Structural insights into the mechanism of intramolecular proteolysis.

A variety of proteins, including glycosylasparaginase, have recently been found to activate functions by self-catalyzed peptide bond rearrangements from single-chain precursors. Here we present the 1.9 A crystal structures of glycosylasparaginase precursors that are able to autoproteolyze via an N --> O acyl shift. Several conserved residues are aligned around the scissile peptide bond that is in a highly strained trans peptide bond configuration. The structure illustrates how a nucleophilic side chain may attack the scissile peptide bond at the immediate upstream backbone carbonyl and provides an understanding of the structural basis for peptide bond cleavage via an N --> O or N --> S acyl shift that is used by various groups of intramolecular autoprocessing proteins.     

Recent insights into poliovirus pathogenesis

The development of a mouse model for poliomyelitis that is transgenic for the human poliovirus receptor (hPVR) has made it much easier to investigate the efficiency of the viral dissemination process in a whole organism. These studies have given an insight into the mechanisms of blood-brain barrier permeation and neural transport. Strain-specific neurovirulence levels, however, appear to depend mainly on the replicating capacity of the virus in the central nervous system rather than the dissemination efficiency. Studies of the poliovirus-induced cytopathic effects on neural cells and specific subcellular localization of hPVR isoforms might determine a new course of investigation of poliovirus pathogenesis.     

Structural insights into the SNARE mechanism

Eukaryotic cells distribute materials among intracellular organelles and secrete into the extracellular space through cargo-loaded vesicles. A concluding step during vesicular transport is the fusion of a transport vesicle with a target membrane. SNARE proteins are essential for all vesicular fusion steps, thus they possibly comprise a conserved membrane fusion machinery. According to the "zipper" model, they assemble into stable membrane-bridging complexes that gradually bring membranes in juxtaposition. Hence, complex formation may provide the necessary energy for overcoming the repulsive forces between two membranes. During the last years, detailed structural and functional studies have extended the evidence that SNAREs are mostly in accord with the zipper model. Nevertheless, it remains unclear whether SNARE assembly between membranes directly leads to the merger of lipid bilayers.     

New insights into the import mechanism of the ferredoxin precursor into chloroplasts.

We have investigated the import pathway of the nuclear-encoded chloroplast protein ferredoxin. By using purified precursor protein and washed intact chloroplasts in a defined in vitro uptake system, we show that preferredoxin is fully import-competent by itself. In addition, we show also that the in vitro, in a wheat germ lysate, synthesized preferredoxin is not stably associated with another protein. Import is dependent only on ATP and does not require the presence of cytosolic proteins. Translocation could be largely stimulated by the thiol reducing agent dithiothreitol (DTT). To determine whether DTT acts on the precursor or on the chloroplast, we modified the 5 cysteines in the precursor by a reaction with iodoacetamide, thereby preventing the formation of disulfide bridges in the precursor. The import of this modified precursor was still stimulated by the addition of DTT, indicating that DTT had a stimulating effect on the chloroplast import machinery. In the case of the modified precursor, the import must have taken place without iron-sulfur cluster attachment in the stroma. The modified precursor could be imported with a similar efficiency as the parent precursor showing that import takes place independently from cofactor assembly.     

Recent insights into the actions of IGFBP-6

IGFBP-6 is an O-linked glycoprotein that preferentially binds IGF-II over IGF-I. It is a relatively selective inhibitor of IGF-II actions including proliferation, survival and differentiation of a wide range of cells. IGFBP-6 has recently been shown to have a number of IGF-independent actions, including promotion of apoptosis in some cells and inhibition of angiogenesis. IGFBP-6 also induces migration of tumour cells including rhabdomyosarcomas by an IGF-independent mechanism. This chemotactic effect is mediated by MAP kinases. IGFBP-6 binds to prohibitin-2 on the cell surface and the latter is required for IGFBP-6-induced migration by a mechanism that is independent of MAP kinases. IGFBP-6 may enter the nucleus and modulate cell survival and differentiation. IGFBP-6 expression is decreased in a number of cancer cells and it has been postulated to act as a tumour suppressor. IGFBP-6 expression is increased in a smaller number of cancers, which may reflect a compensatory mechanism to control IGF-II actions or IGF-independent actions. The relative balance of IGF-dependent and IGF-independent actions of IGFBP-6 in vivo together with the related question regarding the roles of IGFBP-6 binding to IGF and non-IGF ligands are keys to understanding the physiological role of this protein.     

Recent insights into the mechanisms of Chlamydia entry

Chlamydia are widespread bacteria that grow in human and animal cells. They enter their host cell, establish an intracellular environment favourable for their multiplication and finally exit the host cell. A combination of host cell factors and of bacterial proteins contribute to pathogen entry. Recent advances have shed new light on the entry mechanism, following attachment. Here we review recent data concerning endocytosis, host cell signalling, proteins secreted by the bacteria, the actin cytoskeleton in entry and the involvement of small GTPases.     

Recent insights into stearoyl-CoA desaturase-1

PURPOSE OF REVIEW: Stearoyl-Coenzyme A (CoA) desaturase is a central lipogenic enzyme catalyzing the synthesis of monounsaturated fatty acids - mainly oleate (C(18:1)). Oleate is the most abundant monounsaturated fatty acid in dietary fat and is therefore readily available. Why, then, is stearoyl-CoA desaturase a highly regulated enzyme? This review summarizes the recent and timely advances concerning the important role of stearoyl-CoA desaturase in metabolism. RECENT FINDINGS: Recent findings using mice that have a naturally occurring mutation in the SCD1 gene isoform as well as a mouse model with a targeted disruption of the stearoyl-CoA desaturase gene-1 (SCD1-/-) have revealed the role of de-novo synthesized oleate and thus the physiological importance of SCD1 expression. In the highlighted references, it is shown that the SCD1-/- mice have reduced body adiposity, increased insulin sensitivity, and are resistant to diet-induced obesity. The expression of several genes of lipid oxidation is upregulated, whereas lipid synthesis genes are downregulated. SCD1 was also found to be a component of the novel metabolic response to the hormone leptin. SUMMARY: SCD1, therefore, appears to be an important metabolic control point, and inhibition of its expression could be of benefit for the treatment of obesity, diabetes and other metabolic diseases.     

Recent insights into the biological roles of mucin-type O-glycosylation

In this special issue of the Glycoconjugate Journal focusing on glycosciences and development, we summarize recent advances in our understanding of the role of mucin-type O-glycans in development and disease. The presence of this widespread protein modification has been known for decades, yet identification of its biological functions has been hampered by the redundancy and complexity of the enzyme family controlling the initiation of O-glycosylation, as well as the diversity of extensions of the core sugar. Recent studies in organisms as diverse as mammals and Drosophila have yielded insights into the function of this highly abundant and evolutionarily-conserved protein modification. Gaining an understanding of mucin-type O-glycans in these diverse systems will elucidate crucial conserved processes underlying many aspects of development and homeostasis.     

Structural insights into the mechanism of PEPCK catalysis

Phosphoenolpyruvate carboxykinase catalyzes the reversible decarboxylation of oxaloacetic acid with the concomitant transfer of the gamma-phosphate of GTP to form PEP and GDP as the first committed step of gluconeogenesis and glyceroneogenesis. The three structures of the mitochondrial isoform of PEPCK reported are complexed with Mn2+, Mn2+-PEP, or Mn2+-malonate-Mn2+ GDP and provide the first observations of the structure of the mitochondrial isoform and insight into the mechanism of catalysis mediated by this enzyme. The structures show the involvement of the hyper-reactive cysteine (C307) in the coordination of the active site Mn2+. Upon formation of the PEPCK-Mn2+-PEP or PEPCK-Mn2+-malonate-Mn2+ GDP complexes, C307 coordination is lost as the P-loop in which it resides adopts a different conformation. The structures suggest that stabilization of the cysteine-coordinated metal geometry holds the enzyme as a catalytically incompetent metal complex and may represent a previously unappreciated mechanism of regulation. A third conformation of the mobile P-loop in the PEPCK-Mn2+-malonate-Mn2+ GDP complex demonstrates the participation of a previously unrecognized, conserved serine residue (S305) in mediating phosphoryl transfer. The ordering of the mobile active site lid in the PEPCK-Mn2+-malonate-Mn2+ GDP complex yields the first observation of this structural feature and provides additional insight into the mechanism of phosphoryl transfer.     

Molecular insights into the antifungal mechanism of bacilysin

Bacilysin is one of the simplest antimicrobial peptides and has drawn great attention for its excellent performance against Candida albicans. In this study, the antifungal mechanism of bacilysin was investigated. The target enzyme glucosamine-6-phosphate synthase (GFA) was expressed heterologously in Escherichia coli and its inhibition by bacilysin and derivatives was studied. It was concluded that bacilysin could be hydrolyzed by a proteinase of C. albicans, and that the released product, anticapsin, then inhibited the aminotransferase activity of GFA. This result was verified by molecular simulation, and the interaction mode of anticapsin with GFA was detailed, which provides data for the development of novel antifungal drugs. Transport of bacilysin into fungal cells was also simulated and it was shown that bacilysin is more readily transported into cells than anticapsin. Thus, our findings support a mechanism whereby bacilysin is transported into fungal pathogens, hydrolyzed to anticapsin, which then inhibits GFA.     

New insights into the mechanism of protein-protein association

Association of a protein complex follows a two-step mechanism, with the first step being the formation of an encounter complex that evolves into the final complex. Here, we analyze recent experimental data of the association of TEM1-beta-lactamase with BLIP using theoretical calculations and simulation. We show that the calculated Debye-Hückel energy of interaction for a pair of proteins during association resembles an energy funnel, with the final complex at the minima. All attraction is lost at inter-protein distances of 20 A, or rotation angles of >60 degrees from the orientation of the final complex. For faster-associating protein complexes, the energy funnel deepens and its volume increases. Mutations with the largest impact on association (hotspots for association) have the largest effect on the size and depth of the energy funnel. Analyzing existing evidence, we suggest that the transition state along the association pathway is the formation of the final complex from the encounter complex. Consequently, pairs of proteins forming an encounter complex will tend to dissociate more readily than to evolve into the final complex. Increasing directional diffusion by increasing favorable electrostatic attraction results in a faster forming and slower dissociating encounter complex. The possible applicability of electrostatic calculations for protein-protein docking is discussed.     

Theoretical insights into the AIE mechanism of assembles

Fluorophores in aggregated state are commonly used in optoelectronic devices, and the molecular packing are complex and diverse, including crystal, amorphous aggregate in solution, thin film, ordered supramolecular assemblies, and highly ordered cell membrane. In addition, the luminous behavior of the aggregated state can be precisely regulated by external stimuli such as hydrostatic pressure. In this review, we summarize the representative progress on the application of multiscale modeling protocol to illustrate the underlying mechanism of fluorescent emission of organic dyes in different assembles. The aim is to obtain the molecular packing in different forms of assembles and then to understand their underlying mechanism of stimuli-responsive fluorescent behavior at the molecular level. This is essential for the rational design, synthesis, and efficient application of fluorescent dyes.     

Structural insights into the mechanism of protein O-fucosylation

Protein O-fucosylation is an essential post-translational modification, involved in the folding of target proteins and in the role of these target proteins during embryonic development and adult tissue homeostasis, among other things. Two different enzymes are responsible for this modification, Protein O-fucosyltransferase 1 and 2 (POFUT1 and POFUT2, respectively). Both proteins have been characterised biologically and enzymatically but nothing is known at the molecular or structural level. Here we describe the first crystal structure of a catalytically functional POFUT1 in an apo-form and in complex with GDP-fucose and GDP. The enzyme belongs to the GT-B family and is not dependent on manganese for activity. GDP-fucose/GDP is localised in a conserved cavity connected to a large solvent exposed pocket, which we show is the binding site of epidermal growth factor (EGF) repeats in the extracellular domain of the Notch Receptor. Through both mutational and kinetic studies we have identified which residues are involved in binding and catalysis and have determined that the Arg240 residue is a key catalytic residue. We also propose a novel S(N)1-like catalytic mechanism with formation of an intimate ion pair, in which the glycosidic bond is cleaved before the nucleophilic attack; and theoretical calculations at a DFT (B3LYP/6-31+G(d,p) support this mechanism. Thus, the crystal structure together with our mutagenesis studies explain the molecular mechanism of POFUT1 and provide a new starting point for the design of functional inhibitors to this critical enzyme in the future.     

Recent insights into iron import by bacteria

Bacteria are confronted with a low availability of iron owing to its insolubility in the Fe3+ form or its being bound to host proteins. The bacteria cope with the iron deficiency by using host heme or siderophores synthesized by themselves or other microbes. In contrast to most other nutrients, iron compounds are tightly bound to proteins at the cell surfaces, from which they are further translocated by highly specific proteins across the cell wall of gram-positive bacteria and the outer membrane of gram-negative bacteria. Once heme and iron siderophores arrive at the cytoplasmic membrane, they are taken up across the cytoplasmic membrane by ABC transporters. Here we present an outline of bacterial heme and iron siderophore transport exemplified by a few selected cases in which recent progress in the understanding of the transport mechanisms has been achieved.