Yeast Two-Hybrid, a Powerful Tool for Systems Biology

A key property of complex biological systems is the presence of interaction networks formed by its different components, primarily proteins. These are crucial for all levels of cellular function, including architecture, metabolism and signalling, as well as the availability of cellular energy. Very stable, but also rather transient and dynamic protein-protein interactions generate new system properties at the level of multiprotein complexes, cellular compartments or the entire cell. Thus, interactomics is expected to largely contribute to emerging fields like systems biology or systems bioenergetics. The more recent technological development of high-throughput methods for interactomics research will dramatically increase our knowledge of protein interaction networks. The two most frequently used methods are yeast two-hybrid (Y2H) screening, a well established genetic in vivo approach, and affinity purification of complexes followed by mass spectrometry analysis, an emerging biochemical in vitro technique. So far, a majority of published interactions have been detected using an Y2H screen. However, with the massive application of this method, also some limitations have become apparent. This review provides an overview on available yeast two-hybrid methods, in particular focusing on more recent approaches. These allow detection of protein interactions in their native environment, as e.g. in the cytosol or bound to a membrane, by using cytosolic signalling cascades or split protein constructs. Strengths and weaknesses of these genetic methods are discussed and some guidelines for verification of detected protein-protein interactions are provided.     

Maternal Embryonic Leucine Zipper Kinase (MELK): A Novel Regulator in Cell Cycle Control,Embryonic Development,and Cancer

Maternal embryonic leucine zipper kinase (MELK) functions as a modulator of intracellular signaling and affects various cellular and biological processes, including cell cycle, cell proliferation, apoptosis, spliceosome assembly, gene expression, embryonic development, hematopoiesis, and oncogenesis. In these cellular processes, MELK functions by binding to numerous proteins. In general, the effects of multiple protein interactions with MELK are oncogenic in nature, and the overexpression of MELK in kinds of cancer provides some evidence that it may be involved in tumorigenic process. In this review, our current knowledge of MELK function and recent discoveries in MELK signaling pathway were discussed. The regulation of MELK in cancers and its potential as a therapeutic target were also described.     

Cellular and Molecular Nature of Fragmentation of Human Embryos

Embryo fragmentation represents a phenomenon generally characterized by the presence of membrane-bound extracellular cytoplasm into the perivitelline space. Recent evidence supports the cellular and molecular heterogeneity of embryo fragments. In this narrative review, we described the different embryo fragment-like cellular structures in their morphology, molecular content, and supposed function and have reported the proposed theories on their origin over the years. We identified articles related to characterization of embryo fragmentation with a specific literature search string. The occurrence of embryo fragmentation has been related to various mechanisms, of which the most studied are apoptotic cell death, membrane compartmentalization of altered DNA, cytoskeletal disorders, and vesicle formation. These phenomena are thought to result in the extrusion of entire blastomeres, release of apoptotic bodies and other vesicles, and micronuclei formation. Different patterns of fragmentation may have different etiologies and effects on embryo competence. Removal of fragments from the embryo before embryo transfer with the aim to improve implantation potential should be reconsidered on the basis of the present observations     

The energy landscape for protein folding and possible connections to function

In this article we review and discuss the state-of-the-art methods using minimalist models in the context of energy landscape theory to study protein folding. As good agreement between computational/theoretical studies and experimental observations in vitro continues to emerge, many research groups are asking how this structural and dynamical information can be used to understand proteins in vivo. This is a non-trivial question drawing from very limited in vivo studies. From the perspective of theory, it is a new horizon for theoreticians to test or revise their theories by making connections to experiments on this matter. We present a short discussion of several recent efforts that include factors reflecting the cellular environment in computer simulations—and that may provide some insight into the behavior of protein dynamics inside the living cell as well as inspire the development of new experimental approaches for a better understanding of the molecular mechanisms for function.     

The Autonomous Glycosylation of Large DNA Viruses

Glycosylation of surface molecules is a key feature of several eukaryotic viruses, which use the host endoplasmic reticulum/Golgi apparatus to add carbohydrates to their nascent glycoproteins. In recent years, a newly discovered group of eukaryotic viruses, belonging to the Nucleo-Cytoplasmic Large DNA Virus (NCLDV) group, was shown to have several features that are typical of cellular organisms, including the presence of components of the glycosylation machinery. Starting from initial observations with the chlorovirus PBCV-1, enzymes for glycan biosynthesis have been later identified in other viruses; in particular in members of the Mimiviridae family. They include both the glycosyltransferases and other carbohydrate-modifying enzymes and the pathways for the biosynthesis of the rare monosaccharides that are found in the viral glycan structures. These findings, together with genome analysis of the newly-identified giant DNA viruses, indicate that the presence of glycogenes is widespread in several NCLDV families. The identification of autonomous viral glycosylation machinery leads to many questions about the origin of these pathways, the mechanisms of glycan production, and eventually their function in the viral replication cycle. The scope of this review is to highlight some of the recent results that have been obtained on the glycosylation systems of the large DNA viruses, with a special focus on the enzymes involved in nucleotide-sugar production.     

Localization of axonal motor molecules machinery in neurodegenerative disorders

Axonal transport and neuronal survival depend critically on active transport and axon integrity both for supplying materials and communication to different domains of the cell body. All these actions are executed through cytoskeleton, transport and regulatory elements that appear to be disrupted in neurodegenerative diseases. Motor-driven transport both supplies and clears distal cellular portions with proteins and organelles. This transport is especially relevant in projection and motor neurons, which have long axons to reach the farthest nerve endings. Thus, any disturbance of axonal transport may have severe consequences for neuronal function and survival. A growing body of literature indicates the presence of alterations to the motor molecules machinery, not only in expression levels and phosphorylation, but also in their subcellular distribution within populations of neurons, which are selectively affected in the course of neurodegenerative diseases. The implications of this altered subcellular localization and how this affects axon survival and neuronal death still remain poorly understood, although several hypotheses have been suggested. Furthermore, cytoskeleton and transport element localization can be selectively disrupted in some disorders suggesting that specific loss of the axonal functionality could be a primary hallmark of the disorder. This can lead to axon degeneration and neuronal death either directly, through the functional absence of essential axonal proteins, or indirectly, through failures in communication among different cellular domains. This review compares the localization of cytoskeleton and transport elements in some neurodegenerative disorders to ask what aspects may be essential for axon survival and neuronal death.     

The Importance of Alpha-Actinin Proteins in Platelet Formation and Function,and Their Causative Role in Congenital Macrothrombocytopenia

The actin cytoskeleton plays a central role in platelet formation and function. Alpha-actinins (actinins) are actin filament crosslinking proteins that are prominently expressed in platelets and have been studied in relation to their role in platelet activation since the 1970s. However, within the past decade, several groups have described mutations in ACTN1/actinin-1 that cause congenital macrothrombocytopenia (CMTP)—accounting for approximately 5% of all cases of this condition. These findings are suggestive of potentially novel functions for actinins in platelet formation from megakaryocytes in the bone marrow and/or platelet maturation in circulation. Here, we review some recent insights into the well-known functions of actinins in platelet activation before considering possible roles for actinins in platelet formation that could explain their association with CMTP. We describe what is known about the consequences of CMTP-linked mutations on actinin-1 function at a molecular and cellular level and speculate how these changes might lead to the alterations in platelet count and morphology observed in CMTP patients. Finally, we outline some unanswered questions in this area and how they might be addressed in future studies.     

New Insight into Molecular and Hormonal Connection in Andrology

Hormones and cytokines are known to regulate cellular functions in the testes. These biomolecules induce a broad spectrum of effects on various level of spermatogenesis, and among them is the modulation of cell junction restructuring between Sertoli cells and germ cells in the seminiferous epithelium. Cytokines and androgens are closely related, and both correct testicular development and the maintenance of spermatogenesis depend on their function. Cytokines also play a crucial role in the immune testicular system, activating and directing leucocytes across the endothelial barrier to the inflammatory site, as well as in increasing their adhesion to the vascular wall. The purpose of this review is to revise the most recent findings on molecular mechanisms that play a key role in male sexual function, focusing on three specific molecular patterns, namely, cytokines, miRNAs, and endothelial progenitor cells. Numerous reports on the interactions between the immune and endocrine systems can be found in the literature. However, there is not yet a multi-approach review of the literature underlying the role between molecular patterns and testicular and sexual function.     

Current methods for the identification and quantitation of ceramides: An overview

Ceramides are key compounds in the metabolism of sphingolipids and are emerging as important second messengers for various cellular processes including cell cycle arrest, differentiation, senescence, apoptosis, and others. Because of their important biological functions, exact analysis of their molecular species and concentrations is crucial for elucidating their function and metabolism. Toward this goal, several methods have been developed for the identification and quantitation of cellular ceramide levels. Methods have been developed utilizing thin-layer or high-performance liquid chromatography. Mass spectrometry also has become increasingly utilized. The Escherichia coli diacylglycerol kinase assay is one of the most frequently used techniques for ceramide quantitation. This review presents a current summary of methods used for the identification and quantitation of ceramides.     

Essential Roles of PPARs in Lipid Metabolism during Mycobacterial Infection

The mycobacterial cell wall is composed of large amounts of lipids with varying moieties. Some mycobacteria species hijack host cells and promote lipid droplet accumulation to build the cellular environment essential for their intracellular survival. Thus, lipids are thought to be important for mycobacteria survival as well as for the invasion, parasitization, and proliferation within host cells. However, their physiological roles have not been fully elucidated. Recent studies have revealed that mycobacteria modulate the peroxisome proliferator-activated receptor (PPAR) signaling and utilize host-derived triacylglycerol (TAG) and cholesterol as both nutrient sources and evasion from the host immune system. In this review, we discuss recent findings that describe the activation of PPARs by mycobacterial infections and their role in determining the fate of bacilli by inducing lipid metabolism, anti-inflammatory function, and autophagy.     

Mitochondrial Processing Peptidases—Structure,Function and the Role in Human Diseases

Mitochondrial proteins are encoded by both nuclear and mitochondrial DNA. While some of the essential subunits of the oxidative phosphorylation (OXPHOS) complexes responsible for cellular ATP production are synthesized directly in the mitochondria, most mitochondrial proteins are first translated in the cytosol and then imported into the organelle using a sophisticated transport system. These proteins are directed mainly by targeting presequences at their N-termini. These presequences need to be cleaved to allow the proper folding and assembly of the pre-proteins into functional protein complexes. In the mitochondria, the presequences are removed by several processing peptidases, including the mitochondrial processing peptidase (MPP), the inner membrane processing peptidase (IMP), the inter-membrane processing peptidase (MIP), and the mitochondrial rhomboid protease (Pcp1/PARL). Their proper functioning is essential for mitochondrial homeostasis as the disruption of any of them is lethal in yeast and severely impacts the lifespan and survival in humans. In this review, we focus on characterizing the structure, function, and substrate specificities of mitochondrial processing peptidases, as well as the connection of their malfunctions to severe human diseases.     

Polymers used to influence cell fate in 3D geometry: New trends

The extracellular matrix (ECM) is a hydrogel-like structure comprised of several different biopolymers, encompassing a wide range of biological, chemical, and mechanical properties. The composition, organization, and assembly of the ECM play a critical role in cell function. Cellular behavior is guided by interactions that occur between cells and their local microenvironment, and this interrelationship plays a significant role in determining physiological functions. Bioengineering approaches have been developed to mimic native tissue microenvironments by fabricating novel bioactive hydrogel scaffolds. This review explores material designs and fabrication approaches that are guiding the design of hydrogels as tissue engineered scaffolds. As the fundamental biology of the cellular microenvironment is often the inspiration for material design, the review focuses on modifications to control bioactive cues such as adhesion molecules and growth factors, and summarizes the current applications of biomimetic scaffolds that have been used in vitro as well as in vivo.     

Extracellular Vesicles: Interplay with the Extracellular Matrix and Modulated Cell Responses

The discovery that cells secrete extracellular vesicles (EVs), which carry a variety of regulatory proteins, nucleic acids, and lipids, has shed light on the sophisticated manner by which cells can communicate and accordingly function. The bioactivity of EVs is not only defined by their internal content, but also through their surface associated molecules, and the linked downstream signaling effects they elicit in target cells. The extracellular matrix (ECM) contains signaling and structural molecules that are central to tissue maintenance and repair. Recently, a subset of EVs residing within the extracellular matrix has been identified. Although some roles have been proposed for matrix-bound vesicles, their role as signaling molecules within the ECM is yet to be explored. Given the close association of EVs and the ECM, it is not surprising that EVs partly mediate repair and regeneration by modulating matrix deposition and degradation through their cellular targets. This review addresses unique EV features that allow them to interact with and navigate through the ECM, describes how their release and content is influenced by the ECM, and emphasizes the emerging role of stem-cell derived EVs in tissue repair and regeneration through their matrix-modulating properties.     

Sarcopenia: Molecular Pathways and Potential Targets for Intervention

Aging is associated with sarcopenia. The loss of strength results in decreased muscle mass and motor function. This process accelerates the progressive muscle deterioration observed in older adults, favoring the presence of debilitating pathologies. In addition, sarcopenia leads to a decrease in quality of life, significantly affecting self-sufficiency. Altogether, these results in an increase in economic resources from the National Health Systems devoted to mitigating this problem in the elderly, particularly in developed countries. Different etiological determinants are involved in the progression of the disease, including: neurological factors, endocrine alterations, as well as nutritional and lifestyle changes related to the adoption of more sedentary habits. Molecular and cellular mechanisms have not been clearly characterized, resulting in the absence of an effective treatment for sarcopenia. Nevertheless, physical activity seems to be the sole strategy to delay sarcopenia and its symptoms. The present review intends to bring together the data explaining how physical activity modulates at a molecular and cellular level all factors that predispose or favor the progression of this deteriorating pathology.     

NADPH Oxidase 4 (NOX4) in Cancer: Linking Redox Signals to Oncogenic Metabolic Adaptation

Cancer cells can survive and maintain their high proliferation rate in spite of their hypoxic environment by deploying a variety of adaptative mechanisms, one of them being the reorientation of cellular metabolism. A key aspect of this metabolic rewiring is the promotion of the synthesis of antioxidant molecules in order to counter-balance the hypoxia-related elevation of reactive oxygen species (ROS) production and thus combat the onset of cellular oxidative stress. However, opposite to their negative role in the inception of oxidative stress, ROS are also key modulatory components of physiological cellular metabolism. One of the major physiological cellular ROS sources is the NADPH oxidase enzymes (NOX-es). Indeed, NOX-es produce ROS in a tightly regulated manner and control a variety of cellular processes. By contrast, pathologically elevated and unbridled NOX-derived ROS production is linked to diverse cancerogenic processes. In this respect, NOX4, one of the members of the NOX family enzymes, is of particular interest. In fact, NOX4 is closely linked to hypoxia-related signaling and is a regulator of diverse metabolic processes. Furthermore, NOX4 expression and function are altered in a variety of malignancies. The aim of this review is to provide a synopsis of our current knowledge concerning NOX4-related processes in the oncogenic metabolic adaptation of cancer cells.     

Aminoacyl-tRNA Synthetase Complexes in Evolution

Aminoacyl-tRNA synthetases are essential enzymes for interpreting the genetic code. They are responsible for the proper pairing of codons on mRNA with amino acids. In addition to this canonical, translational function, they are also involved in the control of many cellular pathways essential for the maintenance of cellular homeostasis. Association of several of these enzymes within supramolecular assemblies is a key feature of organization of the translation apparatus in eukaryotes. It could be a means to control their oscillation between translational functions, when associated within a multi-aminoacyl-tRNA synthetase complex (MARS), and nontranslational functions, after dissociation from the MARS and association with other partners. In this review, we summarize the composition of the different MARS described from archaea to mammals, the mode of assembly of these complexes, and their roles in maintenance of cellular homeostasis.     

Iron Availability in Tissue Microenvironment: The Key Role of Ferroportin

Body iron levels are regulated by hepcidin, a liver-derived peptide that exerts its function by controlling the presence of ferroportin (FPN), the sole cellular iron exporter, on the cell surface. Hepcidin binding leads to FPN internalization and degradation, thereby inhibiting iron release, in particular from iron-absorbing duodenal cells and macrophages involved in iron recycling. Disruption in this regulatory mechanism results in a variety of disorders associated with iron-deficiency or overload. In recent years, increasing evidence has emerged to indicate that, in addition to its role in systemic iron metabolism, FPN may play an important function in local iron control, such that its dysregulation may lead to tissue damage despite unaltered systemic iron homeostasis. In this review, we focus on recent discoveries to discuss the role of FPN-mediated iron export in the microenvironment under both physiological and pathological conditions.     

Mitochondrial Oxidative Stress—A Causative Factor and Therapeutic Target in Many Diseases

The excessive formation of reactive oxygen species (ROS) and impairment of defensive antioxidant systems leads to a condition known as oxidative stress. The main source of free radicals responsible for oxidative stress is mitochondrial respiration. The deleterious effects of ROS on cellular biomolecules, including DNA, is a well-known phenomenon that can disrupt mitochondrial function and contribute to cellular damage and death, and the subsequent development of various disease processes. In this review, we summarize the most important findings that implicated mitochondrial oxidative stress in a wide variety of pathologies from Alzheimer disease (AD) to autoimmune type 1 diabetes. This review also discusses attempts to affect oxidative stress as a therapeutic avenue.