Harmonizing labeling and analytical strategies to obtain protein turnover rates in intact adult animals.

Changes in the abundance of individual proteins in the proteome can be elicited by modulation of protein synthesis (the rate of input of newly synthesized proteins into the protein pool) or degradation (the rate of removal of protein molecules from the pool). A full understanding of proteome changes therefore requires a definition of the roles of these two processes in proteostasis, collectively known as protein turnover. Because protein turnover occurs even in the absence of overt changes in pool abundance, turnover measurements necessitate monitoring the flux of stable isotope labeled precursors through the protein pool such as labeled amino acids or metabolic precursors such as ammonium chloride or heavy water. In cells in culture, the ability to manipulate precursor pools by rapid medium changes is simple, but for more complex systems such as intact animals, the approach becomes more convoluted. Individual methods bring specific complications, and the suitability of different methods has not been comprehensively explored. In this study we compare the turnover rates of proteins across four mouse tissues, obtained from the same inbred mouse strain maintained under identical husbandry conditions, measured using either [13C6]lysine or [2H2]O as the labeling precursor. We show that for long-lived proteins, the two approaches yield essentially identical measures of the first order rate constant for degradation. For short-lived proteins, there is a need to compensate for the slower equilibration of lysine through the precursor pools. We evaluate different approaches to provide that compensation. We conclude that both labels are suitable, but careful determination of precursor enrichment kinetics in amino acid labeling is critical and has a considerable influence on the numerical values of the derived protein turnover rates.

Frequent Manipulation of Resistance Training Variables Promotes Myofibrillar Spacing Changes in Resistance-Trained Individuals

We sought to determine if manipulating resistance training (RT) variables differentially altered the expression of select sarcoplasmic and myofibril proteins as well as myofibrillar spacing in myofibers. Resistance-trained men (n = 20; 26 ± 3 years old) trained for 8 weeks where a randomized leg performed either a standard (CON) or variable RT protocol (VAR: manipulation of load, volume, muscle action, and rest intervals at each RT session). A pre-training (PRE) vastus lateralis biopsy was obtained from a randomized single leg, and biopsies were obtained from both legs 96 h following the last training bout. The sarcoplasmic protein pool was assayed for proteins involved in energy metabolism, and the myofibril protein pool was assayed for relative myosin heavy chain (MHC) and actin protein abundances. Sections were also histologically analyzed to obtain myofibril spacing characteristics. VAR resulted in ~12% greater volume load (VL) compared to CON (p < 0.001). The mean fiber cross-sectional area increased following both RT protocols [CON: 14.6% (775.5 μm2), p = 0.006; VAR: 13.9% (743.2 μm2), p = 0.01 vs. PRE for both], but without significant differences between protocols (p = 0.79). Neither RT protocol affected a majority of assayed proteins related to energy metabolism, but both training protocols increased hexokinase 2 protein levels and decreased a mitochondrial beta-oxidation marker (VLCAD protein; p < 0.05). Citrate synthase activity levels increased with CON RT (p < 0.05), but not VAR RT. The relative abundance of MHC (summed isoforms) decreased with both training protocols (p < 0.05). However, the relative abundance of actin protein (summed isoforms) decreased with VAR only (13.5 and 9.0%, respectively; p < 0.05). A decrease in percent area occupied by myofibrils was observed from PRE to VAR (−4.87%; p = 0.048), but not for the CON (4.53%; p = 0.979). In contrast, there was an increase in percent area occupied by non-contractile space from PRE to VAR (10.14%; p = 0.048), but not PRE to CON (0.72%; p = 0.979). In conclusion, while both RT protocols increased muscle fiber hypertrophy, a higher volume-load where RT variables were frequently manipulated increased non-contractile spacing in resistance-trained individuals.

Path4Drug: Data Science Workflow for Identification of Tissue-Specific Biological Pathways Modulated by Toxic Drugs

The early prediction of drug adverse effects is of great interest to pharmaceutical research, as toxicity is one of the leading reasons for drug attrition. Understanding the cell signaling and regulatory pathways affected by a drug candidate is crucial to the study of drug toxicity. In this study, we present a computational technique that employs the propagation of drug-protein interactions to connect compounds to biological pathways. Target profiles for drugs were built by retrieving drug target proteins from public repositories such as ChEMBL, DrugBank, IUPHAR, PharmGKB, and TTD. Subsequent enrichment test of the protein pool using Reactome revealed potential pathways affected by the drugs. Furthermore, an optional tissue filter utilizing the Human Protein Atlas was applied to identify tissue-specific pathways. The analysis pipeline was implemented in an open-source KNIME workflow called Path4Drug to allow automated data retrieval and reconstruction for any given drug present in ChEMBL. The pipeline was applied to withdrawn drugs and cardio- and hepatotoxic drugs with black box warnings to identify biochemical pathways they affect and to find pathways that can be potentially connected to the toxic events. To complement this approach, drugs used in cardiac therapy without any record of toxicity were also analyzed. The results provide already known associations as well as a large amount of additional potential connections. Consequently, our approach can link drugs to biological pathways by leveraging big data available in public resources. The developed tool is openly available and modifiable to support other systems biology analyses.

What defines the maternal transcriptome?

In somatic cells, RNA polymerase II (Pol II) transcription initiation starts by the binding of the general transcription factor TFIID, containing the TATA-binding protein (TBP) and 13 TBP-associated factors (TAFs), to core promoters. However, in growing oocytes active Pol II transcription is TFIID/TBP-independent, as during oocyte growth TBP is replaced by its vertebrate-specific paralog TBPL2. TBPL2 does not interact with TAFs, but stably associates with TFIIA. The maternal transcriptome is the population of mRNAs produced and stored in the cytoplasm of growing oocytes. After fertilization, maternal mRNAs are inherited by the zygote from the oocyte. As transcription becomes silent after oocyte growth, these mRNAs are the sole source for active protein translation. They will participate to complete the protein pool required for oocyte terminal differentiation, fertilization and initiation of early development, until reactivation of transcription in the embryo, called zygotic genome activation (ZGA). All these events are controlled by an important reshaping of the maternal transcriptome. This procedure combines cytoplasmic readenylation of stored transcripts, allowing their translation, and different waves of mRNA degradation by deadenylation coupled to decapping, to eliminate transcripts coding for proteins that are no longer required. The reshaping ends after ZGA with an almost total clearance of the maternal transcripts. In the past, the murine maternal transcriptome has received little attention but recent progresses have brought new insights into the regulation of maternal mRNA dynamics in the mouse. This review will address past and recent data on the mechanisms associated with maternal transcriptome dynamic in the mouse.

A catalytic-independent function of human DNA polymerase Kappa controls the stability and abundance of the Checkpoint Kinase 1

DNA polymerase kappa (Pol κ) has been well documented thus far for its specialized DNA synthesis activity during translesion replication, progression of replication forks through regions difficult to replicate, restart of stalled forks and replication checkpoint efficiency. Pol κ is also required for the stabilization of stalled forks although the mechanisms are poorly understood. Here we unveiled an unexpected role for Pol κ in controlling the stability and abundance of Chk1, an important actor for the replication checkpoint and fork stabilization. We found that loss of Pol κ decreased the Chk1 protein level in the nucleus of four human cell lines. Pol κ and not the other Y‐family polymerase members is required to maintain the Chk1 protein pool all along the cell cycle. We showed that Pol κ depletion affected the protein stability of Chk1 and protected it from proteasome degradation. Importantly, we also observed that the fork restart defects observed in Pol κ-depleted cells could be overcome by the re-expression of Chk1. Strikingly, this new function of Pol κ does not require its catalytic activity. We propose that Pol κ could contribute to the protection of stalled forks through Chk1 stability.

Dynamics of autophagy and ubiquitin proteasome system coordination and interplay in skeletal muscle atrophy

: Skeletal muscles are considered the largest reservoirs of the protein pool in the body and are critical for the maintenances of body homeostasis. Skeletal muscle atrophy is supported by various physiopathological conditions that lead to loss of muscle mass and contractile capacity of the skeletal muscle. Lysosomal mediated autophagy and ubiquitin-proteasomal system (UPS) concede the major intracellular systems of muscle protein degradation that result in the loss of mass and strength. Both systems recognize ubiquitination as a signal of degradation through different mechanisms, a sign of dynamic interplay between systems. Hence, growing shreds of evidence suggest the interdependency of autophagy and UPS in the progression of skeletal muscle atrophy under various pathological conditions. Therefore, understanding the molecular dynamics as well associated factors responsible for their interdependency is a necessity for the new therapeutic insights to counteract the muscle loss. Based on current literature, the present review summarizes the factors interplay in between the autophagy and UPS in favor of enhanced proteolysis of skeletal muscle and how they affect the anabolic signaling pathways under various conditions of skeletal muscle atrophy.

Melanin, a potential new matrix material for recombinant His-tag protein purification

Nickel-Sepharose (Ni-Sepharose) has been being applied as the most common matrix in purifying His-tag proteins based on the affinity interaction between histidine residues and Ni2+ ion. However, Sepharose still comes at high cost for this purification purpose, especially in developing countries as Vietnam. Here, we show for the first time that melanin from ink sacs of squids which is considered as biowaste in the food industry, can be used as a new potential matrix material instead of Sepharose. We utilized either melanin or melanin charged with metal ions as the stationary phase of affinity purification of His-tag proteins. The results showed that a recombinant His-tag protein VP28 in a protein pool was captured by melanin and Ni2+/Fe3+/Zn2+ chelated melanin. Experiments for releasing VP28 were performed only on the melanin and Ni2+-melanin matrices. The result showed that VP28 was quite selectively eluted when applying elution buffer of 250 mM imidazole overnight. The relative efficiency in releasing VP28 of melanin and Ni-melanin matrices roughly compared to Ni-Sepharose were about 38 and 18% respectively. Further optimization of this process may allow higher efficiency in the purification of His-tag proteins.

Activation of regulatory T cells triggers specific changes in glycosylation associated with Siglec-1-dependent inflammatory responses

Background: Siglec-1 is a macrophage lectin-like receptor that mediates sialic acid-dependent cellular interactions. Its upregulation on macrophages in autoimmune disease was shown previously to promote inflammation through suppressing the expansion of regulatory T cells (Tregs). Here we investigate the molecular basis for Siglec-1 binding to Tregs using in vitro-induced cells as a model system. Methods: Glycosylation changes that affect Siglec‑1 binding were studied by comparing activated and resting Tregs using RNA-Seq, glycomics, proteomics and binding of selected antibodies and lectins. A proximity labelling and proteomics strategy was used to identify Siglec-1 counter-receptors expressed on activated Tregs. Results: Siglec-1 binding was strongly upregulated on activated Tregs, but lost under resting conditions. Glycomics revealed changes in N-glycans and glycolipids following Treg activation and we observed changes in expression of multiple ‘glycogenes’ that could lead to the observed increase in Siglec-1 binding. Proximity labelling of intact, living cells identified 49 glycoproteins expressed by activated Tregs that may function as Siglec-1 counter-receptors. These represent ~5% of the total membrane protein pool and were mainly related to T cell activation and proliferation. We demonstrate that several of these counter-receptors were upregulated following activation of Tregs and provide initial evidence that their altered glycosylation may also be important for Siglec-1 binding. Conclusions: We provide the first comprehensive analysis of glycan changes that occur in activated Tregs, leading to recognition by the macrophage lectin, Siglec-1 and suppression of Treg expansion. We furthermore provide insights into glycoprotein counter-receptors for Siglec-1 expressed by activated Tregs that are likely to be important for suppressing Treg expansion.