Introduction to the blood coagulation cascade and cloning of blood coagulation factors

The coagulation cascade that occurs in mammalian plasma involves a large number of plasma proteins that participate in a stepwise manner and eventually give rise to the formation of thrombin. This enzyme then converts fibrinogen to an insoluble fibrin clot. This series of reactions involves a number of glycoproteins that particupate as enzymes as well as cofactors. These proteins that circulate in the blood in a precursor or zymogen form are multifunctional proteins that share many common segments or domains. One group includes the vitamin K-dependent glycoproteins (prothrombin, factor IX, factor X, and protein C) that show considerable homology in both their amino acid sequences and their gene structures. The proteins that participate in the contact or early phase of the blood coagulation cascade include plasma prekallikrein, factor XII, and factor IX. The amino-terminal regions of both factor XI and plasma prekallikrein contain four tandem repeats of about 90 amino acids, and these tandem repeats show considerable amino acid sequence homology. Factor XII contains four different domains in the amino-terminai region of the protein, including a kringle structure, two growth factor domains, and type I and type II finger domains. The finger domains were first identified in fibronectin. The carboxyl-terminal portion of plasma prekallikrein, factor XII, and factor XI contains the serine or protease portion of the molecule. These various plasma proteins that share common domains appear to have evolved by gene shuffling that may have, in some cases, involved introns.     

Immune responses to stress proteins: Applications to infectious disease and cancer

Heat shock proteins, or stress proteins have been identified as part of a highly conserved cellular defence mechanism mediated by multiple, distinct gene familes and corresponding gene products. As intracellular chaperones, stress proteins participate in many essential biochemical pathways of protein maturation and function active during times of stress and during normal cellular homeostasis. In addition to their well-characterized role as protein chaperones, stress proteins are now realized to possess another important biological property: immunogenicity. Stress proteins are now understood to play a fundamental role in immune surveillance of infection and malignancy and this body of basic research has provided a framework for their clinical application. As key targets of both humoral and cellular immunity during infection, stress proteins have accordingly received considerable research interest as prophylactic vaccines for infectious disease applications. The unique and potent immunostimulatory properties of stress proteins have similarly been applied to the development of new approaches to cancer therapy, including both protein and gene-based modalities.     

Do caveolins regulate cells by actions outside of caveolae?

Caveolae (caveolin-containing lipid rafts) are plasma membrane domains that scaffold and organize a variety of important proteins in eukaryotic cells. Recent work shows that caveolins can act independently of caveolae, both in cells that lack caveolae (e.g. neurons and leukocytes) and in non-caveolar regions of cells that have caveolae (e.g. cardiac myocytes and fibroblasts). Phosphorylation of caveolins can influence the scaffolding of protein partners, and caveolins appear to participate in the protection and trafficking of proteins to and from the plasma membrane. Together, these results suggest that, despite their name, caveolins should now be thought of as proteins that scaffold signaling and other proteins in both caveolar and non-caveolar regions.     

Golgins and GRASPs: Holding the Golgi together

The GRASP and golgin families of proteins have emerged as key components of the Golgi apparatus, with major roles in both the structural organisation of this organelle and the trafficking that occurs there. Both types of protein participate in membrane tethering events that occur upstream of membrane fusion as well as contributing to the structural scaffold that defines Golgi architecture, referred to as the Golgi matrix. The importance of these proteins is highlighted by their targeting in mitosis, apoptosis, and pathogenic infections that cause dramatic structural and functional reorganisation of the Golgi apparatus. In this review we will discuss our current understanding of GRASP and golgin function, highlighting some of the common themes that have emerged as well as describing previously unsuspected roles for these proteins in various cellular processes.     

Visualizing protein movement on DNA at the single-molecule level using DNA curtains

A fundamental feature of many nucleic-acid binding proteins is their ability to move along DNA either by diffusion-based mechanisms or by ATP-hydrolysis driven translocation. For example, most site-specific DNA-binding proteins must diffuse to some extent along DNA to either find their target sites, or to otherwise fulfill their biological roles. Similarly, nucleic-acid translocases such as helicases and polymerases must move along DNA to fulfill their functions. In both instances, the proteins must also be capable of moving in crowded environments while navigating through DNA-bound obstacles. These types of behaviors can be challenging to analyze by bulk biochemical methods because of the transient nature of the interactions, and/or heterogeneity of the reaction intermediates. The advent of single-molecule methodologies has overcome some of these problems, and has led to many new insights into the mechanisms that contribute to protein motion along DNA. We have developed DNA curtains as a tool to facilitate single molecule observations of protein-nucleic acid interactions, and we have applied these new research tools to systems involving both diffusive-based motion as well as ATP directed translocation. Here we highlight these studies by first discussing how diffusion contributes to target searches by proteins involved in post-replicative mismatch repair. We then discuss DNA curtain assays of two different DNA translocases, RecBCD and FtsK, which participate in homologous DNA recombination and site-specific DNA recombination, respectively.     

Discovering high mobility group A molecular partners in tumour cells

DNA-based activities rely on an extremely coordinated sequence of events performed by several chromatin-associated proteins which act in concert. High Mobility Group A (HMGA) proteins are non-histone architectural nuclear factors that participate in the regulation of specific genes but they are also believed to have a more general role in chromatin dynamics. The peculiarity of these proteins is their flexibility, both in terms of DNA-binding and in protein-protein interactions. Since these proteins act as core elements in the assembly of multiprotein complexes called enhanceosomes, and have already displayed the ability to interact with several different proteins, we started a proteomic approach for the systematic identification of their molecular partners. By a combination of affinity chromatography, two-dimensional gel electrophoresis and mass spectrometry we have identified about twenty putative HMGA interactors which could be roughly assigned to three different classes: mRNA processing proteins, chromatin remodelling related factors and structural proteins. Direct HMGA interaction with some of these proteins was confirmed by glutathione-S-transferase pull-down assays and the HMGA domain involved was mapped. Blot-overlay experiments reveal that members of the HMGA family share most of their molecular partners but, interestingly, it seems that there are some cell-type specific partners. Taken together, these experimental data indicate that HMGA proteins are highly connected nodes in the chromatin protein network. Since these proteins are strongly implicated with cancer development, the identification of molecules able to perturb the HMGA molecular network could be a possible tool to interfere with their oncogenic activity.     

Translating G protein subunit diversity into functional specificity

Historically, it has been assumed that the functional roles of G proteins in receptor recognition and effector regulation are specified by their diverse alpha subunits. However, the discovery of similarly diverse betagamma subunits that participate in both of these functional processes has called this assumption into question; recent work suggests that G proteins function as heterotrimers whose roles in particular receptor signaling pathways are determined by their specific alphabetagamma subunit combinations. Although much remains to be learned, the assembly of specific alphabetagamma subunit combinations seems to involve both structural and spatial factors.     

The RGK family of GTP-binding proteins: regulators of voltage-dependent calcium channels and cytoskeleton remodeling

RGK proteins constitute a novel subfamily of small Ras-related proteins that function as potent inhibitors of voltage-dependent (VDCC) Ca(2+) channels and regulators of actin cytoskeletal dynamics. Within the larger Ras superfamily, RGK proteins have distinct regulatory and structural characteristics, including nonconservative amino acid substitutions within regions known to participate in nucleotide binding and hydrolysis and a C-terminal extension that contains conserved regulatory sites which control both subcellular localization and function. RGK GTPases interact with the VDCC beta-subunit (Ca(V)beta) and inhibit Rho/Rho kinase signaling to regulate VDCC activity and the cytoskeleton respectively. Binding of both calmodulin and 14-3-3 to RGK proteins, and regulation by phosphorylation controls cellular trafficking and the downstream signaling of RGK proteins, suggesting that a complex interplay between interacting protein factors and trafficking contribute to their regulation.     

Glucocorticoids and acute phase proteins

Glucocorticoids as well as acute phase proteins participate in non-specific host defence as well as in restoring host integrity after injury. Plasma levels of both compounds augment during the inflammatory reaction. However, glucocorticoids also have physiological effects that share similar molecular mechanisms with the family of steroids. During the inflammatory reaction, and for participating in host defense, glucocorticoids, together with augmented cytokines, use new signalling pathways. In doing so, they participate in the positive or negative control of inflammatory mediator synthesis. For example, they induce the synthesis of acute phase proteins in synergy with interleukin 6, interleukin 1 and TNF alpha.     

Do unconventional myosins exert functions in dynamics of membrane compartments?

Unconventional myosins have now been identified in amoeba as well as in higher eucaryotic cells. Their cellular localization, their ability to bind membrane vesicles and their ability to produce in vitro movement suggest that they can generate forces on the plasma membrane relative to actin filaments as well as on membrane compartments relative to actin. Genetic approaches and biochemical analysis of cells over-producing nonfunctional domains of unconventional myosins have provided direct evidence for a role of unconventional myosins in movement of intracellular vesicles and have allowed us to formulate hypotheses about the possible mechanisms by which unconventional myosins could participate in the intracellular transport of membrane proteins and secretory proteins.     

Recombinant interleukin-2 analogs. Dynamic probes for receptor structure.

Interleukin-2 (IL-2) and its receptor complex have become one of the most studied members of a growing family of protein hormones characterized by structural similarities in both ligands and their receptors. Structure-function studies of IL-2 have been complicated by the multimeric nature of its receptor. Two receptor subunits (55- and 75-kDa type I cell surface proteins) can participate to form the high affinity binding site. Although the IL-2 is apparently unique in some respects, similar subunit cooperativity has now been shown to be a common feature for other members of this receptor family. The availability of cell lines expressing the individual IL-2 receptor subunits has allowed detailed analysis of subunit binding characteristics. Results regarding the relationship of molecular recognition at each subunit to the mechanism of ligand binding at the high affinity site, however, have led to different interpretations. In this study we have employed previously prepared C-terminal IL-2 mutant proteins to examine receptor binding at all three classes using a variety of equilibrium and kinetic techniques. These results indicate that the high affinity IL-2 receptor complex includes the p55/p75 heterodimer prior to IL-2 binding and that both receptor subunits participate simultaneously in ligand capture.     

Drivers and passengers wanted! the role of kinesin-associated proteins

Members of the kinesin superfamily of proteins participate in a wide variety of cellular processes. Although much attention has been devoted to the structural and biophysical properties of the force-generating motor domain of kinesins, the factors controlling the functional specificity of each kinesin have only recently been examined. Genetic and biochemical approaches have identified two classes of proteins that associate physically with the diverse non-motor domains of kinesins. These proteins can be divided into two general classes: first, those that form tight complexes with the kinesin and are instrumental in directing the distinct function of the motor (i.e. drivers) and, second, those proteins that might transiently interact with the motor or be an integral part of the motor's cargo (i.e. passengers). Here, we discuss known kinesin-binding proteins, and how they might participate in the activity of their motor partners.     

Proteins in the chlamydial inclusion membrane

The chlamydiae are obligate intracellular pathogens that occupy a nonacidified vacuole (the inclusion) during their entire developmental cycle. Several proteins have recently been identified that are localized to the inclusion membrane. The following is a discussion of how inclusion membrane proteins might participate in the chlamydial developmental process.     

CARD games in apoptosis and immunity

A bewildering array of proteins containing the caspase recruitment domain (CARD) have now been identified. Previously, CARD-CARD interactions have been shown to be involved in the assembly of protein complexes that promote caspase processing and activation in the context of apoptosis. However, as the family of CARD-containing proteins has grown, it has become apparent that the majority of these proteins do not recruit caspases or promote caspase activation. Instead, many participate in NF-kappaB signalling pathways associated with innate or adaptive immune responses. Here, we suggest a simplified classification of the CARD proteins based upon their domain structures and discuss the divergent roles of these proteins in the context of host defence.     

Protein-water interactions in ribonuclease A and angiogenin: a molecular dynamics study

It is known that water molecules play an important role in the biological functioning of proteins. The members of the ribonuclease A (RNase A) family of proteins, which are sequentially and structurally similar, are known to carry out the obligatory function of cleaving RNA and individually perform other diverse biological functions. Our focus is on elucidating whether the sequence and structural similarity lead to common hydration patterns, what the common hydration sites are and what the differences are. Extensive molecular dynamics simulations followed by a detailed analysis of protein-water interactions have been carried out on two members of the ribonuclease A superfamily-RNase A and angiogenin. The water residence times are analyzed and their relationship with the characteristic properties of the protein polar atoms, such as their accessible surface area and mean hydration, is studied. The capacity of the polar atoms to form hydrogen bonds with water molecules and participate in protein-water networks are investigated. The locations of such networks are identified for both proteins.     

The ribosomal subunit assembly line

Recent proteomic studies in Saccharomyces cerevisiae have identified nearly 200 proteins, other than the structural ribosomal proteins, that participate in the assembly of ribosomal subunits and their transport from the nucleus. In a separate line of research, proteomic studies of mature plant ribosomes have revealed considerable variability in the protein composition of individual ribosomes.