Hydrophilic residues 526 KNDAAD 531 in the flexible C-terminal region of the chaperonin GroEL are critical for substrate protein folding within the central cavity

The final 23 residues in the C-terminal region of Escherichia coli GroEL are invisible in crystallographic analyses due to high flexibility. To probe the functional role of these residues in the chaperonin mechanism, we generated and characterized C-terminal truncated, double ring, and single ring mutants of GroEL. The ability to assist the refolding of substrate proteins rhodanese and malate dehydrogenase decreased suddenly when 23 amino acids were truncated, indicating that a sudden change in the environment within the central cavity had occurred. From further experiments and analyses of the hydropathy of the C-terminal region, we focused on the hydrophilicity of the sequence region (26 KNDAAD 531 and generated two GroEL mutants where these residues were changed to a neutral hydropathy sequence (526 GGGAAG 531) and a hydrophobic sequence (526 IGIAAI 531), respectively. Very interestingly, the two mutants were found to be defective in function both in vitro and in vivo. Deterioration of function was not observed in mutants where this region was replaced by a scrambled (526 NKADDA 531) or homologous (526 RQEGGE 531) sequence, indicating that the hydrophilicity of this sequence was important. These results highlight the importance of the hydrophilic nature of 526 KNDAAD 531 residues in the flexible C-terminal region for proper protein folding within the central cavity of GroEL.     

The V protein of mumps virus plays a critical role in pathogenesis

Mumps virus (MuV) causes an acute infection in humans characterized by a wide array of symptoms ranging from relatively mild manifestations, such as parotitis, to more-severe complications, such as meningitis and encephalitis. Widespread mumps vaccination has reduced mumps incidence dramatically; however, outbreaks still occur in vaccinated populations. The V protein of MuV, when expressed in cell culture, blocks interferon (IFN) expression and signaling and interleukin-6 (IL-6) signaling. In this work, we generated a recombinant MuV incapable of expressing the V protein (rMuVΔV). The rescued MuV was derived from a clinical wild-type isolate from a recent outbreak in the United States (MuV(Iowa/US/06), G genotype). Analysis of the virus confirmed the roles of V protein in blocking IFN expression and signaling and IL-6 signaling. We also found that the rMuV(Iowa/US/06)ΔV virus induced high levels of IL-6 expression in vitro, suggesting that V plays a role in reducing IL-6 expression. In vivo, the rMuV(Iowa/US/06)ΔV virus was highly attenuated, indicating that the V protein plays an essential role in viral virulence.     

Topologies of a substrate protein bound to the chaperonin GroEL

The chaperonin GroEL assists polypeptide folding through sequential steps of binding nonnative protein in the central cavity of an open ring, via hydrophobic surfaces of its apical domains, followed by encapsulation in a hydrophilic cavity. To examine the binding state, we have classified a large data set of GroEL binary complexes with nonnative malate dehydrogenase (MDH), imaged by cryo-electron microscopy, to sort them into homogeneous subsets. The resulting electron density maps show MDH associated in several characteristic binding topologies either deep inside the cavity or at its inlet, contacting three to four consecutive GroEL apical domains. Consistent with visualization of bound polypeptide distributed over many parts of the central cavity, disulfide crosslinking could be carried out between a cysteine in a bound substrate protein and cysteines substituted anywhere inside GroEL. Finally, substrate binding induced adjustments in GroEL itself, observed mainly as clustering together of apical domains around sites of substrate binding.     

Atypical protein kinase C plays a critical role in protein transport from pre-Golgi intermediates

The small GTPase Rab2 requires atypical protein kinase C iota/lambda (PKCiota/lambda) kinase activity to promote vesicle budding from normal rat kidney cell microsomes (Tisdale, E. J. (2000) Traffic 1, 702-712). The released vesicles lack anterograde-directed cargo but contain coat protein I (COPI) and the recycling protein p53/p58, suggesting that the vesicles traffic in the retrograde pathway. In this study, we have directly characterized the role of PKCiota/lambda in the early secretory pathway. A peptide corresponding to the unique PKCiota/lambda pseudosubstrate domain was introduced into an in vitro assay that efficiently reconstitutes transport of vesicular stomatitis virus glycoprotein from the endoplasmic reticulum to the cis-medial Golgi compartments. This peptide blocked transport in a dose-dependent manner. Moreover, normal rat kidney cells incubated with Rab2 and the pseudosubstrate peptide displayed abundant swollen or dilated vesicles that contained Rab2, PKCiota/lambda, beta-COP, and p53/p58. Because Rab2, beta-COP, and p53/p58 are marker proteins for pre-Golgi intermediates (vesicular tubular clusters,VTCs), most probably the swollen vesicles are derived from VTCs. Similar results were obtained when the assays were supplemented with kinase-dead PKCiota/lambda (W274K). Both the pseudosubstrate peptide and kinase-dead PKCiota/lambda in tandem with Rab2 caused sustained membrane association of PKCiota/lambda, suggesting that reverse translocation was inhibited. Importantly, the inhibitory phenotype of kinase-dead PKCiota/lambda was reversed by PKCiota/lambda wild type. These combined results indicate that PKCiota/lambda is essential for protein transport in the early secretory pathway and suggest that PKCiota/lambda kinase activity is required to promote Rab2-mediated vesicle budding at a VTC subcompartment enriched in recycling cargo.     

Endocytosis plays a critical role in proteolytic processing of the Hendra virus fusion protein

The Hendra virus fusion (F) protein is synthesized as a precursor protein, F(0), which is proteolytically processed to the mature form, F(1) + F(2). Unlike the case for the majority of paramyxovirus F proteins, the processing event is furin independent, does not require the addition of exogenous proteases, is not affected by reductions in intracellular Ca(2+), and is strongly affected by conditions that raise the intracellular pH (C. T. Pager, M. A. Wurth, and R. E. Dutch, J. Virol. 78:9154-9163, 2004). The Hendra virus F protein cytoplasmic tail contains a consensus motif for endocytosis, YXXPhi. To analyze the potential role of endocytosis in the processing and membrane fusion promotion of the Hendra virus F protein, mutation of tyrosine 525 to alanine (Hendra virus F Y525A) or phenylalanine (Hendra virus F Y525F) was performed. The rate of endocytosis of Hendra virus F Y525A was significantly reduced compared to that of the wild-type (wt) F protein, confirming the functional importance of the endocytosis motif. An intermediate level of endocytosis was observed for Hendra virus F Y525F. Surprisingly, dramatic reductions in the rate of proteolytic processing were observed for Hendra virus F Y525A, although initial transport to the cell surface was not affected. The levels of surface expression for both Hendra virus F Y525A and Hendra virus F Y525F were higher than that of the wt protein, and these mutants displayed enhanced syncytium formation. These results suggest that endocytosis is critically important for Hendra virus F protein cleavage, representing a new paradigm for proteolytic processing of paramyxovirus F proteins.     

The unfolding action of GroEL on a protein substrate

A molecular dynamics simulation of the active unfolding of denatured rhodanese by the chaperone GroEL is presented. The compact denatured protein is bound initially to the cis cavity and forms stable contacts with several of the subunits. As the cis ring apical domains of GroEL undergo the transition from the closed to the more open (ATP-bound) state, they exert a force on rhodanese that leads to the increased unfolding of certain loops. The contacts between GroEL and rhodanese are analyzed and their variation during the GroEL transition is shown. The major contacts, which give rise to the stretching force, are found to be similar to those observed in crystal structures of peptides bound to the apical domains. The results of the simulation show that multidomain interactions play an essential role, in accord with experiments. Implications of the results for mutation experiments and for the action of GroEL are discussed.     

Glycosylphosphatidylinositol-anchored high-density lipoprotein-binding protein 1 plays a critical role in the lipolytic processing of chylomicrons

The triglycerides in chylomicrons are hydrolyzed by lipoprotein lipase (LpL) along the luminal surface of the capillaries. However, the endothelial cell molecule that facilitates chylomicron processing by LpL has not yet been defined. Here, we show that glycosylphosphatidylinositol-anchored high-density lipoprotein-binding protein 1 (GPIHBP1) plays a critical role in the lipolytic processing of chylomicrons. Gpihbp1-deficient mice exhibit a striking accumulation of chylomicrons in the plasma, even on a low-fat diet, resulting in milky plasma and plasma triglyceride levels as high as 5000 mg/dl. Normally, Gpihbp1 is expressed highly in heart and adipose tissue, the same tissues that express high levels of LpL. In these tissues, GPIHBP1 is located on the luminal face of the capillary endothelium. Expression of GPIHBP1 in cultured cells confers the ability to bind both LpL and chylomicrons. These studies strongly suggest that GPIHBP1 is an important platform for the LpL-mediated processing of chylomicrons in capillaries.     

Epstein-Barr virus transforming protein LMP1 plays a critical role in virus production

The Epstein-Barr virus (EBV) latent membrane protein 1 (LMP1), which is critical for EBV-induced B-cell transformation, is also abundantly expressed during the lytic cycle of viral replication. However, the biological significance of this strong LMP1 induction remains unknown. We engineered a bacterial artificial chromosome clone containing the entire genome of Akata strain EBV to specifically disrupt the LMP1 gene. Akata cell clones harboring the episomes of LMP1-deleted EBV were established, and the effect of LMP1 loss on virus production was investigated. We found that the degree of viral DNA amplification and the expression levels of viral late gene products were unaffected by LMP1 loss, demonstrating that the LMP1-deleted EBV entered the lytic replication cycle as efficiently as the wild-type counterpart. This was confirmed by our electron microscopic observation that nucleocapsid formation inside nuclei occurred even in the absence of LMP1. By contrast, loss of LMP1 severely impaired virus release into culture supernatants, resulting in poor infection efficiency. The expression of truncated LMP1 in Akata cells harboring LMP1-deleted EBV rescued the virus release into the culture supernatant and the infectivity, and full-length LMP1 partially rescued the infectivity. These results indicate that inducible expression of LMP1 during the viral lytic cycle plays a critical role in virus production.     

Structural changes in GroEL effected by binding a denatured protein substrate

In the absence of nucleotides or cofactors, the Escherichia coli chaperonin GroEL binds select proteins in non-native conformations, such as denatured glutamine synthetase (GS) monomers, preventing their aggregation and spontaneous renaturation. The nature of the GroEL-GS complexes thus formed, specifically the effect on the conformation of the GroEL tetradecamer, has been examined by electron microscopy. We find that specimens of GroEL-GS are visibly heterogeneous, due to incomplete loading of GroEL with GS. Images contain particles indistinguishable from GroEL alone, and also those with consistent identifiable differences. Side-views of the modified particles reveal additional protein density at one end of the GroEL-GS complex, and end-views display chirality in the heptameric projection not seen in the unliganded GroEL. The coordinate appearance of these two projection differences suggests that binding of GS, as representative of a class of protein substrates, induces or stabilizes a conformation of GroEL that differs from the unliganded chaperonin. Three-dimensional reconstruction of the GroEL-GS complex reveals the location of the bound protein substrate, as well as complex conformational changes in GroEL itself, both cis and trans with respect to the bound GS. The most apparent structural alterations are inward movements of the apical domains of both GroEL heptamers, protrusion of the substrate protein from the cavity of the cis ring, and a narrowing of the unoccupied opening of the trans ring.     

A tyrosine-based signal plays a critical role in the targeting and function of adenovirus RIDalpha protein

Early region 3 genes of human adenoviruses contribute to the virus life cycle by altering the trafficking of cellular proteins involved in adaptive immunity and inflammatory responses. The ability of early region 3 genes to target specific molecules suggests that they could be used to curtail pathological processes associated with these molecules and treat human disease. However, this approach requires genetic dissection of the multiple functions attributed to early region 3 genes. The purpose of this study was to determine the role of targeting on the ability of the early region 3-encoded protein RIDalpha to downregulate the EGF receptor. A fusion protein between the RIDalpha cytoplasmic tail and glutathione S-transferase was used to isolate clathrin-associated adaptor 1 and adaptor 2 protein complexes from mammalian cells. Deletion and site-directed mutagenesis studies showed that residues 71-AYLRH of RIDalpha are necessary for in vitro binding to both adaptor complexes and that Tyr72 has an important role in these interactions. In addition, RIDalpha containing a Y72A point mutation accumulates in the trans-Golgi network and fails to downregulate the EGF receptor when it is introduced into mammalian cells as a transgene. Altogether, our data suggest a model where RIDalpha is trafficked directly from the trans-Golgi network to an endosomal compartment, where it intercepts EGF receptors undergoing constitutive recycling to the plasma membrane and redirects them to lysosomes.     

The conserved ubiquitin-like protein Hub1 plays a critical role in splicing in human cells

Different from canonical ubiquitin-like proteins, Hub1 does not form covalent conjugates with substrates but binds proteins noncovalently. In Socchoromyces cerevisioe, Hub1 associates with spUceosomes and mediates alternative splicing of SRCI, without affecting pre-mRNA splicing generaity. Human Hub1 is highty similar to its yeast homotog, but its cellular function remains largely unexplored. Here, we show that human Hub1 binds to the spliceosomal protein Snu66 as in yeast; however, unlike its 5. cerevisioe homolos, human Hub1 is essential for viability. Prolonged in vivo depletion of human Hub1 leads to various cellular defects, including splicing speckle abnormalities, partial nuclear retention of mRNAs, mitotic catastrophe, and consequently cell death by apoptosis. Early consequences of Hub1 depletion are severe splicing defects, however, only for specific splice sites leading to exon skipping and intron retention. Thus, the ubiquitin-iike protein Hub1 is not a canonlcal spliceosomal factor needed generally for splicing, but rather a modulator of spliceosome performance and facilitator of alternative splicing.     

Tyr-326 plays a critical role in controlling SecA-preprotein interaction

SecA is an essential ATP-dependent motor protein that interacts with the preprotein and translocon to drive protein translocation across the eubacterial plasma membrane. A region containing residues 267-340 has been proposed to comprise the preprotein binding site of Escherichia coli SecA. To elucidate the function of this region further, we isolated mutants using a combination of region-specific polymerase chain reaction (PCR) mutagenesis and a genetic and biochemical screening procedure. Although this region displayed considerable plasticity based on phylogenetic and genetic analysis, Tyr-326 was found to be critical for SecA function. secA mutants with non-conservative substitutions at Tyr-326 showed strong protein secretion defects in vivo and were completely defective for SecA-dependent translocation ATPase activity in vitro. The SecA-Y326 mutant proteins were normal in their membrane, SecYE and nucleotide-binding properties. However, they exhibited a reduced affinity for preprotein and were defective in preprotein release, as assessed by several biochemical assays. Our results indicate that the region containing Tyr-326 functions as a conformational response element to regulate the preprotein binding and release cycle of SecA.     

The AKT-mTOR pathway plays a critical role in the development of leiomyosarcomas

We analyzed the PI3K-AKT signaling cascade in a cohort of sarcomas and found a marked induction of insulin receptor substrate-2 (IRS2) and phosphorylated AKT and a concomitant upregulation of downstream effectors in most leiomyosarcomas. To determine the role of aberrant PI3K-AKT signaling in leiomyosarcoma pathogenesis, we genetically inactivated Pten in the smooth muscle cell lineage by cross-breeding Pten(loxP/loxP) mice with Tagln-cre mice. Mice carrying homozygous deletion of Pten alleles developed widespread smooth muscle cell hyperplasia and abdominal leiomyosarcomas, with a very rapid onset and elevated incidence (approximately 80%) compared to other animal models. Constitutive mTOR activation was restricted to the leiomyosarcomas, revealing the requirement for additional molecular events besides Pten loss. The rapamycin derivative everolimus substantially decelerated tumor growth on Tagln-cre/Pten(loxP/loxP) mice and prolonged their lifespan. Our data show a new and critical role for the AKT-mTOR pathway in smooth muscle transformation and leiomyosarcoma genesis, and support treatment of selected sarcomas by the targeting of this pathway with new compounds or combinations of these with conventional chemotherapy agents.     

Thiamine plays a critical role in the acid tolerance of Listeria monocytogenes

Understanding the molecular basis of acid tolerance in the food-borne pathogen Listeria monocytogenes is important as this property contributes to survival in the food-chain and enhances survival within infected hosts. The aim of this study was to identify genes contributing to acid tolerance in L.?monocytogenes using transposon mutagenesis and subsequently to elucidate the physiological role of these genes in acid tolerance. One mutant harboring a Tn917 insertion in the thiT gene (formerly lmo1429), which encodes a thiamine (vitamin B1) uptake system, was found to be highly sensitive to acid. The acid-sensitive phenotype associated with loss of this gene was confirmed with an independently isolated mutant, from which the thiT gene was deleted (?thiT). Cells of both wild-type and ?thiT mutant that were thiamine depleted were found to be significantly more acid sensitive than control cultures. Thiamine-depleted cultures failed to produce significant concentrations of acetoin, consistent with the known thiamine dependence of acetolactate synthase, an enzyme required for acetoin synthesis from pyruvate. As acetoin synthesis is a proton-consuming process, we suggest that the acid sensitivity observed in thiamine-depleted cultures may be owing to an inability to produce acetoin.     

Target DNA structure plays a critical role in RAG transposition

Antigen receptor gene rearrangements are initiated by the RAG1/2 protein complex, which recognizes specific DNA sequences termed RSS (recombination signal sequences). The RAG recombinase can also catalyze transposition: integration of a DNA segment bounded by RSS into an unrelated DNA target. For reasons that remain poorly understood, such events occur readily in vitro, but are rarely detected in vivo. Previous work showed that non-B DNA structures, particularly hairpins, stimulate transposition. Here we show that the sequence of the four nucleotides at a hairpin tip modulates transposition efficiency over a surprisingly wide (>100-fold) range. Some hairpin targets stimulate extraordinarily efficient transposition (up to 15%); one serves as a potent and specific transposition inhibitor, blocking capture of targets and destabilizing preformed target capture complexes. These findings suggest novel regulatory possibilities and may provide insight into the activities of other transposases.     

Chaperonin GroEL meets the substrate protein as a "load" of the rings

Chaperonin GroEL is an essential molecular chaperone that assists protein folding in the cell. With the aid of cochaperonin GroES and ATP, double ring-shaped GroEL encapsulates non-native substrate proteins inside the cavity of the GroEL-ES complex. Although extensive studies have revealed the outline of GroEL mechanism over the past decade, central questions remain: What are the in vivo substrate proteins? How does GroEL encapsulate the substrates inside the cavity in spite of an apparent entropic difficulty? Is the folding inside the GroEL-ES cavity the same as bulk spontaneous folding? In this review I summarize the recent progress on in vivo and in vitro aspects of GroEL. In particular, emerging evidence shows that the substrate protein itself influences the chaperonin GroEL structure and reaction cycle. Finally I propose the mechanistic similarity between GroEL and kinesin, a molecular motor that moves along a microtubule in an ATP-dependent manner.     

N-WASP plays a critical role in fibroblast adhesion and spreading

N-WASP (Neural Wiskott Aldrich Syndrome Protein) regulates actin polymerization by activating the Arp2/3 complex and promotes the formation of actin-rich structures such as filopodia. Such actin-rich structures play critical roles in cell adhesion and cell motility. Analysis of the adhesion properties of N-WASP^+/+ and N-WASP^−/− mouse embryonic fibroblasts to extracellular matrix proteins revealed that N-WASP is critical for cell adhesion to fibronectin. There was no significant difference in the localization of paxillin in the two cell lines, however the vinculin patches in WASP^+/+ cells were thicker and more prominent than those in N-WASP^−/− cells. The β1 integrins in N-WASP^+/+ cells were found in large clusters, while β1 integrins were more dispersed in N-WASP^−/− cells. The N-WASP^−/− cells migrated more rapidly than N-WASP^+/+ cells in a scratch migration assay. Thus, our data suggest that N-WASP deficiency leads to reduced adhesion to fibronectin and increased cell motility.     

Functional characterization of an archaeal GroEL/GroES chaperonin system: significance of substrate encapsulation

In all three kingdoms of life chaperonins assist the folding of a range of newly synthesized proteins. As shown recently, Archaea of the genus Methanosarcina contain both group I (GroEL/GroES) and group II (thermosome) chaperonins in the cytosol. Here we report on a detailed functional analysis of the archaeal GroEL/GroES system of Methanosarcina mazei (Mm) in comparison to its bacterial counterpart from Escherichia coli (Ec). We find that the groESgroEL operon of M. mazei is unable to functionally replace groESgroEL in E. coli. However, the MmGroES protein can largely complement a mutant EcGroES protein in vivo. The ATPase rate of MmGroEL is very low and the dissociation of MmGroES from MmGroEL is 15 times slower than for the EcGroEL/GroES system. This slow ATPase cycle results in a prolonged enclosure time for model substrate proteins, such as rhodanese, in the MmGroEL:GroES folding cage before their release into the medium. Interestingly, optimal functionality of MmGroEL/GroES and its ability to encapsulate larger proteins, such as malate dehydrogenase, requires the presence of ammonium sulfate in vitro. In the absence of ammonium sulfate, malate dehydrogenase fails to be encapsulated by GroES and rather cycles on and off the GroEL trans ring in a non-productive reaction. These results indicate that the archaeal GroEL/GroES system has preserved the basic encapsulation mechanism of bacterial GroEL and suggest that it has adjusted the length of its reaction cycle to the slower growth rates of Archaea. Additionally, the release of only the folded protein from the GroEL/GroES cage may prevent adverse interactions of the GroEL substrates with the thermosome, which is not normally located within the same compartment.     

FtsH11 protease plays a critical role in Arabidopsis thermotolerance

Plants, as sessile organisms, employ multiple mechanisms to adapt to the seasonal and daily temperature fluctuations associated with their habitats. Here, we provide genetic and physiological evidence that the FtsH11 protease of Arabidopsis contributes to the overall tolerance of the plant to elevated temperatures. To identify the various mechanisms of thermotolerance in plants, we isolated a series of Arabidopsis thaliana thermo-sensitive mutants (atts) that fail to acquire thermotolerance after pre-conditioning at 38 degrees C. Two allelic mutants, atts244 and atts405, were found to be both highly susceptible to moderately elevated temperatures and defective in acquired thermotolerance. The growth and development of the mutant plants at all stages examined were arrested after exposure to temperatures above 30 degrees C, which are permissive conditions for wild-type plants. The affected gene in atts244 was identified through map-based cloning and encodes a chloroplast targeted FtsH protease, FtsH11. The Arabidopsis genome contains 12 predicted FtsH protease genes, with all previously characterized FtsH genes playing roles in the alleviation of light stress through the degradation of unassembled thylakoid membrane proteins and photodamaged photosystem II D1 protein. Photosynthetic capability, as measured by chlorophyll content (chl a/b ratios) and PSII quantum yield, is greatly reduced in the leaves of FtsH11 mutants when exposed to the moderately high temperature of 30 degrees C. Under high light conditions, however, FtsH11 mutants and wild-type plants showed no significant difference in photosynthesis capacity. Our results support a direct role for the A. thaliana FtsH11-encoded protease in thermotolerance, a function previously reported for bacterial and yeast FtsH proteases but not for those from plants.