Non-histone protein methylation as a regulator of cellular signalling and function.

Methylation of Lys and Arg residues on non-histone proteins has emerged as a prevalent post-translational modification and as an important regulator of cellular signal transduction mediated by the MAPK, WNT, BMP, Hippo and JAK-STAT signalling pathways. Crosstalk between methylation and other types of post-translational modifications, and between histone and non-histone protein methylation frequently occurs and affects cellular functions such as chromatin remodelling, gene transcription, protein synthesis, signal transduction and DNA repair. With recent advances in proteomic techniques, in particular mass spectrometry, the stage is now set to decode the methylproteome and define its functions in health and disease.

SnapShot: Lysine Methylation beyond Histones.

Lysine methylation is a prevalent post-translational modification (PTM) used by the cell to reversibly regulate protein function. Although it has been extensively studied in the context of histones and the associated chromatin, the remaining methyllysine proteome remains largely unexplored. This SnapShot provides an overview of the current state of lysine methylation research and its emergence as a dynamic PTM occurring on histone and non-histone proteins.

A crucial RNA-binding lysine residue in the Nab3 RRM domain undergoes SET1 and SET3-responsive methylation.

The Nrd1-Nab3-Sen1 (NNS) complex integrates molecular cues to direct termination of noncoding transcription in budding yeast. NNS is positively regulated by histone methylation as well as through Nrd1 binding to the initiating form of RNA PolII. These cues collaborate with Nrd1 and Nab3 binding to target RNA sequences in nascent transcripts through their RRM RNA recognition motifs. In this study, we identify nine lysine residues distributed amongst Nrd1, Nab3 and Sen1 that are methylated, suggesting novel molecular inputs for NNS regulation. We identify mono-methylation of one these residues (Nab3-K363me1) as being partly dependent on the H3K4 methyltransferase, Set1, a known regulator of NNS function. Moreover, the accumulation of Nab3-K363me1 is essentially abolished in strains lacking SET3, a SET domain containing protein that is positively regulated by H3K4 methylation. Nab3-K363 resides within its RRM and physically contacts target RNA. Mutation of Nab3-K363 to arginine (Nab3-K363R) decreases RNA binding of the Nab3 RRM in vitro and causes transcription termination defects and slow growth. These findings identify SET3 as a potential contextual regulator of Nab3 function through its role in methylation of Nab3-K363. Consistent with this hypothesis, we report that SET3 exhibits genetic activation of NAB3 that is observed in a sensitized context.

Phosphorylation-dependent substrate selectivity of protein kinase B (AKT1).

Protein kinase B (AKT1) is a central node in a signaling pathway that regulates cell survival. The diverse pathways regulated by AKT1 are communicated in the cell via the phosphorylation of perhaps more than 100 cellular substrates. AKT1 is itself activated by phosphorylation at Thr-308 and Ser-473. Despite the fact that these phosphorylation sites are biomarkers for cancers and tumor biology, their individual roles in shaping AKT1 substrate selectivity are unknown. We recently developed a method to produce AKT1 with programmed phosphorylation at either or both of its key regulatory sites. Here, we used both defined and randomized peptide libraries to map the substrate selectivity of site-specific, singly and doubly phosphorylated AKT1 variants. To globally quantitate AKT1 substrate preferences, we synthesized three AKT1 substrate peptide libraries: one based on 84 "known" substrates and two independent and larger oriented peptide array libraries (OPALs) of ∼1011 peptides each. We found that each phospho-form of AKT1 has common and distinct substrate requirements. Compared with pAKT1T308, the addition of Ser-473 phosphorylation increased AKT1 activities on some, but not all of its substrates. This is the first report that Ser-473 phosphorylation can positively or negatively regulate kinase activity in a substrate-dependent fashion. Bioinformatics analysis indicated that the OPAL-activity data effectively discriminate known AKT1 substrates from closely related kinase substrates. Our results also enabled predictions of novel AKT1 substrates that suggest new and expanded roles for AKT1 signaling in regulating cellular processes.

Identification of in vitro JMJD lysine demethylase candidate substrates via systematic determination of substrate preference.

A major regulatory influence over gene expression is the dynamic post translational methylation of histone proteins, with major implications from both lysine methylation and demethylation. The KDM5/JARID1 sub-family of Fe(II)/2-oxoglutarate dependent lysine-specific demethylases is, in part, responsible for the removal of tri/dimethyl modifications from lysine 4 of histone H3 (i.e., H3K4me3/2), a mark associated with active gene expression. Although the relevance of KDM5 activity to disease progression has been primarily established through its ability to regulate gene expression via histone methylation, there is evidence that these enzymes may also target non-histone proteins. To aid in the identification of new non-histone substrates, we examined KDM5A in vitro activity towards a library of 180 permutated peptide substrates derived from the H3K4me3 sequence. From this data, a recognition motif was identified and used to predict candidate KDM5A substrates from the methyllysine proteome. High-ranking candidate substrates were then validated for in vitro KDM5A activity using representative trimethylated peptides. Our approach correctly identified activity towards 90% of high-ranked substrates. Here, we have demonstrated the usefulness of our method in identifying candidate substrates that is applicable to any Fe(II)- and 2-oxoglutarate dependent demethylase.

Genetic code expansion and live cell imaging reveal that Thr-308 phosphorylation is irreplaceable and sufficient for Akt1 activity.

The proto-oncogene Akt/protein kinase B (PKB) is a pivotal signal transducer for growth and survival. Growth factor stimulation leads to Akt phosphorylation at two regulatory sites (Thr-308 and Ser-473), acutely activating Akt signaling. Delineating the exact role of each regulatory site is, however, technically challenging and has remained elusive. Here, we used genetic code expansion to produce site-specifically phosphorylated Akt1 to dissect the contribution of each regulatory site to Akt1 activity. We achieved recombinant production of full-length Akt1 containing site-specific pThr and pSer residues for the first time. Our analysis of Akt1 site-specifically phosphorylated at either or both sites revealed that phosphorylation at both sites increases the apparent catalytic rate 1500-fold relative to unphosphorylated Akt1, an increase attributable primarily to phosphorylation at Thr-308. Live imaging of COS-7 cells confirmed that phosphorylation of Thr-308, but not Ser-473, is required for cellular activation of Akt. We found in vitro and in the cell that pThr-308 function cannot be mimicked with acidic residues, nor could unphosphorylated Thr-308 be mimicked by an Ala mutation. An Akt1 variant with pSer-308 achieved only partial enzymatic and cellular signaling activity, revealing a critical interaction between the γ-methyl group of pThr-308 and Cys-310 in the Akt1 active site. Thus, pThr-308 is necessary and sufficient to stimulate Akt signaling in cells, and the common use of phosphomimetics is not appropriate for studying the biology of Akt signaling. Our data also indicate that pThr-308 should be regarded as the primary diagnostic marker of Akt activity.

Characterization of ice recrystallization inhibition activity in the novel freeze-responsive protein Fr10 from freeze-tolerant wood frogs, Rana sylvatica.

Fr10 is a secreted freeze-responsive protein found in the wood frog (Rana sylvatica). This protein has gained notable research attention for its highly dynamic expression in response to seasonal freezing stress, while its over-expression has been documented to enhance freeze tolerance in cold-susceptible cultured cells. This study further characterizes the properties of this novel protein with regards to thermal stability and ice recrystallization inhibition (i.e. IRI) activity. Thermal stability was assessed using differential scanning fluorimetry, with an experimental Tm value of 50.8 ±â€¯0.1 °C. Potential IRI activity of Fr10 was evaluated using a recently developed nanoparticle-based colorimetric assay, where Fr10 displayed the ability to prevent freeze-induced aggregation of gold nanoparticles. Based upon this assay, Fr10 protein appeared to have a low level of IRI activity and it was therefore predicted that one of Fr10's biological functions may be to inhibit ice crystal growth via recrystallization. A SPLAT cooling assay was then employed to directly characterize the IRI properties of Fr10 and provide further insight into this hypothesis. In the presence of 30 µM of Fr10, a 40% reduction in the mean grain size of ice crystals relative to the control samples was observed, thus introducing the possibility of Fr10 to inhibit ice recrystallization. Collectively, the results from this study provide new insight into the potential of further exploring the potential of this vertebrate freeze-responsive protein in cryoprotection.

A framework for improving microRNA prediction in non-human genomes.

The prediction of novel pre-microRNA (miRNA) from genomic sequence has received considerable attention recently. However, the majority of studies have focused on the human genome. Previous studies have demonstrated that sensitivity (correctly detecting true miRNA) is sustained when human-trained methods are applied to other species, however they have failed to report the dramatic drop in specificity (the ability to correctly reject non-miRNA sequences) in non-human genomes. Considering the ratio of true miRNA sequences to pseudo-miRNA sequences is on the order of 1:1000, such low specificity prevents the application of most existing tools to non-human genomes, as the number of false positives overwhelms the true predictions. We here introduce a framework (SMIRP) for creating species-specific miRNA prediction systems, leveraging sequence conservation and phylogenetic distance information. Substantial improvements in specificity and precision are obtained for four non-human test species when our framework is applied to three different prediction systems representing two types of classifiers (support vector machine and Random Forest), based on three different feature sets, with both human-specific and taxon-wide training data. The SMIRP framework is potentially applicable to all miRNA prediction systems and we expect substantial improvement in precision and specificity, while sustaining sensitivity, independent of the machine learning technique chosen.

Exploration of low temperature microRNA function in an anoxia tolerant vertebrate ectotherm, the red eared slider turtle (Trachemys scripta elegans).

As a model for vertebrate long-term survival in oxygen-restricted environments, the red-eared slider turtle (Trachemys scripta elegans) can adapt at the biochemical level to survive in oxygen-free (anoxic) cold water (<10°C). This impressive ability is enabled through a coordinated suppression of energy-expensive, non-essential, cell processes. This study explored the anoxia-responsive expression of several microRNA species (miR-1a, -133, -17, -107, -148a, -21, -103, -210, -20a, -365 and -29b) in adult turtles exposed to 5h and 20h anoxia (at 5±1°C). Furthermore, since microRNA target binding is regularly defined only by microRNA-mRNA interactions at 37°C, the possibility of unique low temperature-selective microRNA targeting interactions with mRNA was explored in this ectotherm. Approximately twice as many microRNA-mRNA interactions were predicted at 5°C versus 37°C with particular enrichment of mRNA targets involved in biological processes known to be part of the stress response. Hence, the results suggest that the influence of temperature should be considered for the prediction of microRNA targets (and their follow-up) in poikilothermic animals and that interacting effects of low body temperature and anoxia on microRNA expression could potentially be important to achieve the profound metabolic rate depression that characterizes turtle hibernation underwater during the winter.

Analysis of microRNA expression during the torpor-arousal cycle of a mammalian hibernator, the 13-lined ground squirrel.

Hibernation is a highly regulated stress response that is utilized by some mammals to survive harsh winter conditions and involves a complex metabolic reprogramming at the cellular level to maintain tissue protections at low temperature. In this study, we profiled the expression of 117 conserved microRNAs in the heart, muscle, and liver of the 13-lined ground squirrel (Ictidomys tridecemlineatus) across four stages of the torpor-arousal cycle (euthermia, early torpor, late torpor, and interbout arousal) by real-time PCR. We found significant differential regulation of numerous microRNAs that were both tissue specific and torpor stage specific. Among the most significant regulated microRNAs was miR-208b, a positive regulator of muscle development that was found to be upregulated by fivefold in the heart during late torpor (13-fold during arousal), while decreased by 3.7-fold in the skeletal muscle, implicating a potential regulatory role in the development of cardiac hypertrophy and skeletal muscle atrophy in the ground squirrels during torpor. In addition, the insulin resistance marker miR-181a was upregulated by 5.7-fold in the liver during early torpor, which supports previous suggestions of hyperinsulinemia in hibernators during the early stages of the hibernation cycle. Although microRNA expression profiles were largely unique between the three tissues, GO annotation analysis revealed that the putative targets of upregulated microRNAs tend to enrich toward suppression of progrowth-related processes in all three tissues. These findings implicate microRNAs in the regulation of both tissue-specific processes and general suppression of cell growth during hibernation.

Insight into post-transcriptional gene regulation: stress-responsive microRNAs and their role in the environmental stress survival of tolerant animals.

Living animals are constantly faced with various environmental stresses that challenge normal life, including: oxygen limitation, very low or high temperature, as well as restriction of water and food. It has been well established that in response to these stresses, tolerant organisms regularly respond with a distinct suite of cellular modifications that involve transcriptional, translational and post-translational modification. In recent years, a new mechanism of rapid and reversible transcriptome regulation, via the action of non-coding RNA molecules, has emerged into post-transcriptional regulation and has since been shown to be part of the survival response. However, these RNA-based mechanisms by which tolerant organisms respond to stressed conditions are not well understood. Recent studies have begun to show that non-coding RNAs control gene expression and translation of mRNA to protein, and can also have regulatory influence over major cellular processes. For example, select microRNAs have been shown to have regulatory influence over the cell cycle, apoptosis, signal transduction, muscle atrophy and fatty acid metabolism during periods of environmental stress. As we are on the verge of dissecting the roles of non-coding RNA in environmental stress adaptation, this Commentary summarizes the hallmark alterations in microRNA expression that facilitate stress survival.

A high-throughput protocol for message RNA quantification using RNA dot-blots.

This study develops a method to rapidly measure the relative abundance of mRNA in total RNA samples using a dot-blotting technique and biotin-labeled detection probes that recognize the polyadenylate tail on mRNA. We demonstrate the effectiveness of this technique by determining the relative total amounts of mRNA in three tissues of turtles (Trachemys scripta elegans) exposed to normoxic versus anoxic conditions. The data emphasize the usefulness of the method for the simple and rapid analysis of relative total mRNA levels for a variety of comparison purposes.

High-throughput amplification of mature microRNAs in uncharacterized animal models using polyadenylated RNA and stem-loop reverse transcription polymerase chain reaction.

This study makes a significant advancement on a microRNA amplification technique previously used for expression analysis and sequencing in animal models without annotated mature microRNA sequences. As research progresses into the post-genomic era of microRNA prediction and analysis, the need for a rapid and cost-effective method for microRNA amplification is critical to facilitate wide-scale analysis of microRNA expression. To facilitate this requirement, we have reoptimized the design of amplification primers and introduced a polyadenylation step to allow amplification of all mature microRNAs from a single RNA sample. Importantly, this method retains the ability to sequence reverse transcription polymerase chain reaction (RT-PCR) products, validating microRNA-specific amplification.

Expression of freeze-responsive proteins, Fr10 and Li16, from freeze-tolerant frogs enhances freezing survival of BmN insect cells.

To date, two novel freeze-responsive proteins, Fr10 and Li16, have been discovered in the wood frog, Rana sylvatica, and likely support freezing survival. Although previous studies have established tissue distribution of each protein, there have been no studies that explore their functional consequences in intolerant cells. To assess the ability of Fr10 and Li16 to confer freeze tolerance, we transfected each protein into a freeze-intolerant silkworm cell line (BmN). Selected controls were the transfection of an unrelated protein (CAT) and a no-transfection sample. Li16 and Fr10 showed 1.8 ± 0.1- and 1.7 ± 0.2-fold, respectively, greater survival after freezing at -6°C for 1 h than did transfection controls. To investigate how these novel proteins protect cells from freezing damage, protein structures were predicted from primary amino acid sequences. Analysis of the structures indicated that Fr10 is a secreted protein and may be a new type IV antifreeze protein, whereas Li16 may have intracellular membrane associated functions. This study shows that freezing protection can be provided to intolerant cells by the overexpression of transfected Li16 and Fr10 frog proteins. Results from this study will provide new insights into adapting intolerant cells for medical organ cryoprotection using a natural vertebrate model of tolerance.

Systematic in vitro optimization of antimicrobial peptides against Escherichia coli.

Objectives: Antimicrobial resistance is a growing concern and claims over 1 million lives per year. The discovery of new antimicrobial drugs is expensive and often generates low profitability, with very low success rates. One way to combat this is by the improvement of known antimicrobials, such as antimicrobial peptides (AMPs). The aim of this study was to improve the antimicrobial activities of two known AMPs, UyCT3 and indolicidin, with the use of peptide libraries and growth curves. Methods: Peptide permutation libraries were synthesized for two AMPs, indolicidin and UyCT3, which included 520 peptides. These peptides were subsequently tested against MG1655-K12, to which subsequent peptide design was performed, then tested against three clinically Gram-negative relevant drug-resistant isolates. Best-performing candidates were subjected to a haemolysis assay for toxicity validation. Results: Single amino acid permutations of UyCT3 and indolicidin were sufficient to inhibit growth of MG1655-K12, and subsequent generations of peptide design were able to inhibit growth of clinical isolates at concentrations as low as 5 µM. Our best-performing AMP, UyCT3I5A, W6Y, K10I, F13I, was not seen to be toxic towards sheep RBCs. Conclusions: The efficacy of the AMPs improved with the use of our peptide library technology, whereby an AMP was found that inhibited bacterial growth of clinical Gram-negative isolates 4-fold better than its WT counterpart.

Unraveling the battle for lysine: A review of the competition among post-translational modifications.

Proteins play a critical role as key regulators in various biological systems, influencing crucial processes such as gene expression, cell cycle progression, and cellular proliferation. However, the functions of proteins can be further modified through post-translational modifications (PTMs), which expand their roles and contribute to disease progression when dysregulated. In this review, we delve into the methodologies employed for the characterization of PTMs, shedding light on the techniques and tools utilized to help unravel their complexity. Furthermore, we explore the prevalence of crosstalk and competition that occurs between different types of PTMs, specifically focusing on both histone and non-histone proteins. The intricate interplay between different modifications adds an additional layer of regulation to protein function and cellular processes. To gain insights into the competition for lysine residues among various modifications, computational systems such as MethylSight have been developed, allowing for a comprehensive analysis of the modification landscape. Additionally, we provide an overview of the exciting developments in the field of inhibitors or drugs targeting PTMs, highlighting their potential in combatting prevalent diseases. The discovery and development of drugs that modulate PTMs present promising avenues for therapeutic interventions, offering new strategies to address complex diseases. As research progresses in this rapidly evolving field, we anticipate remarkable advancements in our understanding of PTMs and their roles in health and disease, ultimately paving the way for innovative treatment approaches.

Insights into a Cancer-Target Demethylase: Substrate Prediction through Systematic Specificity Analysis for KDM3A.

Jumonji C (JmjC) lysine demethylases (KDMs) catalyze the removal of methyl (-CH3) groups from modified lysyl residues. Several JmjC KDMs promote cancerous properties and these findings have primarily been in relation to histone demethylation. However, the biological roles of these enzymes are increasingly being shown to also be attributed to non-histone demethylation. Notably, KDM3A has become relevant to tumour progression due to recent findings of this enzyme's role in promoting cancerous phenotypes, such as enhanced glucose consumption and upregulated mechanisms of chemoresistance. To aid in uncovering the mechanism(s) by which KDM3A imparts its oncogenic function(s), this study aimed to unravel KDM3A substrate specificity to predict high-confidence substrates. Firstly, substrate specificity was assessed by monitoring activity towards a peptide permutation library of histone H3 di-methylated at lysine-9 (i.e., H3K9me2). From this, the KDM3A recognition motif was established and used to define a set of high-confidence predictions of demethylation sites from within the KDM3A interactome. Notably, this led to the identification of three in vitro substrates (MLL1, p300, and KDM6B), which are relevant to the field of cancer progression. This preliminary data may be exploited in further tissue culture experiments to decipher the avenues by which KDM3A imparts cancerous phenotypes.

Assessing sequence-based protein-protein interaction predictors for use in therapeutic peptide engineering.

Engineering peptides to achieve a desired therapeutic effect through the inhibition of a specific target activity or protein interaction is a non-trivial task. Few of the existing in silico peptide design algorithms generate target-specific peptides. Instead, many methods produce peptides that achieve a desired effect through an unknown mechanism. In contrast with resource-intensive high-throughput experiments, in silico screening is a cost-effective alternative that can prune the space of candidates when engineering target-specific peptides. Using a set of FDA-approved peptides we curated specifically for this task, we assess the applicability of several sequence-based protein-protein interaction predictors as a screening tool within the context of peptide therapeutic engineering. We show that similarity-based protein-protein interaction predictors are more suitable for this purpose than the state-of-the-art deep learning methods publicly available at the time of writing. We also show that this approach is mostly useful when designing new peptides against targets for which naturally-occurring interactors are already known, and that deploying it for de novo peptide engineering tasks may require gathering additional target-specific training data. Taken together, this work offers evidence that supports the use of similarity-based protein-protein interaction predictors for peptide therapeutic engineering, especially peptide analogs.

Characterization of KDM5 lysine demethylase family substrate preference and identification of novel substrates.

The KDM5/JARID1 sub-family are 2-oxoglutarate and Fe(II)-dependent lysine-specific histone demethylases that are characterized by their Jumonji catalytic domains. The KDM5 family is known to remove tri-/di-methyl modifications from lysine-4 of histone H3 (i.e. H3-K4me2/3), a mark associated with active gene expression. As a result, studies to date have revolved around the influence of KDM5 on disease through their ability to regulate H3-K4me2/3. Recent evidence demonstrates that KDM5 may influence disease beyond H3-K4 demethylation, making it critical to further investigate KDM5-mediated demethylation of non-histone proteins. To help identify potential non-histone substrates for the KDM5 family, we developed a library of 180 permutated peptide substrates, with sequences that are systematically altered from the wild-type H3-K4me3 substrate. From this library, we characterized recombinant KDM5A/B/C/D substrate preference and developed recognition motifs for each KDM5 demethylase. The recognition motifs developed were used to predict potential substrates for KDM5A/B/C/D and profiled to generate a list of high-ranking and medium/low-ranking substrates for further in vitro validation. Through this approach, we identified 66 high-ranking substrates in which KDM5 demethylases displayed significant in vitro activity towards.

PeSA 2.0: A software tool for peptide specificity analysis implementing positive and negative motifs and motif-based peptide scoring.

There are a vast number of molecular interactions that occur at the cellular level. Among these molecular interactions, interactions between multiple proteins are a widely studied area of research due to the importance of these interactions in cellular function and their potential in drug development. PeSA is a desktop application developed to facilitate the in vitro peptide study analysis to predict protein-protein interactions. PeSA can effortlessly generate visual outputs like motifs, bar charts, and visual matrices. Our implementation of PeSA version 2.0 includes additional tools, including the ability to further score peptide lists for consensus amongst interactions. The software is also able to design de novo peptides based on sequence motifs (sequence generator), which can be used to help design additional experiments for motif validation. Further, the efficacy of the sequence generator was validated using the lysine methyltransferase, SETD8, to identify new substrates of methylation based on motif-based predictions developed using PeSA2.0.