

Sequence preferences for key functional regions of the PGM1 paralogs.

<p>Selected regions of paralog-specific multiple sequence alignments are shown for the phosphoserine (P-Ser), metal-binding, sugar-binding, and PO₄-binding loops. Identical residues are highlighted with red background; similar residues are shown in red font. Asterisk (*) indicates the catalytic serine residue.</p

Residues affected by disease-related mutants mapped onto the structures of (A) PGM1 (PDB ID 5EPC) and (B) a homolog of PGM3 (PDB ID 2DKD).

<p>Residues affected by PGM1 missense variants are highlighted in yellow; those in PGM3 in green.</p

Tissue-specific protein expression profiles of the PGM1 paralogs.

<p>Data are from ProteomicsDB [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0183563#pone.0183563.ref035" target="_blank">35</a>] for 30 human tissues. (<b>A)</b> Protein expression levels are shown on a log scale using their iBAQ scores. Brackets at left indicate clustering of tissues into groups with similar protein expression patterns. (<b>B)</b> RNA expression levels. Data are normalized across each row by Z-score, emphasizing the relative RNA expression for the paralogs in each tissue. (Z-score is the standard deviation from the mean per row). Gray cells indicate that the corresponding tissues are missing in the respective database.</p

Known and predicted disease-associated missense variants in the PGM1 paralogs.

<p>Known and predicted disease-associated missense variants in the PGM1 paralogs.</p