Development of a Certification Exam to Assess Undergraduate Students' Proficiency in Biochemistry and Molecular Biology Core Concepts.

With support from the American Society for Biochemistry and Molecular Biology (ASBMB), a community of biochemistry and molecular biology (BMB) scientist-educators has developed and administered an assessment instrument designed to evaluate student competence across four core concept and skill areas fundamental to BMB. The four areas encompass energy and metabolism; information storage and transfer; macromolecular structure, function, and assembly; and skills including analytical and quantitative reasoning. First offered in 2014, the exam has now been administered to nearly 4000 students in ASBMB-accredited programs at more than 70 colleges and universities. Here, we describe the development and continued maturation of the exam program, including the organic role of faculty volunteers as drivers and stewards of all facets: content and format selection, question development, and scoring.

Correction: Conserved mRNA-granule component Scd6 targets Dhh1 to repress translation initiation and activates Dcp2-mediated mRNA decay in vivo.

[This corrects the article DOI: 10.1371/journal.pgen.1007806.].

Conserved mRNA-granule component Scd6 targets Dhh1 to repress translation initiation and activates Dcp2-mediated mRNA decay in vivo.

Scd6 protein family members are evolutionarily conserved components of translationally silent mRNA granules. Yeast Scd6 interacts with Dcp2 and Dhh1, respectively a subunit and a regulator of the mRNA decapping enzyme, and also associates with translation initiation factor eIF4G to inhibit translation in cell extracts. However, the role of Scd6 in mRNA turnover and translational repression in vivo is unclear. We demonstrate that tethering Scd6 to a GFP reporter mRNA reduces mRNA abundance via Dcp2 and suppresses reporter mRNA translation via Dhh1. Thus, in a dcp2Δ mutant, tethered Scd6 reduces GFP protein expression with little effect on mRNA abundance, whereas tethered Scd6 has no impact on GFP protein or mRNA expression in a dcp2Δ dhh1Δ double mutant. The conserved LSm domain of Scd6 is required for translational repression and mRNA turnover by tethered Scd6. Both functions are enhanced in a ccr4Δ mutant, suggesting that the deadenylase function of Ccr4-Not complex interferes with a more efficient repression pathway enlisted by Scd6. Ribosome profiling and RNA-Seq analysis of scd6Δ and dhh1Δ mutants suggests that Scd6 cooperates with Dhh1 in translational repression and turnover of particular native mRNAs, with both processes dependent on Dcp2. Our results suggest that Scd6 can (i) recruit Dhh1 to confer translational repression and (ii) activate mRNA decapping by Dcp2 with attendant degradation of specific mRNAs in vivo, in a manner dependent on the Scd6 LSm domain and modulated by Ccr4.

Removal of Abnormal Myofilament O-GlcNAcylation Restores Ca2+ Sensitivity in Diabetic Cardiac Muscle.

Contractile dysfunction and increased deposition of O-linked ß-N-acetyl-d-glucosamine (O-GlcNAc) in cardiac proteins are a hallmark of the diabetic heart. However, whether and how this posttranslational alteration contributes to lower cardiac function remains unclear. Using a refined ß-elimination/Michael addition with tandem mass tags (TMT)-labeling proteomic technique, we show that CpOGA, a bacterial analog of O-GlcNAcase (OGA) that cleaves O-GlcNAc in vivo, removes site-specific O-GlcNAcylation from myofilaments, restoring Ca(2+) sensitivity in streptozotocin (STZ) diabetic cardiac muscles. We report that in control rat hearts, O-GlcNAc and O-GlcNAc transferase (OGT) are mainly localized at the Z-line, whereas OGA is at the A-band. Conversely, in diabetic hearts O-GlcNAc levels are increased and OGT and OGA delocalized. Consistent changes were found in human diabetic hearts. STZ diabetic hearts display increased physical interactions of OGA with α-actin, tropomyosin, and myosin light chain 1, along with reduced OGT and increased OGA activities. Our study is the first to reveal that specific removal of O-GlcNAcylation restores myofilament response to Ca(2+) in diabetic hearts and that altered O-GlcNAcylation is due to the subcellular redistribution of OGT and OGA rather than to changes in their overall activities. Thus, preventing sarcomeric OGT and OGA displacement represents a new possible strategy for treating diabetic cardiomyopathy.

O-GlcNAc cycling enzymes associate with the translational machinery and modify core ribosomal proteins.

Protein synthesis is globally regulated through posttranslational modifications of initiation and elongation factors. Recent high-throughput studies have identified translation factors and ribosomal proteins (RPs) as substrates for the O-GlcNAc modification. Here we determine the extent and abundance of O-GlcNAcylated proteins in translational preparations. O-GlcNAc is present on many proteins that form active polysomes. We identify twenty O-GlcNAcylated core RPs, of which eight are newly reported. We map sites of O-GlcNAc modification on four RPs (L6, L29, L32, and L36). RPS6, a component of the mammalian target of rapamycin (mTOR) signaling pathway, follows different dynamics of O-GlcNAcylation than nutrient-induced phosphorylation. We also show that both O-GlcNAc cycling enzymes OGT and OGAse strongly associate with cytosolic ribosomes. Immunofluorescence experiments demonstrate that OGAse is present uniformly throughout the nucleus, whereas OGT is excluded from the nucleolus. Moreover, nucleolar stress only alters OGAse nuclear staining, but not OGT staining. Lastly, adenovirus-mediated overexpression of OGT, but not of OGAse or GFP control, causes an accumulation of 60S subunits and 80S monosomes. Our results not only establish that O-GlcNAcylation extensively modifies RPs, but also suggest that O-GlcNAc play important roles in regulating translation and ribosome biogenesis.

The intersections between O-GlcNAcylation and phosphorylation: implications for multiple signaling pathways.

A paradigm-changing discovery in biology came about when it was found that nuclear and cytosolic proteins could be dynamically glycosylated with a single O-linked beta-N-acetylglucosamine (O-GlcNAc) moiety. O-GlcNAcylation is akin to phosphorylation: it occurs on serine and/or threonine side chains of proteins, and cycles rapidly upon cellular activation. O-GlcNAc and phosphate show a complex interplay: they can either competitively occupy a single site or proximal sites, or noncompetitively occupy different sites on a substrate. Phosphorylation regulates O-GlcNAc-cycling enzymes and, conversely, O-GlcNAcylation controls phosphate-cycling enzymes. Such crosstalk is evident in all compartments of the cell, a finding that is congruent with the fundamental role of O-GlcNAc in regulating nutrient- and stress-induced signal transduction. O-GlcNAc transferase is recruited to the plasma membrane in response to insulin and is targeted to substrates by forming transient holoenzyme complexes that have different specificities. Cytosolic O-GlcNAcylation is important for the proper transduction of signaling cascades such as the NFkappaB pathway, whereas nuclear O-GlcNAc is crucial for regulating the activity of numerous transcription factors. This Commentary focuses on recent findings supporting an emerging concept that continuous crosstalk between phosphorylation and O-GlcNAcylation is essential for the control of vital cellular processes and for understanding the mechanisms that underlie certain neuropathologies.

HSP70 interacts with ribosomal subunits of thermotolerant cells.

The expression of heat shock or stress proteins (hsps) is a widespread response to stress that results in the protection of cells from subsequent insults, coined stress tolerance. Stress tolerance is apparently due to the preservation of several cellular structures and processes, such as translation. Protection of protein synthesis has been correlated with the presence of Hsp70. In the present study, Hsp70 was found to interact with translating ribosomes. This interaction is due to the preferential binding of Hsp70 to the 40S ribosomal subunit. Additionally, Hsp70 seems to interact weakly with nascent polypeptides within the 60S subunit. The interaction between Hsp70 and ribosomal subunits could also be observed in vitro conditions. Binding of Hsp70 to ribosomes was salt resistant, suggesting that this protein is not bound to transiently associated translational factors. Moreover, protection of protein synthesis requires new gene expression. We speculate that the binding of Hsp70 to ribosomes is part of a mechanism to guarantee the rapid and abundant protein synthesis during stress, particularly the translation of mRNAs encoding for hsps.