Noise-Induced Acceleration of Single Molecule Kinesin-1.

The movement of single kinesin molecules was observed while applying noisy external forces that mimic intracellular active fluctuations. We found kinesin accelerates under noise, especially when a large hindering load is added. The behavior quantitatively conformed to a theoretical model that describes the kinesin movement with simple two-state reactions. The universality of the kinetic theory suggests that intracellular enzymes share a similar noise-induced acceleration mechanism, i.e., active fluctuations in cells are not just noise but are utilized to promote various physiological processes.

Integrating abiotic chemical catalysis and enzymatic catalysis in living cells.

Life emerges from networks of multiple chemical reactions mediated by enzymes. If abiotic chemical catalysis is implanted into the reaction network of life, such an integration would produce organisms generating unique secondary metabolites and value-added materials from feedstocks or even air, or new diagnostics and therapeutics against diseases. In this review, we introduce selected papers in this emerging field of catalysis research.

Temporal Sampling of Enzymes from Live Cells by Localized Electroporation and Quantification of Activity by SAMDI Mass Spectrometry.

Measuring changes in enzymatic activity over time from small numbers of cells remains a significant technical challenge. In this work, a method for sampling the cytoplasm of cells is introduced to extract enzymes and measure their activity at multiple time points. A microfluidic device, termed the live cell analysis device (LCAD), is designed, where cells are cultured in microwell arrays fabricated on polymer membranes containing nanochannels. Localized electroporation of the cells opens transient pores in the cell membrane at the interface with the nanochannels, enabling extraction of enzymes into nanoliter-volume chambers. In the extraction chambers, the enzymes modify immobilized substrates, and their activity is quantified by self-assembled monolayers for matrix-assisted laser desorption/ionization (SAMDI) mass spectrometry. By employing the LCAD-SAMDI platform, protein delivery into cells is demonstrated. Next, it is shown that enzymes can be extracted, and their activity measured without a loss in viability. Lastly, cells are sampled at multiple time points to study changes in phosphatase activity in response to oxidation by hydrogen peroxide. With this unique sampling device and label-free assay format, the LCAD with SAMDI enables a powerful new method for monitoring the dynamics of cellular activity from small populations of cells.

Enzyme promiscuity and evolution in light of cellular metabolism.

This Special Issue is composed of 10 reviews that delve into the intricacies behind enzyme promiscuity and evolution, an area that is of increasing interest in the biological research community. In particular, the reviews in this Special Issue explore enzyme promiscuity and evolution in the context of cellular metabolism, as discussed in this introductory Editorial. It is our hope that you enjoy these fascinating and informative reviews and we wish to thank the authors for their compelling contributions to The FEBS Journal. doi: 10.1111/febs.12650.

Enzyme-based protein-tagging systems for site-specific labeling of proteins in living cells.

Various protein-labeling methods based on the specific interactions between genetically encoded tags and synthetic probes have been proposed to complement fluorescent protein-based labeling. In particular, labeling methods based on enzyme reactions have been intensively developed by taking advantage of the highly specific interactions between enzymes and their substrates. In this approach, the peptides or proteins are genetically attached to the target proteins as a tag, and the various labels are then incorporated into the tags by enzyme reactions with the substrates carrying those labels. On the other hand, we have been developing an enzyme-based protein-labeling system distinct from the existing ones. In our system, the substrate protein is attached to the target proteins as a tag, and the labels are incorporated into the tag by post-translational modification with an enzyme carrying those labels followed by tight complexation between the enzyme and the substrate protein. In this review, I summarize the enzyme-based protein-labeling systems with a focus on several typical methods and then describe our labeling system based on tight complexation between the enzyme and the substrate protein.

Murburn concept: a paradigm shift in cellular metabolism and physiology.

Two decades of evidence-based exploratory pursuits in heme-flavin enzymology led to the formulation of a new biological electron/moiety transfer paradigm, called murburn concept. Murburn is a novel literary abstraction from "mured burning" or "mild unrestricted burning". This concept was invoked to explain the longstanding conundrum of maverick physiological dose responses and also applied to remodel the prevailing understanding of drug metabolism and cellular respiration. A conglomeration of simple ideas grounded in the known principles of thermodynamics and reaction chemistry, murburn concept invokes catalytic/functional roles for diffusible reactive species or radicals. Hitherto, diffusible reactive species were primarily seen as toxic agents of chaos, non-conducible to the maintenance of life-order. Since the murburn paradigm offers a distinctly different perspective for several biological phenomena, researchers holding conventional views of cellular metabolism pose a direct conflict of interests to the advancement of murburn concept. Murburn schemes are poised to integrate numerous metabolic motifs with holistic physiological outcomes; redefining pursuits in biology and medicine. To advance this agenda, I present a brief account of murburn concept and point out how redundant ideas are still advocated in some prestigious journals.

The E3 ubiquitin ligase CHIP in normal cell function and in disease conditions.

In eukaryotic cells, ubiquitination and proteasomal degradation is an essential mechanism for regulating protein functions. For example, critical signaling proteins play their roles by controlling different cellular functions. Once a signaling protein has been activated, its activity needs to be quickly downregulated by different mechanisms, including ubiquitination/proteasome regulation. Failure to regulate the activity or expression levels of these proteins may cause human diseases. Protein ubiquitination involves a cascade of biochemical processes and requires three types of ubiquitin enzymes: E1 activating enzyme, E2 conjugating enzyme, and E3 ligase. Among these enzymes, E3 ubiquitin ligases play a specific role in recognizing specific protein substrates. There are several structurally diverse groups of E3 ubiquitin ligases in eukaryotic cells, and one type of these E3 ligases is the U-box ubiquitin ligases. Carboxyl terminus of HSP70-interacting protein (CHIP) is a member of a family of U-box E3 ligases. It plays critical roles in multiple organs and tissues in the body. In this review article, we provide an update on some of the most recent discoveries about CHIP in normal physiological function and in disease.

Intracellular Delivery and Sensing System Based on Electroplated Conductive Nanostraw Arrays.

One-dimensional nanoneedle-like arrays have emerged as an attractive tool for penetrating the cell membrane to achieve intracellular applications including drug delivery, electrical recording, and biochemical detection. Hollow nanoneedles, also called nanostraws (NSs), combined with nanoelectroporation have been demonstrated as a powerful platform for intracellular drug delivery and extraction of intracellular contents. However, the fabrication technique of nanostraws still requires complicated and expensive atomic layer deposition and etching processes and fails to produce conductive nanostraws. Herein, we developed a commonly accessible and versatile electrodeposition approach to controllably fabricate conductive nanostraw arrays based on various types of metal or conductive polymer materials. Representatively, Pt nanostraws (Pt NSs) with 400 nm diameter were further integrated with a low-voltage nanoelectroporation system to achieve cell detection, intracellular drug delivery, and sensing of intracellular enzymes. Both theoretical simulations and experimental results revealed that the conductive nanostraws in direct contact with cells could induce high-efficiency cell electroporation at relatively low voltage (∼5 V). Efficient delivery of reagents into live cells with spatial control and repeated extraction of intracellular enzymes (e.g., caspase-3) for temporal monitoring from the same set of cells were demonstrated. This work not only pioneers a new avenue for universal production of conductive nanostraws on a large scale but also presents great potential for developing nanodevices to achieve a variety of biomedical applications including cell re-engineering, cell-based therapy, and signaling pathway monitoring.

Caspases interplay with kinases and phosphatases to determine cell fate.

Cellular differentiation is one of the critical processes in the life of multicellular organisms. In this phenomenon, a non-specialized cell is converted to a specialized one with its own specific function and morphology. One of the requirements for specialization is silencing of the pathways involved in cell proliferation in parallel with turning on the molecular mechanisms involved in differentiation. Similar to other biological phenomena, the change in cellular state from the proliferative to the differentiated needs molecular switches to persuade the change in response to the internal or external inducers. The quiddity of these molecular switches has not been identified, yet. However, there exists a growing body of evidence showing that the same agents involved in apoptosis have a broad contribution to differentiation progression. To our knowledge, this evidence is still ambiguous because it has raised fundamental questions that require more proof to be answered. The most important questions are: How can two totally different cellular fates act through a similar pathway? What is the separating edge? What forces a cell to choose one of them (death or differentiation)? To address these issues, we will concentrate on three groups of molecules; caspases as the key players of apoptosis, protein kinases, and phosphatases as the major regulators of many cellular and biochemical processes. The evidence reveals a triangle of caspases, kinases, and phosphatases in which their communication leads to the fine-tuning of caspases and consequently they determine cell fate.

Class II PI3K Functions in Cell Biology and Disease.

The phosphoinositide 3-kinases (PI3Ks) are a family of lipid kinases that phosphorylate inositol phospholipids, thereby controlling membrane lipid composition and regulating a wide range of intracellular processes, including vesicular trafficking and signal transduction. Despite the vast knowledge on class I PI3Ks, recent studies are only now revealing the importance of class II PI3Ks in cell proliferation, survival, and migration. Increasing evidence suggests that the three class II PI3Ks isoforms (PI3K-C2α, PI3K-C2ß, and PI3K-C2γ) have distinct and non-overlapping cellular roles. Here, we focus on the cellular functions of class II PI3Ks in different cell systems and underline the emerging importance of these enzymes in various physiological and pathological contexts.

The impact of phosphoinositide 5-phosphatases on phosphoinositides in cell function and human disease.

Phosphoinositides (PIs) are recognized as major signaling molecules in many different functions of eukaryotic cells. PIs can be dephosphorylated by multiple phosphatase activities at the 5-, 4-, and 3- positions. Human PI 5-phosphatases belong to a family of 10 members. Except for inositol polyphosphate 5-phosphatase A, they all catalyze the dephosphorylation of PI(4,5)P2 and/or PI(3,4,5)P3 at the 5- position. PI 5-phosphatases thus directly control the levels of PI(3,4,5)P3 and participate in the fine-tuning regulatory mechanisms of PI(3,4)P2 and PI(4,5)P2 Second messenger functions have been demonstrated for PI(3,4)P2 in invadopodium maturation and lamellipodia formation. PI 5-phosphatases can use several substrates on isolated enzymes, and it has been challenging to establish their real substrate in vivo. PI(4,5)P2 has multiple functions in signaling, including interacting with scaffold proteins, ion channels, and cytoskeleton proteins. PI 5-phosphatase isoenzymes have been individually implicated in human diseases, such as the oculocerebrorenal syndrome of Lowe, through mechanisms that include lipid control. Oncogenic and tumor-suppressive functions of PI 5-phosphatases have also been reported in different cell contexts. The mechanisms responsible for genetic diseases and for oncogenic or tumor-suppressive functions are not fully understood. The regulation of PI 5-phosphatases is thus crucial in understanding cell functions.

Accurate quantification of ß-hexosaminidase released from laboratory of allergic diseases 2 cells via liquid chromatography tandem mass spectrometry method.

ß-Hexosaminidase is one of the enzymes that is secreted from mast cells via antigen-induced degranulation and has frequently been used as an indicator of anaphylactic reactions. The main method for determining ß-hexosaminidase is indirect, "substrate-based" and shows limitations. Hence, development of an accurate detecting method is particularly important and urgently needed. In this study we developed a liquid chromatography tandem mass spectrometry (LC-MS/MS) method for measuring ß-hexosaminidase. Laboratory of allergic diseases 2 (LAD2) cells were stimulated with compound 48/80 (C48/80), the supernatant was collected and subjected to in-solution protein digestion; the obtained peptides were desalted, concentrated, separated and analyzed with an LC-tandem MS instrument. A peptide with the sequence "LAPGTIVEVWK" was selected for quantitative analysis, and four other peptides for qualitative research. The time-effect relationship curve was studied, and the results of the LC-MS/MS method were found to be almost consistent with those obtained via the conventional method. The method was then employed to measure ß-hexosaminidase released from LAD2 cells stimulated with potential allergens, and the results showed that it can be applied to determine the potential allergenicity of drugs. This new method showed good specificity, high sensitivity and a wide application range. It could be used to evaluate allergic reactions, providing a guide for medication safety during clinical testing.

An activity-dependent proximity ligation platform for spatially resolved quantification of active enzymes in single cells.

Integration of chemical probes into proteomic workflows enables the interrogation of protein activity, rather than abundance. Current methods limit the biological contexts that can be addressed due to sample homogenization, signal-averaging, and bias toward abundant proteins. Here we report a platform that integrates family-wide chemical probes with proximity-dependent oligonucleotide amplification and imaging to quantify enzyme activity in native contexts with high spatial resolution. Application of this method, activity-dependent proximity ligation (ADPL), to serine hydrolase and cysteine protease enzymes enables quantification of differential enzyme activity resulting from endogenous changes in localization and expression. In a competitive format, small-molecule target engagement with endogenous proteins in live cells can be quantified. Finally, retention of sample architecture enables interrogation of complex environments such as cellular co-culture and patient samples. ADPL should be amenable to diverse probe and protein families to detect active enzymes at scale and resolution out of reach with current methods.

Noise slows the rate of Michaelis-Menten reactions.

Microscopic randomness and the small volumes of living cells combine to generate random fluctuations in molecule concentrations called "noise". Here I investigate the effect of noise on biochemical reactions obeying Michaelis-Menten kinetics, concluding that substrate noise causes these reactions to slow. I derive a general expression for the time evolution of the joint probability density of chemical species in arbitrarily connected networks of non-linear chemical reactions in small volumes. This equation is a generalization of the chemical master equation (CME), a common tool for investigating stochastic chemical kinetics, extended to reaction networks occurring in small volumes, such as living cells. I apply this equation to a generalized Michaelis-Menten reaction in an open system, deriving the following general result: 〈p〉≤p¯ and 〈s〉≥s¯, where s¯ and p¯ denote the deterministic steady-state concentration of reactant and product species, respectively, and 〈s〉 and 〈p〉 denote the steady-state ensemble average over independent realizations of a stochastic reaction. Under biologically realistic conditions, namely when substrate is degraded or diluted by cell division, 〈p〉≤p¯. Consequently, noise slows the rate of in vivo Michaelis-Menten reactions. These predictions are validated by extensive stochastic simulations using Gillespie's exact stochastic simulation algorithm. I specify the conditions under which these effects occur and when they vanish, therefore reconciling discrepancies among previous theoretical investigations of stochastic biochemical reactions. Stochastic slowdown of reaction flux caused by molecular noise in living cells may have functional consequences, which the present theory may be used to quantify.

[Over-expression of uracil DNA glycosylase 2 (UNG2) enhances the resistance to oxidative damage in HepG2 cells].

Objective To investigate the uracil glycosidic enzyme activity of uracil DNA glycosylase 2 (UNG2) and study the role of UNG2 in the resistance of antioxidant stress of HepG2 cells. Methods The UNG2-expressing vector was built. Western blotting was used to detect the expression of UNG2. Immunofluorescence staining was performed to observe the cellular location of UNG2. Oligonucleotide was used as substrate for the determination of the UNG2 glycosidic enzyme activity. H2O2 toxicity assay was done to study the function of UNG2 in the antioxidant resistance of hepatocellular carcinoma HepG2 cells. Results UNG2 was successfully over-expressed in HEK293FT cells, and UNG2 was found to be mainly located in nucleus. Enzyme activity assay showed that UNG2 had significant oligonucleotide dU glycosidic enzyme activity. H2O2 toxicity assay showed that over-expressed UNG2 could remarkably increase the survival of HepG2 cells after exposed to H2O2. Conclusion UNG2 possesses specific DNA glycosidic enzyme activity, and it can protect HepG2 cells against oxidative stress damage.

Mechanisms for initiating cellular DNA replication.

Cellular DNA replication factories depend on ring-shaped hexameric helicases to aid DNA synthesis by processively unzipping the parental DNA helix. Replicative helicases are loaded onto DNA by dedicated initiator, loader, and accessory proteins during the initiation of DNA replication in a tightly regulated, multistep process. We discuss here the molecular choreography of DNA replication initiation across the three domains of life, highlighting similarities and differences in the strategies used to deposit replicative helicases onto DNA and to melt the DNA helix in preparation for replisome assembly. Although initiators and loaders are phylogenetically related, the mechanisms they use for accomplishing similar tasks have diverged considerably and in an unpredictable manner.

Proanthocyanidins purified from fruit pericarp of Clausena lansium (Lour.) Skeels as efficient tyrosinase inhibitors: structure evaluation, inhibitory activity and molecular mechanism.

Fruit pericarp of Clausena lansium (Lour.) Skeels, a food waste, was selected as a raw material for proanthocyanidins. The proanthocyanidins' structures were integrally analyzed using three methods: matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), high performance liquid chromatography electrospray ionization mass spectrometry (HPLC-ESI-MS) and 13C nuclear magnetic resonance (NMR). The results elucidated that these compounds were composed of prodelphinidin (75%) and procyanidin (25%) with a degree of polymerization (DP) up to the 20-mers. They were proved to be remarkable, reversible and mixed competitive inhibitors of tyrosinase according to results from enzyme experiments. The IC50 values were calculated to be 23.6 ± 1.2 and 7.0 ± 0.2 µg mL-1 for the monophenolase and diphenolase activities, respectively. In addition, the proanthocyanidins had a good inhibitory effect on cell proliferation, cellular tyrosinase activity and melanin production of B16 mouse melanoma cells. Chelation between the hydroxyl group on the B ring of the proanthocyanidins and dicopper irons of the enzyme provided one of the feasible mechanisms for the inhibition on the basis of fluorescence quenching and molecular docking analyses. This research would supply the scientific basis to these compounds application in the pharmaceutical, insecticides, and preservative fields.

Multi-level regulation of cellular glycosylation: from genes to transcript to enzyme to structure.

Glycosylation is a ubiquitous mammalian post-translational modification that both decorates a majority of expressed proteins and regulates their function. Cellular glycan biosynthesis is facilitated by a few hundred enzymes that are collectively termed 'glycoenzymes'. The expression and activity of these enzymes is controlled at the transcription, translation and post-translation levels. New wet-lab advances are providing analytical methods to collect large-scale data at these multiple levels, relational databases are starting to collate these results, and computer models are beginning to integrate this information across scales in order to gain new knowledge. These activities are likely to enable the qualitative and quantitative mapping of pathways regulating glycan production and function in proteins, cells and tissue.

Critical telomerase activity for uncontrolled cell growth.

The lengths of the telomere regions of chromosomes in a population of cells are modelled using a chemical master equation formalism, from which the evolution of the average number of cells of each telomere length is extracted. In particular, the role of the telomere-elongating enzyme telomerase on these dynamics is investigated. We show that for biologically relevant rates of cell birth and death, one finds a critical rate, R crit, of telomerase activity such that the total number of cells diverges. Further, R crit is similar in magnitude to the rates of mitosis and cell death. The possible relationship of this result to replicative immortality and its associated hallmark of cancer is discussed.

Transglutaminase 2 has opposing roles in the regulation of cellular functions as well as cell growth and death.

Transglutaminase 2 (TG2) is primarily known as the most ubiquitously expressed member of the transglutaminase family with Ca(2+)-dependent protein crosslinking activity; however, this enzyme exhibits multiple additional functions through GTPase, cell adhesion, protein disulfide isomerase, kinase, and scaffold activities and is associated with cell growth, differentiation, and apoptosis. TG2 is found in the extracellular matrix, plasma membrane, cytosol, mitochondria, recycling endosomes, and nucleus, and its subcellular localization is an important determinant of its function. Depending upon the cell type and stimuli, TG2 changes its subcellular localization and biological activities, playing both anti- and pro-apoptotic roles. Increasing evidence indicates that the GTP-bound form of the enzyme (in its closed form) protects cells from apoptosis but that the transamidation activity of TG2 (in its open form) participates in both facilitating and inhibiting apoptosis. A difficulty in the study and understanding of this enigmatic protein is that opposing effects have been reported regarding its roles in the same physiological and/or pathological systems. These include neuroprotective or neurodegenerative effects, hepatic cell growth-promoting or hepatic cell death-inducing effects, exacerbating or having no effect on liver fibrosis, and anti- and pro-apoptotic effects on cancer cells. The reasons for these discrepancies have been ascribed to TG2's multifunctional activities, genetic variants, conformational changes induced by the immediate environment, and differences in the genetic background of the mice used in each of the experiments. In this article, we first report that TG2 has opposing roles like the protagonist in the novel Dr. Jekyll and Mr. Hyde, followed by a summary of the controversies reported, and finally discuss the possible reasons for these discrepancies.