Macropinocytosis: searching for an endocytic identity and role in the uptake of cell penetrating peptides

Macropinocytosis defines a series of events initiated by extensive plasma membrane reorganization or ruffling to form an external macropinocytic structure that is then enclosed and internalized. The process is constitutive in some organisms and cell types but in others it is only pronounced after growth factor stimulation. Internalized macropinosomes share many features with phagosomes and both are distinguished from other forms of pinocytic vesicles by their large size, morphological heterogeneity and lack of coat structures. A paucity of information is available on other distinguishing features for macropinocytosis such as specific marker proteins and drugs that interfere with its mechanism over other endocytic processes. This has hampered efforts to characterize the dynamics of this pathway and to identify regulatory proteins that are expressed in order to allow it to proceed. Upon internalization, macropinosomes acquire regulatory proteins common to other endocytic pathways, suggesting that their identities as unique structures are short-lived. There is however less consensus regarding the overall fate of the macropinosome cargo or its limiting membrane and processes such as fusion, tubulation, recycling and regulated exocytosis have all been implicated in shaping the macropinosome and directing cargo traffic. Macropinocytosis has also been implicated in the internalization of cell penetrating peptides that are of significant interest to researchers aiming to utilize their translocation abilities to deliver therapeutic entities such as genes and proteins into cells. This review focuses on recent findings on the regulation of macropinocytosis, the intracellular fate of the macropinosome and discusses evidence for the role of this pathway as a mechanism of entry for cell penetrating peptides.     

Use of detergents in two-dimensional crystallization of membrane proteins

Structure determination at high resolution is actually a difficult challenge for membrane proteins and the number of membrane proteins that have been crystallized is still small and far behind that of soluble proteins. Because of their amphiphilic character, membrane proteins need to be isolated, purified and crystallized in detergent solutions. This makes it difficult to grow the well-ordered three-dimensional crystals that are required for high resolution structure analysis by X-ray crystallography. In this difficult context, growing crystals confined to two dimensions (2D crystals) and their structural analysis by electron crystallography has opened a new way to solve the structure of membrane proteins. However, 2D crystallization is one of the major bottlenecks in the structural studies of membrane proteins. Advances in our understanding of the interaction between proteins, lipids and detergents as well as development and improvement of new strategies will facilitate the success rate of 2D crystallization. This review deals with the various available strategies for obtaining 2D crystals from detergent-solubilized intrinsic membrane proteins. It gives an overview of the methods that have been applied and gives details and suggestions of the physical processes leading to the formation of the ordered arrays which may be of help for getting more proteins crystallized in a form suitable for high resolution structural analysis by electron crystallography.     

Global Network Analysis of Lipid-Raft-Related Proteins Reveals Their Centrality in the Network and Their Roles in Multiple Biological Processes

Lipid rafts are specialized cholesterol-enriched microdomains in the cell membrane. They have been known as a platform for protein-protein interactions and to take part in multiple biological processes. Nevertheless, how lipid rafts influence protein properties at the proteomic level is still an open question for researchers using traditional biochemical approaches. Here, by annotating the lipid raft localization of proteins in human protein-protein interaction networks, we performed a systematic analysis of the function of proteins related to lipid rafts. Our results demonstrated that lipid raft proteins and their interactions were critical for the structure and stability of the whole network, and that the interactions between them were significantly enriched. Furthermore, for each protein in the network, we calculated its “lipid raft dependency (LRD),” which indicates how close it is topologically associated with lipid rafts, and we then uncovered the connection between LRD and protein functions. Proteins with high LRD tended to be essential for mammalian development, and malfunction of these proteins was inclined to cause human diseases. Coordinated with their neighbors, high-LRD proteins participated in multiple biological processes and targeted many pathways in diseases pathogenesis. High-LRD proteins were also found to have tissue specificity of expression. In summary, our network-based analysis denotes that lipid raft proteins have higher centrality in the network, and that lipid-raft-related proteins have multiple functions and are probably concerned with many biological processes in disease development.     

Seeking the determinants of the elusive functions of Sco proteins

Sco proteins are present in all types of organisms, including the vast majority of eukaryotes and many prokaryotes. It is well established that Sco proteins in eukaryotes are involved in the assembly of the Cu(A) cofactor of mitochondrial cytochrome c oxidase; however their precise role in this process has not yet been elucidated at the molecular level. In particular, some but not all eukaryotes including humans possess two Sco proteins whose individual functions remain unclear. There is evidence that eukaryotic Sco proteins are also implicated in other cellular processes such as redox signalling and regulation of copper homeostasis. The range of physiological functions of Sco proteins appears to be even wider in prokaryotes, where Sco-encoding genes have been duplicated many times during evolution. While some prokaryotic Sco proteins are required for the biosynthesis of cytochrome c oxidase, others are most likely to take part in different processes such as copper delivery to other enzymes and protection against oxidative stress. The detailed understanding of the multiplicity of roles ascribed to Sco proteins requires the identification of the subtle determinants that modulate the two properties central to their known and potential functions, i.e. copper binding and redox properties. In this review, we provide a comprehensive summary of the current knowledge on Sco proteins gained by genetic, structural and functional studies on both eukaryotic and prokaryotic homologues, and propose some hints to unveil the elusive molecular mechanisms underlying their functions.     

Cytoskeleton and plant organogenesis

The functions of microtubules and actin filaments during various processes that are essential for the growth, reproduction and survival of single plant cells have been well characterized. A large number of plant structural cytoskeletal or cytoskeleton-associated proteins, as well as genes encoding such proteins, have been identified. Although many of these genes and proteins have been partially characterized with respect to their functions, a coherent picture of how they interact to execute cytoskeletal functions in plant cells has yet to emerge. Cytoskeleton-controlled cellular processes are expected to play crucial roles during plant cell differentiation and organogenesis, but what exactly these roles are has only been investigated in a limited number of studies in the whole plant context. The intent of this review is to discuss the results of these studies in the light of what is known about the cellular functions of the plant cytoskeleton, and about the proteins and genes that are required for them. Directions are outlined for future work to advance our understanding of how the cytoskeleton contributes to plant organogenesis and development.     

GTP-Binding Regulatory Proteins in Higher Plant Cells

The exsitence of GTP-binding regulatory proteins (for short term, often refered as G-proteins) in higher plant cells is certain. G-proteins are classified into two groups based on their molecular structures, which are the heterotrimeric G-proteins (big G-proteins) that contain three different subunits and the small G-proteins that have only one subunit (monomeric G-proteins). All G-proteins are characterized by their properties to bind with and hydrolyze GTP, by which G-proteins function as transmembrane and intracellular signalling molecules. As a distinguished participant in signal transduction, G-proteins directly and/or indirectly regulate a number of physiological processes, such as regulation of phytochrome-related physiological processes and gene expression, involvement in blue-light response, K+-channel regulation, stomatal movement, hormone regulation, protein phosphrylation dephosphorylation, etc. Although G-proteins in plant cells have not been purified, the genes for a subunit of heterotrimeric G-proteins have been cloned. More evidences for the importance of G-proteins in plant signalling processes are rapidly accumulating.     

A functional genomic yeast screen to identify pathogenic bacterial proteins

Many bacterial pathogens promote infection and cause disease by directly injecting into host cells proteins that manipulate eukaryotic cellular processes. Identification of these translocated proteins is essential to understanding pathogenesis. Yet, their identification remains limited. This, in part, is due to their general sequence uniqueness, which confounds homology-based identification by comparative genomic methods. In addition, their absence often does not result in phenotypes in virulence assays limiting functional genetic screens. Translocated proteins have been observed to confer toxic phenotypes when expressed in the yeast Saccharomyces cerevisiae. This observation suggests that yeast growth inhibition can be used as an indicator of protein translocation in functional genomic screens. However, limited information is available regarding the behavior of non-translocated proteins in yeast. We developed a semi-automated quantitative assay to monitor the growth of hundreds of yeast strains in parallel. We observed that expression of half of the 19 Shigella translocated proteins tested but almost none of the 20 non-translocated Shigella proteins nor approximately 1,000 Francisella tularensis proteins significantly inhibited yeast growth. Not only does this study establish that yeast growth inhibition is a sensitive and specific indicator of translocated proteins, but we also identified a new substrate of the Shigella type III secretion system (TTSS), IpaJ, previously missed by other experimental approaches. In those cases where the mechanisms of action of the translocated proteins are known, significant yeast growth inhibition correlated with the targeting of conserved cellular processes. By providing positive rather than negative indication of activity our assay complements existing approaches for identification of translocated proteins. In addition, because this assay only requires genomic DNA it is particularly valuable for studying pathogens that are difficult to genetically manipulate or dangerous to culture.     

Spatial cycles in G‐protein crowd control

The nature of living systems and their apparent resilience to the second law of thermodynamics has been the subject of extensive investigation and imaginative speculation. The segregation and compartmentalization of proteins is one manifestation of this departure from equilibrium conditions; the effect of which is now beginning to be elucidated. This should not come as a surprise, as even a cursory inspection of cellular processes reveals the large amount of energetic cost borne to maintain cell‐scale patterns, separations and gradients of molecules. The G‐proteins, kinases, calcium‐responsive proteins have all been shown to contain reaction cycles that are inherently coupled to their signalling activities. G‐proteins represent an important and diverse toolset used by cells to generate cellular asymmetries. Many small G‐proteins in particular, are dynamically acylated to modify their membrane affinities, or localized in an activity‐dependent manner, thus manipulating the mobility modes of these proteins beyond pure diffusion and leading to finely tuned steady state partitioning into cellular membranes. The rates of exchange of small G‐proteins over various compartments, as well as their steady state distributions enrich and diversify the landscape of possibilities that GTPase‐dependent signalling networks can display over cellular dimensions. The chemical manipulation of spatial cycles represents a new approach for the modulation of cellular signalling with potential therapeutic benefits.     

Effects of endogenous ligands on the biological role of human serum albumin in S-nitrosylation

Many proteins have been identified as targets for S-nitrosylation, including structural and signaling proteins, and ion channels. S-nitrosylation plays an important role in regulating their activity and function. We used human serum albumin (HSA), a major endogenous NO traffic protein, and studied the effect of mediators on S-nitrosylation processes which control NO bioactivity. By using NOC-7, S-nitrosoglutathione, and activated RAW264.7 cells as NO-donors we found that high-affinity binding of endogenous ligands (Cu²⁺, bilirubin and fatty acid) can affect these processes. It is likely that the same effects take place in many clinical situations characterized by increased fatty acid concentrations in plasma such as type II diabetes and the metabolic syndrome. Thus, endogenous ligands, changing their plasma concentrations, could be a novel type of mediator of S-nitrosylation not only in the case of HSA but also for other target proteins.     

Experimental and computational approaches for the study of calmodulin interactions

Ca²⁺, a universal messenger in eukaryotes, plays a major role in signaling pathways that control many growth and developmental processes in plants as well as their responses to various biotic and abiotic stresses. Cellular changes in Ca²⁺ in response to diverse signals are recognized by protein sensors that either have their activity modulated or that interact with other proteins and modulate their activity. Calmodulins (CaMs) and CaM-like proteins (CMLs) are Ca²⁺ sensors that have no enzymatic activity of their own but upon binding Ca²⁺ interact and modulate the activity of other proteins involved in a large number of plant processes. Protein-protein interactions play a key role in Ca²⁺/CaM-mediated in signaling pathways. In this review, using CaM as an example, we discuss various experimental approaches and computational tools to identify protein-protein interactions. During the last two decades hundreds of CaM-binding proteins in plants have been identified using a variety of approaches ranging from simple screening of expression libraries with labeled CaM to high-throughput screens using protein chips. However, the high-throughput methods have not been applied to the entire proteome of any plant system. Nevertheless, the data provided by these screens allows the development of computational tools to predict CaM-interacting proteins. Using all known binding sites of CaM, we developed a computational method that predicted over 700 high confidence CaM interactors in the Arabidopsis proteome. Most (>600) of these are not known to bind calmodulin, suggesting that there are likely many more CaM targets than previously known. Functional analyses of some of the experimentally identified Ca²⁺ sensor target proteins have uncovered their precise role in Ca²⁺-mediated processes. Further studies on identifying novel targets of CaM and CMLs and generating their interaction network - “calcium sensor interactome” - will help us in understanding how Ca²⁺ regulates a myriad of cellular and physiological processes.     

The ubiquitin-proteasome system in cardiac dysfunction

Since proteins play crucial roles in all biological processes, the finely tuned equilibrium between their synthesis and degradation regulates cellular homeostasis. Controlling the quality of proteome informational content is essential for cell survival and function. After initial synthesis, membrane and secretory proteins are modified, folded, and assembled in the endoplasmic reticulum, whereas other proteins are synthesized and processed in the cytosol. Cells have different protein quality control systems, the molecular chaperones, which help protein folding and stabilization, and the ubiquitin-proteasome system (UPS) and lysosomes, which degrade proteins. It has generally been assumed that UPS and lysosomes are regulated independently and serve distinct functions. The UPS degrades both cytosolic, nuclear proteins, and myofibrillar proteins, whereas the lysosomes degrade most membrane and extracellular proteins by endocytosis as well as cytosolic proteins and organelles via autophagy. Over the last two decades, the UPS has been increasingly recognized as a major system in several biological processes including cell proliferation, adaptation to stress and cell death. More recently, activation or impairment of the UPS has been reported in cardiac disease and recent evidence indicate that autophagy is a key mechanism to maintain cardiac structure and function. This review mainly focuses on the UPS and its various components in healthy and diseased heart, but also summarizes recent data suggesting parallel activation of the UPS and autophagy in cardiac disease.     

Fluorescent proteins as proteomic probes

Protein complexes mediate the majority of cellular processes. Knowledge of the localization and composition of such complexes provides key insights into their functions. Although green fluorescent protein (GFP) has been widely applied for in vivo visualization of proteins, it has been relatively little used as a tool for the isolation of protein complexes. Here we describe the use of the standard GFP tag to both visualize proteins in living cells and capture their interactions via a simple immunoaffinity purification procedure. We applied this method to the analysis of a variety of endogenous protein complexes from different eukaryotic cells. We show that efficient isolations can be achieved in 5-60 min. This rapid purification helps preserve protein complexes close to their original state in the cell and minimizes nonspecific interactions. Given the wide use and availability of GFP-tagged protein reagents, the present method should greatly facilitate the elucidation of many cellular processes.     

Identification of NaCl stress-responsive apoplastic proteins in rice shoot stems by 2D-DIGE

Plants have evolved sophisticated systems to cope with adverse environmental conditions such as cold, drought, and salinity. Although a number of stress response networks have been proposed, the role of plant apoplast in plant stress response has been ignored. To investigate the role of apoplastic proteins in the salt stress response, 10-day old rice plants were treated with 200mM NaCl for 1, 6 or 12h, and the soluble apoplast proteins of rice shoot stems were extracted for differential analysis, compared with untreated controls, by 2-D DIGE saturation labeling techniques. One hundred twenty-two significantly changed spots were identified by LC-MS/MS, and 117 spots representing 69 proteins have been identified. Of these proteins, 37 are apoplastic proteins according to the bioinformatic analysis. These proteins are mainly involved in the processes of carbohydrate metabolism, oxido-reduction, and protein processing and degradation. According to their functional categories and cluster analysis, a stress response model of apoplastic proteins has been proposed. These data indicate that the apoplast is important in plant stress signal reception and response.     

The molecular bases for copper uptake and distribution: lessons from yeast

Copper exists in two oxidation states, cuprous (Cu1+) and cupric (Cu2+), which, respectively, can donate or accept electrons. The fact that copper has two readily interconvertible redox states makes it a catalytic co-factor for many important enzymes. Over the past years, work in a number of laboratories has clearly demonstrated that studies in yeast have served as a springboard for identifying cellular components and processes involved in copper uptake and distribution. In several cases, it has been shown that mammalian proteins are capable of functionally replacing yeast proteins, thereby revealing their remarkable functional conservation. For high-affinity copper transport into cells, it has been shown that copper transporters of the Ctr family are required. Upon entering the cell, copper is partitioned to different proteins and into different compartments within the cell. Given the potential toxicity of copper, specialized proteins bind copper after it enters the cell and subsequently donate the bound copper to their corresponding recipient proteins. Three copper-binding proteins, Ccs1, Cox17, and Atx1, have been identified that serve as "copper chaperones" to deliver copper. double dagger.