Protease and DNase Activities of a Very Stable High-Molecular-Mass Multiprotein Complex from Sea Cucumber Eupentacta fraudatrix

Only some human organs, including the liver, are capable of very weak self-regeneration. Some marine echinoderms are very useful for studying the self-regeneration processes of organs and tissues. For example, sea cucumbers Eupentacta fraudatrix (holothurians) demonstrate complete restoration of all organs and the body within several weeks after their division into two parts. Therefore, these cucumbers are a prospective model for studying the general mechanisms of self-regeneration. However, there is no data available yet concerning biomolecules of holothurians, which can stimulate the processes of organ and whole-body regeneration. Investigation of these restoration mechanisms is very important for modern medicine and biology because it can help to understand which hormones, nucleic acids, proteins, enzymes, or complexes play an essential role in self-regeneration. It is possible that stable, polyfunctional, high-molecular-weight protein complexes play an essential role in these processes. It has recently been shown that sea cucumbers Eupentacta fraudatrix contain a very stable multiprotein complex of about 2000 kDa. The first analysis of possible enzymatic activities of a stable protein complex was carried out in this work, revealing that the complex possesses several protease and DNase activities. The complex metalloprotease is activated by several metal ions (Zn²⁺ > Mn²⁺ > Mg²⁺). The relative contribution of metalloproteases (~63.4%), serine-like protease (~30.5%), and thiol protease (~6.1%) to the total protease activity of the complex was estimated. Metal-independent proteases of the complex hydrolyze proteins at trypsin-specific sites (after Lys and Arg). The complex contains both metal-dependent and metal-independent DNases. Mg²⁺, Mn²⁺, and Co²⁺ ions were found to strongly increase the DNase activity of the complex.     

Hofmeister Series: From Aqueous Solution of Biomolecules to Single Cells and Nanovesicles

Hofmeister effects play a critical role in numerous physicochemical and biological phenomena, including the solubility and/or accumulation of proteins, the activities of enzymes, ion transport in biochannels, the structure of lipid bilayers, and the dynamics of vesicle opening and exocytosis. This minireview focuses on how ionic specificity affects the physicochemical properties of biomolecules to regulate cellular exocytosis, vesicular content, and nanovesicle opening. We summarize recent progress in further understanding Hofmeister effects on biomacromolecules and their applications in biological systems. These important steps have increased our understanding of the Hofmeister effects on cellular exocytosis, vesicular content, and nanovesicle opening. Increasing evidence is firmly establishing that the ions along the Hofmeister series play an important role in living organisms that has often been ignored.     

Reversible Modulation of Aptamer-Ligand Binding in RNA Light-Up Aptamers Containing G-Quadruplex Using Chemical Stimuli

Regulating a system in equilibrium transiently to out-of-equilibrium by using certain stimuli is the strategy used by natural biomolecules to function. Herein, we showed that the interaction of synthetic RNA aptamers, having a G-quadruplex core structure, with their corresponding ligands could be regulated from their equilibrium state to non-equilibrium state in a reversible manner using simple chemical stimuli (Ag⁺ and cysteine). The approach would be useful for designing aptamer regulators that work in a dynamic nucleic acid network, where a strict control on aptamer-ligand interaction is needed. In addition, to the best of our knowledge, this is the first report which shows that RNA G-quadruplexes can be disrupted by the addition of silver ions. This would be useful not only in designing RNA-based sensors or regulators but would also be useful for understanding the role of metal ions in RNA folding and catalysis.     

Two Unconventional Metallothioneins in the Apple Snail Pomacea bridgesii Have Lost Their Metal Specificity during Adaptation to Freshwater Habitats

Metallothioneins (MTs) are a diverse group of proteins responsible for the control of metal homeostasis and detoxification. To investigate the impact that environmental conditions might have had on the metal-binding abilities of these proteins, we have characterized the MTs from the apple snail Pomacea bridgesii, a gastropod species belonging to the class of Caenogastropoda with an amphibious lifestyle facing diverse situations of metal bioavailability. P. bridgesii has two structurally divergent MTs, named PbrMT1 and PbrMT2, that are longer than other gastropod MTs due to the presence of extra sequence motifs and metal-binding domains. We have characterized the Zn(II), Cd(II), and Cu(I) binding abilities of these two MTs after their heterologous expression in E. coli. Our results have revealed that despite their structural differences, both MTs share an unspecific metal-binding character, and a great ability to cope with elevated amounts of different metal ions. Our analyses have also revealed slight divergences in their metal-binding features: PbrMT1 shows a more pronounced Zn(II)-thionein character than PbrMT2, while the latter has a stronger Cu(I)-thionein character. The characterization of these two unconventional PbrMTs supports the loss of the metal-binding specificity during the evolution of the MTs of the Ampullariid family, and further suggests an evolutionary link of this loss with the adaptation of these gastropod lineages to metal-poor freshwater habitats.     

VDACs Post-Translational Modifications Discovery by Mass Spectrometry: Impact on Their Hub Function

VDAC (voltage-dependent anion selective channel) proteins, also known as mitochondrial porins, are the most abundant proteins of the outer mitochondrial membrane (OMM), where they play a vital role in various cellular processes, in the regulation of metabolism, and in survival pathways. There is increasing consensus about their function as a cellular hub, connecting bioenergetics functions to the rest of the cell. The structural characterization of VDACs presents challenging issues due to their very high hydrophobicity, low solubility, the difficulty to separate them from other mitochondrial proteins of similar hydrophobicity and the practical impossibility to isolate each single isoform. Consequently, it is necessary to analyze them as components of a relatively complex mixture. Due to the experimental difficulties in their structural characterization, post-translational modifications (PTMs) of VDAC proteins represent a little explored field. Only in recent years, the increasing number of tools aimed at identifying and quantifying PTMs has allowed to increase our knowledge in this field and in the mechanisms that regulate functions and interactions of mitochondrial porins. In particular, the development of nano-reversed phase ultra-high performance liquid chromatography (nanoRP-UHPLC) and ultra-sensitive high-resolution mass spectrometry (HRMS) methods has played a key role in this field. The findings obtained on VDAC PTMs using such methodologies, which permitted an in-depth characterization of these very hydrophobic trans-membrane pore proteins, are summarized in this review.     

Inorganic nanoparticles as carriers for efficient cellular delivery

Cellular delivery involving the transfer of various drugs and bio-active molecules (peptides, proteins and DNAs, etc.) through the cell membrane into cells has attracted increasing attention because of its importance in medicine and drug delivery. This topic has been extensively reviewed. The direct delivery of drugs and biomolecules, however, is generally inefficient and suffering from problems such as enzymic degradation of DNAs. Therefore, searching for efficient and safe transport vehicles (carriers) to delivery genes or drugs into cells has been challenging yet exciting area of research. In past decades, many carriers have been developed and investigated extensively which can be generally classified into four major groups: viral carriers, organic cationic compounds, recombinant protiens and inorganic nanoparticles. Many inorganic materials, such as calcium phosphate, gold, carbon materials, silicon oxide, iron oxide and layered double hydroxide (LDH), have been studied. Inorganic nanoparticles show low toxicity and promise for controlled delivery properties, thus presenting a new alternative to viral carriers and cationic carriers. Inorganic nanoparticles generally possess versatile properties suitable for cellular delivery, including wide availability, rich functionality, good biocompatibility, potential capability of targeted delivery (e.g. selectively destroying cancer cells but sparing normal tissues) and controlled release of carried drugs. This paper reviews the latest advances in inorganic nanoparticle applications as cellular delivery carriers and highlights some key issues in efficient cellular delivery using inorganic nanoparticles. Critical properties of inorganic nanoparticles, surface functionalisation (modification), uptake of biomolecules, the driving forces for delivery, and release of biomolecules will be reviewed systematically. Selected examples of promising inorganic nanoparticle delivery systems, including gold, fullerences and carbon nanotubes, LDH and various oxide nanoparticles in particular their applications for gene delivery will be discussed. The fundamental understanding of properties of inorganic nanoparticles in relation to cellular delivery efficiency as the most paramount issue will be highlighted.     

Metal‐Assisted Folding of Prolinomycin Allows Facile Design of Functional Peptides

Cyclic peptides have been proposed as privileged scaffolds that might mimic the folding and function of natural proteins. However, simple cyclic peptides typically cannot fold into well‐defined structures. Herein, we describe a foldable cyclic peptide scaffold on which functional side chains can be displayed for targeted recognition of biomolecules. The foldable scaffold is based on prolinomycin, a proline‐rich analogue of valinomycin. We report synthetic mutants of prolinomycin that retain the metal‐assisted folding behavior under physiological conditions. The predictable structure formation of prolinomycin makes it a powerful platform to enable the development of synthetic receptors for biomolecules of interest. We demonstrate the potential of this scaffold by creating prolinomycin mutants that selectively bind anionic vesicles and bacterial cells.     

Phase Separation in cGAS-STING Signaling: Cytosolic DNA Sensing and Regulatory Functions

Phase separation is a crucial biophysical process that governs cellular signaling and function. This process allows biomolecules to separate and form membraneless compartments in response to both extra- and intra-cellular stimuli. Recently, the identification of phase separation in different immune signaling pathways, including the cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) signaling pathway, has shed light on its tight association with pathological processes such as viral infections, cancers, and inflammatory diseases. In this review, we present the phase separation in cGAS-STING signaling, along with its related cellular regulatory functions. Furthermore, we discuss the introduction of therapeutics targeting cGAS-STING signaling, which plays a pivotal role in cancer progression.     

Selective Covalent Targeting of Anti-apoptotic BFL-1 by a Sulfonium-Tethered Peptide

Anti-apoptotic B cell lymphoma 2 (BCL-2) family proteins are proven targets for human cancers. Targeting the BH3-binding pockets of these anti-apoptotic proteins could reactivate apoptosis in BCL-2-depedent cancers. BFL-1 is a BCL-2 family protein overexpressed in various chemoresistant cancers. A unique cysteine at the binding interface of the BH3 and BFL-1 was previously proven to be an intriguing targeting site to irreversibly inhibit BFL-1 functions with stabilized cyclic peptide bearing a covalent warhead. Recently, we developed a sulfonium-tethered peptide cyclization strategy to construct peptide ligands that could selectively and efficiently react with the cysteine(s) of target proteins near the interacting interface. Using this method, we constructed a BFL-1 peptide inhibitor, B4-MC, that could selectively conjugate with BFL-1 both in vitro and in cell. B4-MC showed good cellular uptake, colocalized with BFL-1 on mitochondria, and showed obvious growth inhibition of BFL-1 over-expressed cancer cell lines.     

Secretome and Tunneling Nanotubes: A Multilevel Network for Long Range Intercellular Communication between Endothelial Cells and Distant Cells

As a cellular interface between the blood and tissues, the endothelial cell (EC) monolayer is involved in the control of key functions including vascular tone, permeability and homeostasis, leucocyte trafficking and hemostasis. EC regulatory functions require long-distance communications between ECs, circulating hematopoietic cells and other vascular cells for efficient adjusting thrombosis, angiogenesis, inflammation, infection and immunity. This intercellular crosstalk operates through the extracellular space and is orchestrated in part by the secretory pathway and the exocytosis of Weibel Palade Bodies (WPBs), secretory granules and extracellular vesicles (EVs). WPBs and secretory granules allow both immediate release and regulated exocytosis of messengers such as cytokines, chemokines, extracellular membrane proteins, coagulation or growth factors. The ectodomain shedding of transmembrane protein further provide the release of both receptor and ligands with key regulatory activities on target cells. Thin tubular membranous channels termed tunneling nanotubes (TNTs) may also connect EC with distant cells. EVs, in particular exosomes, and TNTs may contain and transfer different biomolecules (e.g., signaling mediators, proteins, lipids, and microRNAs) or pathogens and have emerged as a major triggers of horizontal intercellular transfer of information.     

Structural Bioinformatics and Deep Learning of Metalloproteins: Recent Advances and Applications

All living organisms require metal ions for their energy production and metabolic and biosynthetic processes. Within cells, the metal ions involved in the formation of adducts interact with metabolites and macromolecules (proteins and nucleic acids). The proteins that require binding to one or more metal ions in order to be able to carry out their physiological function are called metalloproteins. About one third of all protein structures in the Protein Data Bank involve metalloproteins. Over the past few years there has been tremendous progress in the number of computational tools and techniques making use of 3D structural information to support the investigation of metalloproteins. This trend has been boosted by the successful applications of neural networks and machine/deep learning approaches in molecular and structural biology at large. In this review, we discuss recent advances in the development and availability of resources dealing with metalloproteins from a structure-based perspective. We start by addressing tools for the prediction of metal-binding sites (MBSs) using structural information on apo-proteins. Then, we provide an overview of the methods for and lessons learned from the structural comparison of MBSs in a fold-independent manner. We then move to describing databases of metalloprotein/MBS structures. Finally, we summarizing recent ML/DL applications enhancing the functional interpretation of metalloprotein structures.     

Exchange of Proteins in Liposomes through Streptolysin O Pores

Liposomes, which are vesicles surrounded by lipid membranes, can be used as biochemical reactors by encapsulating various reactions. Accordingly, they are useful for studying cellular functions under controlled conditions that mimic the environment within a cell. However, one of the shortcomings of liposomes as biochemical reactors is the difficulty of introducing or removing proteins due to the impermeability of the membrane. In this study, we established a method for exchanging proteins in liposomes by forming reversible pores in the membrane. We used the toxic protein streptolysin O (SLO); this forms pores in membranes made of phospholipids containing cholesterol that can be closed by the addition of calcium ions. After optimizing the experimental procedure and lipid composition, we observed the exchange of fluorescent proteins (transferrin Alexa Fluor 488 and 647) in 9.9 % of liposomes. We also introduced T7 RNA polymerase, a 98-kDa enzyme, and observed RNA synthesis in ∼8 % of liposomes. Our findings establish a new method for controlling the internal protein composition of liposomes, thereby increasing their utility as bioreactors.     

The Chemistry of Reactive Oxygen Species (ROS) Revisited: Outlining Their Role in Biological Macromolecules (DNA,Lipids and Proteins) and Induced Pathologies

Living species are continuously subjected to all extrinsic forms of reactive oxidants and others that are produced endogenously. There is extensive literature on the generation and effects of reactive oxygen species (ROS) in biological processes, both in terms of alteration and their role in cellular signaling and regulatory pathways. Cells produce ROS as a controlled physiological process, but increasing ROS becomes pathological and leads to oxidative stress and disease. The induction of oxidative stress is an imbalance between the production of radical species and the antioxidant defense systems, which can cause damage to cellular biomolecules, including lipids, proteins and DNA. Cellular and biochemical experiments have been complemented in various ways to explain the biological chemistry of ROS oxidants. However, it is often unclear how this translates into chemical reactions involving redox changes. This review addresses this question and includes a robust mechanistic explanation of the chemical reactions of ROS and oxidative stress.     

Protein Dynamics: From Molecules,to Interactions,to Biology

Proteins have a remarkably rich diversity of dynamical behaviors, and the articles in this issue of the International Journal of Molecular Sciences are a testament to that fact. From the picosecond motions of single sidechains probed by NMR or fluorescence spectroscopy, to aggregation processes at interfaces that take months, all time scales play a role. Proteins are functional molecules, so by their nature they always interact with their environment. This environment includes water, other biomolecules, or larger cellular structures. In a sense, it also includes the protein molecule itself: proteins are large enough to fold and interact with themselves. These interactions have been honed by evolution to produce behaviors completely different from those of random polymers.     

Recent Advances in Bifunctional Paramagnetic Chelates for MRI

Magnetic resonance imaging (MRI) is a non-invasive diagnostic modality routinely employed in modern clinical medicine to collect images of the internal organs of the human body. Contrast agents (CA) are usually administered to patients to improve the sensitivity of this technique and nowadays these agents are gadolinium complexes. Bifunctional chelating agents (BFCAs) are dual molecules containing a multidentate ligand, able to strongly coordinate a metal ion, and a reactive moiety for conjugation purposes. With BFCAs an easy labelling of biomolecules or vectors with paramagnetic ions is possible, allowing the in vivo MRI visualization of diseases at cellular or molecular level.     

Cell-to-Cell Communication by Host-Released Extracellular Vesicles in the Gut: Implications in Health and Disease

Communication between cells is crucial to preserve body homeostasis and health. Tightly controlled intercellular dialog is particularly relevant in the gut, where cells of the intestinal mucosa are constantly exposed to millions of microbes that have great impact on intestinal homeostasis by controlling barrier and immune functions. Recent knowledge involves extracellular vesicles (EVs) as mediators of such communication by transferring messenger bioactive molecules including proteins, lipids, and miRNAs between cells and tissues. The specific functions of EVs principally depend on the internal cargo, which upon delivery to target cells trigger signal events that modulate cellular functions. The vesicular cargo is greatly influenced by genetic, pathological, and environmental factors. This finding provides the basis for investigating potential clinical applications of EVs as therapeutic targets or diagnostic biomarkers. Here, we review current knowledge on the biogenesis and cargo composition of EVs in general terms. We then focus the attention to EVs released by cells of the intestinal mucosa and their impact on intestinal homeostasis in health and disease. We specifically highlight their role on epithelial barrier integrity, wound healing of epithelial cells, immunity, and microbiota shaping. Microbiota-derived EVs are not reviewed here.     

The Metallothionein/Thionein System: An Oxidoreductive Metabolic Zinc Link

Metallothioneins (MTs) were discovered more than 50 years ago and identified as low‐molecular weight, sulfhydryl‐rich proteins that were subsequently found to bind zinc predominantly. The binding of seemingly redox inactive zinc ions allows MT to play a central role in oxidoreductive cellular metabolism, cellular zinc distribution and homeostasis. In this interpretive study, we discuss the interaction of MT with physiologically relevant molecules and its effect on zinc? thiolate bonds. These interactions are linked to recent progress in the functional role of MT in cellular zinc transport, energy production, and protection of the organism against oxidative stress and neurodegenerative diseases.