The immune system rapidly responds to intracellular infections by detecting MHC class I restricted T-cell epitopes presented on infected cells. It was originally thought that viral peptides are liberated during constitutive protein turnover, but this conflicts with the observation that viral epitopes are detected within minutes of their synthesis even when their source proteins exhibit half-lives of days. The DRiPs hypothesis proposes that epitopes derive from Defective Ribosomal Products (DRiPs), rather than degradation of mature protein products. One potential source of DRiPs is premature translation termination. If this is a major source of DRiPs, this should be reflected in positional bias towards the N-terminus. By contrast, if downstream initiation is a major source of DRiPs, there should be positional bias towards the C-terminus. Here, we systematically assessed positional bias of epitopes in viral antigens, exploiting the large set of data available in the Immune Epitope Database and Analysis Resource. We show a statistically significant degree of positional skewing among epitopes; epitopes from both ends of antigens tend to be under-represented. Centric-skewing correlates with a bias towards class I binding peptides being over-represented in the middle, in parallel with a higher degree of evolutionary conservation.
To defend the host from an infection, the immune system continuously scans cell surfaces for foreign objects. Specifically, a virus inside a cell exploits the host to make copies of its proteins; viral proteins are broken up into peptide fragments; and the fragments are displayed on the infected cell's surface, thereby allowing detection and cell-killing. How these peptide fragments for cell-surface presentation are generated remains unknown. An understanding of this step will lead to rational design of vaccines and insights into tumor immunosurveillance and autoimmunity. One possible mechanism is that the peptide fragments come from defective proteins missing either the beginning or end regions, which may result in a bias. Here, we analyzed locations of a large set of known viral epitopes, peptide fragments recognized by the immune system, within their proteins. We find that all regions of proteins are represented well by the immune system. However, there is a statistically significant bias in the central regions of proteins, which correlate with a pattern of conservation spanning the length of viral proteins. Our results suggest a combined effect of conservation and enhancement of immune responses through repeated exposures in shaping the distribution of known viral epitopes.
Defective Ribosomal Products (DRiPs) are a subset of rapidly degraded polypeptides that provide peptide ligands for MHC class I molecules. Here, I review recent progress in understanding DRiP biogenesis. These findings place DRiPs at the center of the MHC class I antigen processing pathway, linking immunosurveillance of viruses and tumors to mechanisms of specialized translation and cellular compartmentalization. DRiPs enable the immune system to rapidly and sensitively detect alterations in cellular gene expression.
The defective ribosomal product (DRiP) hypothesis of endogenous Ag processing posits that rapidly degraded forms of nascent proteins are a major source of peptide ligands for MHC class I molecules. Although there is broad experimental support for the DRiP hypothesis, careful kinetic analysis of the generation of defined peptide class I complexes has been limited to studies of recombinant vaccinia viruses expressing genes derived from other organisms. In this study, we show that insertion of the SIIN-FEKL peptide into the stalk of influenza A virus neuraminidase (NA) does not detectably modify NA folding, degradation, transport, or sp. act. when expressed in its natural context of influenza A virus infection. Using the 25-D1.16 mAb specific for Kb-SIINFEKL to precisely quantitate cell surface complexes by flow cytometry, we demonstrate that SIINFEKL is generated in complete lockstep with initiation and abrogation of NA biosynthesis in both L-Kb fibroblast cells and DC2.4 dendritic/monocyte cells. SIINFEKL presentation requires active proteasomes and TAP, consistent with its generation from a cytosolic DRiP pool. From the difference in the shutoff kinetics of Kb-SIINFEKL complex expression following protein synthesis versus proteasome inhibition, we estimate that the t1/2 of the biosynthetic source of NA peptide is ~5 min. These observations extend the relevance of the DRiP hypothesis to viral proteins generated in their natural context.
Following viral infection, cells rapidly present peptides from newly synthesized viral proteins on MHC class I molecules, likely from rapidly degraded forms of nascent proteins. The nature of these defective ribosomal products (DRiPs) remains largely undefined. Using inhibitors of RNA polymerase II that block influenza A virus neuraminidase (NA) mRNA export from the nucleus and inhibit cytoplasmic NA translation, we demonstrate a surprising disconnect between levels of NA translation and generation of SIINFEKL peptide genetically inserted into the NA stalk. A 33-fold reduction in NA expression is accompanied by only a 5-fold reduction in Kb-SIINFEKL complex cell-surface expression, resulting in a net 6-fold increase in the overall efficiency of Ag presentation. Although the proteasome inhibitor MG132 completely blocked Kb-SIINFEKL complex generation, we were unable to biochemically detect a MG132-dependent cohort of NA DRiPs relevant for Ag processing, suggesting that a minute population of DRiPs is a highly efficient source of antigenic peptides. These data support the idea that Ag processing uses compartmentalized translation, perhaps even in the nucleus itself, to increase the efficiency of the generation of class I peptide ligands.
It has been 15 years since we proposed the defective ribosomal product (DRiP) hypothesis to explain the rapid presentation of viral peptides by MHC class I molecules on the surface of infected cells. Here, we review the evidence for the contribution of DRiPs to antigen processing, pointing to the uncertainties regarding the physical nature of DRiPs, and emphasizing recent findings suggesting that peptide generation is a specialized process involving compartmentalized translation.
Antigen processing; MHC class I; Proteasome; Translation; Virus
CD8+ T cells rapidly recognize virus-infected cells due to the generation of antigenic peptides from defective ribosomal products (DRiPs) that are encoded by standard open reading frames (ORFs). New data now show that alternative reading frame (ARF) DRiPs can also induce robust CD8+ T cell responses. ARF-specific T cells control retroviral replication and select for viral escape in monkeys, providing the most compelling evidence to date for the biological relevance of ARF immunosurveillance.
Cytotoxic T cells detect intracellular pathogens by surveying peptide loaded MHC class I molecules (pMHC I) on the cell surface. Effective immune surveillance also requires infected cells to present pMHC I promptly before viral progeny can escape. Rapid pMHC I presentation apparently occurs because infected cells can synthesize and present peptides from antigenic precursors called defective ribosomal products (DRiPs). The molecular characteristics of DRiPs are not known.
Here, using a novel method for detecting antigenic precursors and proteolytic intermediates, we tracked the synthesis and processing of Epstein-Barr Virus encoded nuclear antigen 1 (EBNA1). We find that ribosomes initiated translation appropriately, but rapidly produced DRiPs representing ∼120 amino acid truncated EBNA1 polypeptides by premature termination. Moreover, specific sequences in EBNA1 mRNA strongly inhibited the generation of truncated DRiPs and pMHC I presentation.
Our results reveal the first characterization of virus DRiPs as truncated translation products. Furthermore, production of EBNA1-derived DRiPs is down-regulated in cells, possibly limiting the antigenicity of EBNA1.
In response to inflammatory stimulation, dendritic cells (DCs) have a remarkable pattern of differentiation (maturation) that exhibits specific mechanisms to control antigen processing and presentation. One of these mechanisms is the sorting of polyubiquitinated proteins in large cytosolic aggregates called dendritic cell aggresome-like induced structures (DALIS). DALIS formation and maintenance are tightly linked to protein synthesis. Here, we took advantage of an antibody recognizing the antibiotic puromycin to follow the fate of improperly translated proteins, also called defective ribosomal products (DRiPs). We demonstrate that DRiPs are rapidly stored and protected from degradation in DALIS. In addition, we show that DALIS contain the ubiquitin-activating enzyme E1, the ubiquitin-conjugating enzyme E225K, and the COOH terminus of Hsp70-interacting protein ubiquitin ligase. The accumulation of these enzymes in the central area of DALIS defines specific functional sites where initial DRiP incorporation and ubiquitination occur. Therefore, DCs are able to regulate DRiP degradation in response to pathogen-associated motifs, a capacity likely to be important for their immune functions.
DRiPs; DALIS; puromycin; dendritic cells; antigen processing
Here we show that cells expressing genes inserted into Semliki Forest virus (SFV) vectors generate a large fraction of defective ribosomal products (DRiPs) due to frequent initiation on downstream Met residues. In monopolizing the host cell translational machinery, SFV reduces levels of translation eukaryotic initiation factor 4E (eIF4E), diminishes phosphorylation of ribosome subunit S6, and phosphorylates translation initiation factor eIF2α. We show that the last event is required for SFV mistranslation of inserted genes. Downstream initiation is suppressed by fusing inserted genes with the open reading frame encoding the SFV capsid, demonstrating that one function of the capsid element is to enable ribosomes to initiate translation in the proper location. These results show that in modifying translation, viral vectors can unpredictably increase the generation of truncated polypeptides and thereby the DRiP fraction of inserted gene products, which can potentially affect their yield, therapeutic efficacy, and immunogenicity.
We have constructed and expressed a series of mutant influenza virus hemagglutinins, each containing a new consensus site for glycosylation in addition to the seven sites found on the wild-type protein. Oligosaccharide side chains were added with high efficiency at four of the five novel sites, located on areas of the protein's surface that are not normally shielded by carbohydrate. Investigations of the structure, intracellular transport, and biological activities of the mutant hemagglutinin molecules indicated that (a) supernumerary carbohydrate side chains can be used to shield or disrupt functional epitopes on the surface of hemagglutinin, and (b) the presence of an additional oligosaccharide may cause temperature-dependent defects in the transport of the glycoprotein. We discuss the addition of supernumerary oligosaccharides as a general tool for shielding chosen areas of the surface of proteins that enter or traverse the secretory pathway.
Peptide macrocycles are found in many biologically active natural products. Their versatility, resistance to proteolysis and ability to traverse membranes has made them desirable molecules. Although technologies exist to synthesize such compounds, the full extent of diversity found among natural macrocycles has yet to be achieved synthetically. Cyanobactins are ribosomal peptide macrocycles encompassing an extraordinarily diverse range of ring sizes, amino acids and chemical modifications. We report the structure, biochemical characterization and initial engineering of the PatG macrocyclase domain of Prochloron sp. from the patellamide pathway that catalyzes the macrocyclization of linear peptides. The enzyme contains insertions in the subtilisin fold to allow it to recognize a three-residue signature, bind substrate in a preorganized and unusual conformation, shield an acyl-enzyme intermediate from water and catalyze peptide bond formation. The ability to macrocyclize a broad range of nonactivated substrates has wide biotechnology applications.
Chemokine receptors are members of the G protein-coupled receptor (GPCR) family. CCR5 and CXCR4 act as co-receptors for human immunodeficiency virus (HIV) and several efforts have been made to develop ligands to inhibit HIV infection by blocking those receptors. Removal of chemokine receptors from the cell surface using polymorphisms or other means confers some levels of immunity against HIV infection. Up to now, very limited success has been obtained using ligand therapies so we explored potential avenues to regulate chemokine receptor expression at the plasma membrane. We identified a molecular chaperone, DRiP78, that interacts with both CXCR4 and CCR5, but not the heterodimer formed by these receptors. We further characterized the effects of DRiP78 on CCR5 function. We show that the molecular chaperone inhibits CCR5 localization to the plasma membrane. We identified the interaction region on the receptor, the F(x)6LL motif, and show that upon mutation of this motif the chaperone cannot interact with the receptor. We also show that DRiP78 is involved in the assembly of CCR5 chemokine signaling complex as a homodimer, as well as with the Gαi protein. Finally, modulation of DRiP78 levels will affect receptor functions, such as cell migration in cells that endogenously express CCR5. Our results demonstrate that modulation of the functions of a chaperone can affect signal transduction at the cell surface.
We previously reported that autophagy in tumor cells plays a critical role in cross-presentation of tumor antigens and that autophagosomes are efficient antigen carriers for cross-priming of tumor-reactive CD8+ T cells. Here we sought to characterize further the autophagosome-enriched vaccine named DRibble (DRiPs-containing blebs), derived from tumor cells after inhibition of protein degradation and provide insights into the mechanisms responsible for their efficacy as a novel cancer immunotherapy.
DRibbles were characterized by western blot and light or transmission electron microscopy. The efficiency of cross-presentation mediated by DRibbles was first compared with that of whole tumor cells and pure proteins. The mechanisms of antigen cross-presentation by DRibbles were analyzed and the anti-tumor efficacy of the DRibble vaccine was tested in 3LL Lewis lung tumors and B16F10 melanoma.
The DRibbles sequester both long-lived and short-lived proteins, including defective ribosomal products (DRiPs), as well as damage-associated molecular pattern (DAMP) molecules exemplified by HSP90, HSP94, calreticulin, and HMGB1. DRibbles express ligands for CLEC9A, a newly described C-type lectin receptor expressed by a subset of conventional DCs (cDCs) and plasmacytoid DCs (pDCs) and cross-presentation was partially CLEC9A-dependent. Furthermore, this autophagy assisted antigen cross presentation pathway involved both caveolae- and clathrin-mediated endocytosis and ERAD machinery. It depends on proteasome and TAP1, but lysosome functions of APCs. Importantly, DC loaded with autophagosome-enriched DRibbles can eradicate 3LL Lewis lung tumors and significantly delay the growth of B16F10 melanoma.
These data documented the unique characteristics and potent anti-tumor efficacy of the autophagosome-based DRibble vaccine. The efficacy of DRibble cancer vaccine will be further tested in clinical trials.
DRibble; autophagosome; cross-presentation; CLEC9A; tumor regression; cancer vaccine; immunotherapy
The transcriptional activity of the vitamin D receptor (VDR) is regulated by a number of coactivator and corepressor complexes, which bind to the VDR in a ligand (1,25(OH)2D3) dependent (coactivators) or inhibited (corepressors) process. In the keratinocyte the major coactivator complexes include the vitamin D interacting protein (DRIP) complex and the steroid receptor coactivator (SRC) complexes. These coactivator complexes are not interchangeable in their regulation of keratinocyte proliferation and differentiation. We found that the DRIP complex is the main complex binding to VDR in the proliferating keratinocyte, whereas SRC2 and 3 and their associated proteins are the major coactivators binding to VDR in the differentiated keratinocyte. Moreover, we have found a specific role for DRIP205 in the regulation of β-catenin pathways regulating keratinocyte proliferation, whereas SRC3 uniquely regulates the ability of 1,25(OH)2D3 to induce more differentiated functions such as lipid synthesis and processing required for permeability barrier formation and the innate immune response triggered by disruption of the barrier. These findings provide a basis by which we can understand how one receptor (VDR) and one ligand (1,25(OH)2D3) can regulate a large number of genes in a sequential and differentiation specific fashion.
Vitamin D receptor; coactivators; keratinocytes; differentiation; proliferation; β-catenin; innate immunity; permeability barrier
Ubiquitin-dependent proteolysis can initiate at ribosomes for myriad reasons including misfolding of a nascent chain or stalling of the ribosome during translation of mRNA. Clearance of a stalled complex is required to recycle the ribosome for future use. Here we show that the ubiquitin (Ub) pathway segregase Cdc48/p97 and its adaptors Ufd1-Npl4 participate in ribosome-associated degradation (RAD) by mediating the clearance of ubiquitinated, tRNA-linked nascent peptides from ribosomes. Through characterization of both endogenously-generated and heterologous model substrates for the RAD pathway, we conclude that budding yeast Cdc48 functions downstream of the Ub ligases Ltn1 and Ubr1 to release nascent proteins from the ribosome so that they can be degraded by the proteasome. Defective RAD could contribute to the pathophysiology of human diseases caused by mutations in p97.
Ribosomes are complex molecular machines that translate the sequence of bases in a messenger RNA (mRNA) transcript into a polypeptide that subsequently folds to form a protein. Each ribosome is composed of two major subunits: the small subunit reads the mRNA transcript, and the large subunit joins amino acids together to form the polypeptide. This process stops when the ribosome encounters a stop codon and releases the completed polypeptide.
It is critical that cells perform some form of quality control on the polypeptides as they are translated to prevent a build up of incomplete, incorrect or toxic proteins in cells. Problems can occur if a ribosome stalls while translating the mRNA transcript, or if the mRNA transcript is defective. For example, most mRNA transcripts contain a stop codon, but some do not, and these non-stop mRNA transcripts result in a non-stop polypeptide that remains tethered to the ribosome. It is important that the cell identifies and removes these faulty polypeptides so as to leave the ribosome free to translate other (non-faulty) mRNA transcripts. A regulatory protein called ubiquitin is responsible for marking and sending proteins that are faulty, or are no longer needed by the cell, to a molecular machine called the proteasome, where they are degraded by a process called proteolysis. In 2010 researchers identified Ltn1 as the enzyme that attaches ubiquitin to non-stop proteins in yeast.
Now, building on this work, Verma et al. identify additional proteins involved in this process. In particular, an ATPase enzyme called Cdc48 (known as p97 or VCP in human cells) and two co-factors—Ufd1 and Npl4—promote release of the ubiquitinated non-stop polypeptides from the ribosomes, thus committing the marked polypeptide to destruction by the proteasome. Verma et al. also show that the Cdc48-Ufd1-Npl4 complex is involved in other aspects of quality control of newly synthesized proteins within cells. Collectively these processes are known as ribosome-associated degradation.
Mutations of the gene that codes for human p97 can cause a number of diseases, including Paget's disease of the bone and frontotemporal dementia, so an improved understanding of ribosome-associated degradation could provide new insights into these diseases.
ubiquitin; ribosome; Cdc48; S. cerevisiae
Endoproteolysis of the β-amyloid precursor protein (APP) by β- and γ-secretases generates the toxic amyloid β-peptide (Aβ), which accumulates in the brain of Alzheimer's disease (AD) patients. Here, we established a novel approach to regulate production of Aβ based on intracellular expression of single chain antibodies (intrabodies) raised to an epitope adjacent to the β-secretase cleavage site of human APP. The intrabodies rapidly associated, within the endoplasmic reticulum (ER), with newly synthesized APP. One intrabody remained associated during APP transport along the secretory line, shielded the β-secretase cleavage site and facilitated the alternative, innocuous cleavage operated by α-secretase. Another killer intrabody with an ER retention sequence triggered APP disposal from the ER. The first intrabody drastically inhibited and the second almost abolished generation of Aβ. Intrabodies association with specific substrates rather than with enzymes, may modulate intracellular processes linked to disease with highest specificity and may become instrumental to investigate molecular mechanisms of cellular events.
Protein translocation in the mammalian endoplasmic reticulum (ER) occurs cotranslationally and requires the binding of translationally active ribosomes to components of the ER membrane. Three candidate ribosome receptors, p180, p34, and Sec61p, have been identified in binding studies with inactive ribosomes, suggesting that ribosome binding is mediated through a receptor-ligand interaction. To determine if the binding of nascent chain-bearing ribosomes is regulated in a manner similar to inactive ribosomes, we have investigated the ribosome/nascent chain binding event that accompanies targeting. In agreement with previous reports, indicating that Sec61p displays the majority of the ER ribosome binding activity, we observed that Sec61p is shielded from proteolytic digestion by native, bound ribosomes. The binding of active, nascent chain bearing ribosomes to the ER membrane is, however, insensitive to the ribosome occupancy state of Sec61p. To determine if additional, Sec61p independent, stages of the ribosome binding reaction could be identified, ribosome/nascent chain binding was assayed as a function of RM concentration. At limiting RM concentrations, a protease resistant ribosome-membrane junction was formed, yet the nascent chain was salt extractable and cross-linked to Sec61p with low efficiency. At nonlimiting RM concentrations, bound nascent chains were protease and salt resistant and cross-linked to Sec61p with higher efficiency. On the basis of these and other data, we propose that ribosome binding to the ER membrane is a multi-stage process comprised of an initial, Sec61p independent binding event, which precedes association of the ribosome/nascent chain complex with Sec61p.
The public health aspects of the use of wastewater in agriculture and the effects of the drip irrigation method on the contamination of vegetables were studied. The method used was to simulate enteric microorganisms' dissemination by contaminated irrigation water in the field. The vegetables were irrigated with an effluent inoculated with a high titer of traceable microorganisms: poliovirus vaccine and a drug-resistant Escherichia coli. The dissemination of the marker organisms in the field was followed, and the effects of certain manipulations of the drip irrigation method on the contamination of the crops by the effluent were examined. It was shown that drip irrigation under plastic sheet cover with the drip lines placed either on the soil surface or buried at a depth of 10 cm significantly reduced crop contamination from inoculated irrigation water even when massive doses of bacteria and viruses were used. The microbial contamination was found to persist in the irrigation pipes and in the soil for at least 8 and 18 days, respectively. The data indicate that the recovery of the marker organisms was affected by soil texture and environmental conditions.
Peroxisome proliferator-activated receptor γ (PPARγ) plays a major role in adipogenesis. PPARγ binds to DNA as a heterodimer with retinoid X receptor (RXR), and PPARγ-RXR can be activated by ligands specific for either receptor; the presence of both ligands can result in a cooperative effect on the transactivation of target genes. How these ligands mediate transactivation, however, remains unclear. PPARγ is known to interact with both the p160/SRC-1 family of coactivators and the distinct, multisubunit coactivator complex called DRIP. A single DRIP subunit, DRIP205 (TRAP220, PBP), binds directly to PPARγ. Here we report that PPARγ and RXR selectively interacted with DRIP205 and p160 proteins in a ligand-dependent manner. At physiological concentrations, RXR-specific ligands only induced p160 binding to RXR, and PPARγ-specific ligands exclusively recruited DRIP205 but not p160 coactivators to PPARγ. This selectivity was not observed in interaction assays off DNA, implying that the specificity of coactivator binding in response to ligand is strongly influenced by the allosteric effects of DNA-bound heterodimers. These coactivator-selective effects were also observed in transient-transfection assays in the presence of overexpressed p160 or DRIP coactivators. The results suggest that the cooperative effects of PPARγ- and RXR-specific ligands may occur at the level of selective coactivator recruitment.
When liver treated with antibiotics to inhibit microbial growth were held at 10 degrees C, the initial high pH (6.4) declined as lactic acid accumulated throughout the storage period of 10 days. The glycogen content also declined, but the glucose concentration in the tissues remained high. When livers were allowed to spoil at 10 degrees C, distinct but variable floras developed within the tissues, in the drip, and on the upper surface. Deep-tissue floras were composed of anaerobic and facultative organisms (Lactobacillus, Enterobacter, Aeromonas); surface floras were generally dominated by strictly aerobic organisms (Pseudomonas, Acinetobacter); drip floras contained variable proportions of organisms of all three types, but the facultatively anaerobic Enterobacter were usually present as a major component. Spoilage occurred after 4 to 6 days with the development of visible discrete colonies on the upper surface without spoilage odors being evident. Changes in tissue and drip composition due to microbial activity could be detected only when spoilage had reached an advanced stage.
The vitamin D receptor (VDR) is a nuclear hormone receptor that controls transcription of target genes. It exerts its biological effects through transcriptional coactivators. Previously, we identified two distinct classes of VDR coactivators, VDR-interacting protein (DRIP) and steroid receptor coactivator (SRC) at different stages of keratinocyte differentiation. Here, we determined the functions of VDR and coactivators in lipid production and permeability barrier formation. Silencing of either VDR, SRC2, or SRC3 resulted in decreases in specific glucosylceramide (GlcCer) species but not other lipids such as cholesterol and free fatty acids. Their silencing also caused decreased transcription of fatty acid elongase and ceramide glucosyltransferase, which are critical for the synthesis of epidermis-unique GlcCer species, and defects in lamellar body formation associated with decreased expression of the lipid transporter ATP-binding cassette transporter protein 12. VDR null mice exhibit abnormal barrier function with altered lipid composition in vivo. These results demonstrate that VDR and coactivators SRC2 and SRC3, which are also involved in other nuclear receptors as well, are critical for epidermis-specific sphingolipid production and barrier formation. In contrast, DRIP silencing had no apparent effect on these processes indicating that the two classes of coactivators are differentially utilized.
Several protein synthesis inhibitors are known to inhibit ribosome assembly. This may be a consequence of direct binding of the antibiotic to ribosome precursor particles, or it could result indirectly from loss of coordination in the production of ribosomal components due to the inhibition of protein synthesis. Here we demonstrate that erythromycin and chloramphenicol, inhibitors of the large ribosomal subunit, affect the assembly of both the large and small subunits. Expression of a small erythromycin resistance peptide acting in cis on mature ribosomes relieves the erythromycin-mediated assembly defect for both subunits. Erythromycin treatment of bacteria expressing a mixture of erythromycin-sensitive and -resistant ribosomes produced comparable effects on subunit assembly. These results argue in favor of the view that erythromycin and chloramphenicol affect the assembly of the large ribosomal subunit indirectly.
The cytoplasmic surface of Sec61p is the binding site for the ribosome and has been proposed to interact with the signal recognition particle receptor during targeting of the ribosome nascent chain complex to the translocation channel. Point mutations in cytoplasmic loops six (L6) and eight (L8) of yeast Sec61p cause reductions in growth rates and defects in the translocation of nascent polypeptides that use the cotranslational translocation pathway. Sec61 heterotrimers isolated from the L8 sec61 mutants have a greatly reduced affinity for 80S ribosomes. Cytoplasmic accumulation of protein precursors demonstrates that the initial contact between the large ribosomal subunit and the Sec61 complex is important for efficient insertion of a nascent polypeptide into the translocation pore. In contrast, point mutations in L6 of Sec61p inhibit cotranslational translocation without significantly reducing the ribosome-binding activity, indicating that the L6 and L8 sec61 mutants affect different steps in the cotranslational translocation pathway.
Flaviviruses bud into the endoplasmic reticulum and are transported through the secretory pathway, where the mildly acidic environment triggers particle rearrangement and allows furin processing of the prM protein to pr and M. The peripheral pr peptide remains bound to virus at low pH and inhibits virus-membrane interaction. Upon exocytosis, the release of pr at neutral pH completes virus maturation to an infectious particle. Together this evidence suggests that pr may shield the flavivirus fusion protein E from the low pH environment of the exocytic pathway. Here we developed an in vitro system to reconstitute the interaction of dengue virus (DENV) pr with soluble truncated E proteins. At low pH recombinant pr bound to both monomeric and dimeric forms of E and blocked their membrane insertion. Exogenous pr interacted with mature infectious DENV and specifically inhibited virus fusion and infection. Alanine substitution of E H244, a highly conserved histidine residue in the pr-E interface, blocked pr-E interaction and reduced release of DENV virus-like particles. Folding, membrane insertion and trimerization of the H244A mutant E protein were preserved, and particle release could be partially rescued by neutralization of the low pH of the secretory pathway. Thus, pr acts to silence flavivirus fusion activity during virus secretion, and this function can be separated from the chaperone activity of prM. The sequence conservation of key residues involved in the flavivirus pr-E interaction suggests that this protein-protein interface may be a useful target for broad-spectrum inhibitors.
Enveloped viruses infect cells by fusing their membrane with that of the host cell. Dengue virus (DENV) is an important human pathogen whose membrane fusion is triggered by low pH during virus entry into the cell. However, newly synthesized DENV must also transit through a low pH environment during virus exit. DENV is believed to escape premature fusion in the exit pathway via the small viral protein pr, which is processed and associates with virus after biosynthesis, and is released from the virus particle in the neutral pH extracellular environment. Here we have reconstituted the interaction of pr with the DENV fusion protein E using soluble protein components. The interaction has a low pH optimum and inhibits membrane insertion of the fusion protein. The recombinant pr peptide can “add back” to fully infectious mature DENV and block virus fusion and infection. We found that mutation of a critical conserved histidine on the fusion protein inhibits the interaction of E and pr, and makes the virus susceptible to low pH-induced inactivation during exit. This work characterizes the mechanism of pr protection, and suggests that the conserved multifunctional pr-E interaction may be an important target for anti-viral strategies.
Vitamin D-receptor interacting protein (DRIP150) coactivates estrogen receptor α (ERα)-mediated transactivation in breast cancer cell lines transfected with a construct (pERE3) containing three estrogen responsive elements (EREs). In this study, we show that DRIP150 also coactivates ERα/Sp1-mediated transactivation in ZR-75, MCF-7 and MDA-MB-231 breast cancer cells transfected with a construct (pSp13) containing three consensus GC-rich motifs. Studies on coactivation of wild-type and variant ERα/Sp1 by DRIP150 indicates that the DNA-binding domain and helix 12 in the ligand binding domain of ERα are required and the coactivation response is squelched by overexpressing an NR-box peptide that contains two LXXLL motifs from GRIP2. In contrast, coactivation of ERα/Sp1 by wild-type and mutant DRIP150 expression plasmids show that coactivation of ERα/Sp1 by DRIP150 is independent of the NR-boxes. Deletion analysis of DRIP150 demonstrates that coactivation requires an α-helical NIFSEVRVYN (amino acids 795-804) motif within twenty-three amino acid sequence (789-811) in the central region of DRIP150 and similar results were obtained for coactivation of ERα by DRIP150. Thus, although different domains of ERα are required for hormone-dependent activation of ERα and ERα/Sp1, coactivation of these transcription factors by DRIP150 requires the α-helical amino acids 795-804. This is the first report of a coactivator that enhances ERα/Sp1-mediated transactivation in breast cancer cells.
DRIP150; ERα/Sp1; ZR-75 cells; coactivation; NR box-independent