The Golgi complex: a hub of the secretory pathway

The Golgi complex plays a central role in protein secretion by regulating cargo sorting and trafficking. As these processes are of functional importance to cell polarity, motility, growth, and division, there is considerable interest in achieving a comprehensive understanding of Golgi complex biology. However, the unique stack structure of this organelle has been a major hurdle to our understanding of how proteins are secreted through the Golgi apparatus. Herein, we summarize available relevant research to gain an understanding of protein secretion via the Golgi complex. This includes the molecular mechanisms of intra-Golgi trafficking and cargo export in the trans-Golgi network. Moreover, we review recent insights on signaling pathways regulated by the Golgi complex and their physiological significance.     

Morphogenetic surfactants and their role in the formation of aerial hyphae in Streptomyces coelicolor

Withstanding environmental adversity and seeking optimal conditions for reproduction are basic requirements for the survival of all organisms. Filamentous bacteria of the genus Streptomyces produce a remarkable cell type called the aerial hyphae that is central to its ability to meet both of these challenges. Recent advances have brought about a major shift in our understanding of the cell surface proteins that play important roles in the generation of these cells. Here we review our current understanding of one of these groups of proteins, the morphogenetic surfactants, with emphasis on the SapB protein of Streptomyces coelicolor.     

Mammalian target of rapamycin: master regulator of cell growth in the nervous system

The mammalian target of rapamycin (mTOR) is a highly conserved serine/threonine protein kinase that regulates a number of diverse biologic processes important for cell growth and proliferation, including ribosomal biogenesis and protein translation. In this regard, hyperactivation of the mTOR signaling pathway has been demonstrated in numerous human cancers, including a number of inherited cancer syndromes in which individuals have an increased risk of developing benign and malignant tumors. Three of these inherited cancer syndromes (Lhermitte-Duclos disease, neurofibromatosis type 1, and tuberous sclerosis complex) are characterized by significant central nervous system dysfunction and brain tumor formation. Each of these disorders is caused by a genetic mutation that disrupts the expression of proteins which negatively regulate mTOR signaling, indicating that the mTOR signaling pathway is critical for appropriate brain development and function. In this review, we discuss our current understanding of the mTOR signaling pathway and its role in promoting ribosome biogenesis and cell growth. We suggest that studies of this pathway may prove useful in identifying molecular targets for biologically-based therapies of brain tumors associated with these inherited cancer syndromes as well as sporadic central nervous system tumors.     

Mechanism of the eukaryotic chaperonin: protein folding in the chamber of secrets

Chaperonins are key components of the cellular chaperone machinery. These large, cylindrical complexes contain a central cavity that binds to unfolded polypeptides and sequesters them from the cellular environment. Substrate folding then occurs in this central cavity in an ATP-dependent manner. The eukaryotic chaperonin TCP-1 ring complex (TRiC, also called CCT) is indispensable for cell survival because the folding of an essential subset of cytosolic proteins requires TRiC, and this function cannot be substituted by other chaperones. This specificity indicates that TRiC has evolved structural and mechanistic features that distinguish it from other chaperones. Although knowledge of this unique complex is in its infancy, we review recent advances that open the way to understanding the secrets of its folding chamber.     

Molecular screens for inner ear genes

Identification of the genes that encode proteins that are important for proper function of specific inner ear cell types is central to our understanding of the molecular basis of hearing and balance. Whereas the combination of electrophysiology and biophysics has resulted in an exquisite understanding of inner ear function, little is known about the proteins that confer these properties at the cellular level. Furthermore, the genes that control inner ear development, susceptibility to wear and tear, regeneration from damage, and age-related degeneration, are largely unknown. This review discusses tools that have been developed during the past few years to address this imbalance between a thorough physiologic characterization of inner ear function and a detailed understanding at a molecular level of the proteins involved in these functions. Creation of inner ear cDNA libraries has laid the foundation for the discovery of genes that are specifically expressed by cell types of the inner ear and that encode proteins that are important for molecular processes in these cells. In conjunction with expressed sequence tag database analysis, cDNA subtraction, and DNA arrays, functionally important genes, whose specific expression patterns are usually verified by gene expression analysis, can be identified. Discussion of these techniques takes into account the specific characteristics of the inner ear in relation to its study using molecular biological approaches.     

Cytoskeletal components of an invasion machine--the apical complex of Toxoplasma gondii

The apical complex of Toxoplasma gondii is widely believed to serve essential functions in both invasion of its host cells (including human cells), and in replication of the parasite. The understanding of apical complex function, the basis for its novel structure, and the mechanism for its motility are greatly impeded by lack of knowledge of its molecular composition. We have partially purified the conoid/apical complex, identified approximately 200 proteins that represent 70% of its cytoskeletal protein components, characterized seven novel proteins, and determined the sequence of recruitment of five of these proteins into the cytoskeleton during cell division. Our results provide new markers for the different subcompartments within the apical complex, and revealed previously unknown cellular compartments, which facilitate our understanding of how the invasion machinery is built. Surprisingly, the extreme apical and extreme basal structures of this highly polarized cell originate in the same location and at the same time very early during parasite replication.     

Cellulose biosynthesis and deposition in higher plants

The plant cell wall is central to plant development. Cellulose is a major component of plant cell walls, and is the world's most abundant biopolymer. Cellulose contains apparently simple linear chains of glucose residues, but these chains aggregate to form immensely strong microfibrils. It is the physical properties of these microfibrils that, when laid down in an organized manner, are responsible for both oriented cell elongation during plant growth and the strength required to maintain an upright growth habit. Despite the importance of cellulose, only recently have we started to unravel details of its synthesis. Mutational analysis has allowed us to identify some of the proteins involved in its synthesis at the plasma membrane, and to define a set of cellulose synthase enzymes essential for cellulose synthesis. These proteins are organized into a very large plasma membrane-localized protein complex. The way in which this protein complex is regulated and directed is central in depositing cellulose microfibrils in the wall in the correct orientation, which is essential for directional cell growth. Recent developments have given us clues as to how cellulose synthesis and deposition is regulated, an understanding of which is essential if we are to manipulate cell wall composition.     

The NADPH oxidase complex of phagocytic leukocytes: a biochemical and cytochemical view

The NADPH oxidase complex catalyzes the formation of superoxide (O₂ ^–) in phagocytic leukocytes. This paper reviews recent advances in our understanding of this enzyme system. Recent studies have defined conditions for reconstitution of this enzymatic activity with purified proteins in a cell-free system. The role of the individual proteins that make up the active complex, their regulation and the effects of mutations in these proteins are discussed. While these studies represent major achievements, it is clear from cytochemical investigations that additional levels of complexity exist in the modulation of the NADPH oxidase complex in vivo. A major role for cytochemical analysis in understanding the cell biological aspects of the generation of reactive oxygen species is discussed.Portions of this review were presented at the 36th Symposium of the Society for Histochemistry, 21 September 1994, Heidelberg, Germany     

The blueprint of the type-3 injectisome

Type-3 secretion systems are sophisticated syringe-like nanomachines present in many animal and plant Gram-negative pathogens. They are capable of translocating an arsenal of specific bacterial toxins (effector proteins) from the prokaryotic cytoplasm across the three biological membranes directly into the eukaryotic cytosol, some of which modulate host cell mechanisms for the benefit of the pathogen. They populate a particular biological niche, which is maintained by specific, pathogen-dependent effectors. In contrast, the needle complex, which is the central component of this specialized protein delivery machine, is structurally well-conserved. It is a large supramolecular cylindrical structure composed of multiple copies of a relatively small subset of proteins, is embedded in the bacterial membranes and protrudes from the pathogen's surface with a needle filament. A central channel traverses the entire needle complex, and serves as a hollow conduit for proteins destined to travel this secretion pathway. In the past few years, there has been a tremendous increase in an understanding on both the structural and the mechanistic level. This review will thus focus on new insights of this remarkable molecular machine.     

What can proteomic analyses contribute to understanding the molecular biology and clinical behavior of prostate cancer?

Identifying the proteins and their complex interactions that promote and/or sustain the aggressive malignant phenotype is essential for understanding key effectors of the molecular biology of prostate cancer. This is also essential for development of new clinical applications. A variety of proteomic techniques, ranging from mass spectrometry to new methods of multiplexing protein identification, have great potential for rapidly achieving these goals. However, in order to obtain meaningful results, these techniques must be applied within the context of our knowledge of the heterogeneity of prostate tissues and tumors, the impact of specimen processing on both the quality and quantity of proteins detected and a thorough understanding of prostate cell biology. Collaboration between the protein chemist and the prostate cell biologist will expedite progress in this important field.     

Tumor-induced perturbations of cytokines and immune cell networks

Until recently, the intrinsically high level of cross-talk between immune cells, the complexity of immune cell development, and the pleiotropic nature of cytokine signaling have hampered progress in understanding the mechanisms of immunosuppression by which tumor cells circumvent native and adaptive immune responses. One technology that has helped to shed light on this complex signaling network is the cytokine antibody array, which facilitates simultaneous screening of dozens to hundreds of secreted signal proteins in complex biological samples. The combined applications of traditional methods of molecular and cell biology with the high-content, high-throughput screening capabilities of cytokine antibody arrays and other multiplexed immunoassays have revealed a complex mechanism that involves multiple cytokine signals contributed not just by tumor cells but by stromal cells and a wide spectrum of immune cell types. This review will summarize the interactions among cancerous and immune cell types, as well as the key cytokine signals that are required for tumors to survive immunoediting in a dormant state or to grow and spread by escaping it. Additionally, it will present examples of how probing secreted cell–cell signal networks in the tumor microenvironment (TME) with cytokine screens have contributed to our current understanding of these processes and discuss the implications of this understanding to antitumor therapies.     

Caveolin forms a hetero-oligomeric protein complex that interacts with an apical GPI-linked protein: implications for the biogenesis of caveolae

Glycosyl-phosphatidylinositol (GPI)-linked proteins are transported to the apical surface of epithelial cells where they undergo cholesterol- dependent clustering in membrane micro-invaginations, termed caveolae or plasmalemmal vesicles. However, the sorting machinery responsible for this caveolar-clustering mechanism remains unknown. Using transfected MDCK cells as a model system, we have identified a complex of cell surface molecules (80, 50, 40, 22-24, and 14 kD) that interact in a pH- and cholesterol-dependent fashion with an apical recombinant GPI-linked protein. A major component of this hetero-oligomeric protein complex is caveolin, a type II transmembrane protein. As this hetero- oligomeric caveolin complex is detectable almost immediately after caveolin synthesis, our results suggest that caveolae may assemble intracellularly during transport to the cell surface. As such, our studies have implications for understanding both the intracellular biogenesis of caveolae and their subsequent interactions with GPI- linked proteins in epithelia and other cell types.     

DED or alive: assembly and regulation of the death effector domain complexes

Death effector domains (DEDs) are protein–protein interaction domains initially identified in proteins such as FADD, FLIP and caspase-8 involved in regulating apoptosis. Subsequently, these proteins have been shown to have important roles in regulating other forms of cell death, including necroptosis, and in regulating other important cellular processes, including autophagy and inflammation. Moreover, these proteins also have prominent roles in innate and adaptive immunity and during embryonic development. In this article, we review the various roles of DED-containing proteins and discuss recent developments in our understanding of DED complex formation and regulation. We also briefly discuss opportunities to therapeutically target DED complex formation in diseases such as cancer.     

Understand the Functions of Scaffold Proteins in Cell Signaling by a Mesoscopic Simulation Method

Scaffold proteins are central players in regulating the spatial-temporal organization of many important signaling pathways in cells. They offer physical platforms to downstream signaling proteins so that their transient interactions in a crowded and heterogeneous environment of cytosol can be greatly facilitated. However, most scaffold proteins tend to simultaneously bind more than one signaling molecule, which leads to the spatial assembly of multimeric protein complexes. The kinetics of these protein oligomerizations are difficult to quantify by traditional experimental approaches. To understand the functions of scaffold proteins in cell signaling, we developed a, to our knowledge, new hybrid simulation algorithm in which both spatial organization and binding kinetics of proteins were implemented. We applied this new technique to a simple network system that contains three molecules. One molecule in the network is a scaffold protein, whereas the other two are its binding targets in the downstream signaling pathway. Each of the three molecules in the system contains two binding motifs that can interact with each other and are connected by a flexible linker. By applying the new simulation method to the model, we show that the scaffold proteins will promote not only thermodynamics but also kinetics of cell signaling given the premise that the interaction between the two signaling molecules is transient. Moreover, by changing the flexibility of the linker between two binding motifs, our results suggest that the conformational fluctuations in a scaffold protein play a positive role in recruiting downstream signaling molecules. In summary, this study showcases the capability of computational simulation in understanding the general principles of scaffold protein functions.     

Regulation of integrin activity and signalling

The ability of cells to attach to each other and to the extracellular matrix is of pivotal significance for the formation of functional organs and for the distribution of cells in the body. Several molecular families of proteins are involved in adhesion, and recent work has substantially improved our understanding of their structures and functions. Also, these molecules are now being targeted in the fight against disease. However, less is known about how their activity is regulated. It is apparent that among the different classes of adhesion molecules, the integrin family of adhesion receptors is unique in the sense that they constitute a large group of widely distributed receptors, they are unusually complex and most importantly their activities are strictly regulated from the inside of the cell. The activity regulation is achieved by a complex interplay of cytoskeletal proteins, protein kinases, phosphatases, small G proteins and adaptor proteins. Obviously, we are only in the beginning of our understanding of how the integrins function, but already now fascinating details have become apparent. Here, we describe recent progress in the field, concentrating mainly on mechanistical and structural studies of integrin regulation. Due to the large number of articles dealing with integrins, we focus on what we think are the most exciting and rewarding directions of contemporary research on cell adhesion and integrins.     

Immortalized neurons for the study of hypothalamic function

The hypothalamus is a vital part of the central nervous system: it harbors control systems implicated in regulation of a wide range of homeostatic processes, including energy balance and reproduction. Structurally, the hypothalamus is a complex neuroendocrine tissue composed of a multitude of unique neuronal cell types that express a number of neuromodulators, including hormones, classical neurotransmitters, and specific neuropeptides that play a critical role in mediating hypothalamic function. However, neuropeptide and receptor gene expression, second messenger activation, and electrophysiological and secretory properties of these hypothalamic neurons are not yet fully defined, primarily because the heterogeneity and complex neuronal architecture of the neuroendocrine hypothalamus make such studies challenging to perform in vivo. To circumvent this problem, our research group recently generated embryonic- and adult-derived hypothalamic neuronal cell models by utilizing the novel molecular techniques of ciliary neurotrophic factor-induced neurogenesis and SV40 T antigen transfer to primary hypothalamic neuronal cell cultures. Significant research with these cell lines has demonstrated their value as a potential tool for use in molecular genetic analysis of hypothalamic neuronal function. Insights gained from hypothalamic immortalized cells used in conjunction with in vivo models will enhance our understanding of hypothalamic functions such as neurogenesis, neuronal plasticity, glucose sensing, energy homeostasis, circadian rhythms, and reproduction. This review discusses the generation and use of hypothalamic cell models to study mechanisms underlying the function of individual hypothalamic neurons and to gain a more complete understanding of the overall physiology of the hypothalamus.     

Conformational selection in the recognition of phosphorylated substrates by the catalytic domain of human Pin1

Post-translational phosphorylation and the related conformational changes in signaling proteins are responsible for regulating a wide range of subcellular processes. Human Pin1 is central to many of these cell signaling pathways in normal and aberrant subcellular processes, catalyzing cis-trans isomerization of the peptide ω-bond in phosphorylated serine/threonine-proline motifs in many proteins. Pin1 has therefore been identified as a possible drug target in many diseases, including cancer and Alzheimer's. The effects of phosphorylation on Pin1 substrates, and the atomistic basis for Pin1 recognition and catalysis, are not well understood. Here, we determine the conformational consequences of phosphorylation on Pin1 substrate analogues and the mechanism of recognition by the catalytic domain of Pin1 using all-atom molecular dynamics simulations. We show that phosphorylation induces backbone conformational changes on the peptide substrate analogues. We also show that Pin1 recognizes specific conformations of its substrate by conformational selection. Furthermore, dynamical correlated motions in the free Pin1 enzyme are present in the enzyme of the enzyme-substrate complex when the substrate is in the transition state configuration, suggesting that these motions play significant roles during catalytic turnover. These results provide a detailed atomistic picture of the mechanism of Pin1 recognition that can be exploited for drug design purposes and further our understanding of the synergistic complexities of post-translational phosphorylation and cis-trans isomerization.