Additional Lysine Residues Research Articles

Crystal Contact Engineering for Enhanced Cross-Linking Efficiency of HheG Crystals

The generation of cross-linked enzyme crystals is a very attractive method for immobilization of enzymes displaying high crystalizability. However, the commonly used cross-linker glutaraldehyde is not always compatible with enzyme activity. Therefore, we previously reported the engineering of halohydrin dehalogenase HheG from Ilumatobacter coccineus to enable thiol-specific cross-linking during CLEC generation by insertion of cysteine residues in the crystal contact. To broaden the applicability of this approach, herein crystal contact engineering of HheG has been performed to incorporate additional lysine residues as defined cross-linking sites for CLEC generation. Using the primary amine-specific cross-linker dithiobis(succinimidyl propionate) (DSP), CLECs of HheG variant V46K were obtained that displayed a high gain in thermal stability compared to wild-type HheG, while using only a low cross-linker concentration. Moreover, respective V46K CLECs exhibited a 10 K higher reaction temperature optimum as well as significantly improved activity and stability at acidic pH and in the presence of organic co-solvents. Overall, our study demonstrates that lysine-specific cross-linkers can also be used as an alternative to glutaraldehyde for stable CLEC generation of halohydrin dehalogenases, and that cross-linking efficiency is significantly improved upon crystal contact engineering.

An increase in surface hydrophobicity mediates chaperone activity in N-chlorinated RidA

Under physiological conditions, Escherichia coli RidA is an enamine/imine deaminase, which promotes the release of ammonia from reactive enamine/imine intermediates. However, when modified by hypochlorous acid (HOCl), it turns into a potent chaperone-like holdase that can effectively protect E. coli's proteome during oxidative stress. However, it is unknown, which residues need to be chlorinated for activation. Here, we employ a combination of LC-MS/MS analysis, a chemo-proteomic approach, and a mutagenesis study to identify residues responsible for RidA's chaperone-like function. Through LC-MS/MS of digested RidAHOCl, we obtained direct evidence of the chlorination of one arginine residue. To overcome the instability of the N-chloramine modification, we established a chemoproteomic approach using 5-(dimethylamino) naphthalene-1-sulfinic acid (DANSO2H) as a probe to label N-chlorinated lysines. Using this probe, we were able to detect the N-chlorination of six additional lysine residues. Moreover, using a mutagenesis study to genetically probe the role of single arginine and lysine residues, we found that the removal of arginines R105 and/or R128 led to a substantial reduction of RidAHOCl's chaperone activity. These results, together with structural analysis, confirm that the chaperone activity of RidA is concomitant with the loss of positive charges on the protein surface, leading to an increased overall protein hydrophobicity. Molecular modelling of RidAHOCl and the rational design of a RidA variant that shows chaperone activity even in the absence of HOCl further supports our hypothesis. Our data provide a molecular mechanism for HOCl-mediated chaperone activity found in RidA and a growing number of other HOCl-activated chaperones.

High-Resolution Mapping of Amino Acid Residues in DNA-Protein Cross-Links Enabled by Ribonucleotide-Containing DNA.

DNA-protein cross-links have broad applications in mapping DNA-protein interactions and provide structural insights into macromolecular structures. However, high-resolution mapping of DNA-interacting amino acid residues with tandem mass spectrometry remains challenging due to difficulties in sample preparation and data analysis. Herein, we developed a method for identifying cross-linking amino residues in DNA-protein cross-links at single amino acid resolution. We leveraged the alkaline lability of ribonucleotides and designed ribonucleotide-containing DNA to produce structurally defined nucleic acid-peptide cross-links under our optimized ribonucleotide cleavage conditions. The structurally defined oligonucleotide-peptide heteroconjugates improved ionization, reduced the database search space, and facilitated the identification of cross-linking residues in peptides. We applied the workflow to identifying abasic (AP) site-interacting residues in human mitochondrial transcription factor A (TFAM)-DNA cross-links. With sub-nmol sample input, we obtained high-quality fragmentation spectra for nucleic acid-peptide cross-links and identified 14 cross-linked lysine residues with the home-built AP_CrosslinkFinder program. Semi-quantification based on integrated peak areas revealed that K186 of TFAM is the major cross-linking residue, consistent with K186 being the closest (to the AP modification) lysine residue in solved TFAM:DNA crystal structures. Additional cross-linking lysine residues (K69, K76, K136, K154) support the dynamic characteristics of TFAM:DNA complexes. Overall, our combined workflow using ribonucleotide as a chemically cleavable DNA modification together with optimized sample preparation and data analysis offers a simple yet powerful approach for mapping cross-linking sites in DNA-protein cross-links. The method is amendable to other chemical or photo-cross-linking systems and can be extended to complex biological samples.

The yeast mitophagy receptor Atg32 is ubiquitinated and degraded by the proteasome.

Mitophagy, the process that degrades mitochondria selectively through autophagy, is involved in the quality control of mitochondria in cells grown under respiratory conditions. In yeast, the presence of the Atg32 protein on the outer mitochondrial membrane allows for the recognition and targeting of superfluous or damaged mitochondria for degradation. Post-translational modifications such as phosphorylation are crucial for the execution of mitophagy. In our study we monitor the stability of Atg32 protein in the yeast S. cerevisiae and show that Atg32 is degraded under normal growth conditions, upon starvation or rapamycin treatment. The Atg32 turnover can be prevented by inhibition of the proteasome activity, suggesting that Atg32 is also ubiquitinated. Mass spectrometry analysis of purified Atg32 protein revealed that at least lysine residue in position 282 is ubiquitinated. Interestingly, the replacement of lysine 282 with alanine impaired Atg32 degradation only partially in the course of cell growth, suggesting that additional lysine residues on Atg32 might also be ubiquitinated. Our results provide the foundation to further elucidate the physiological significance of Atg32 turnover and the interplay between mitophagy and the proteasome.

Fine-Tuning of Neuronal Ion Channels-Mapping of Residues Involved in Glucose Sensitivity of Recombinant Human Glycine Receptors.

The inhibitory glycine receptor (GlyR) mediates synaptic inhibition in the spinal cord, brain stem, and other regions of the mammalian central nervous system. Glucose was shown to potentiate α1 GlyRs by interacting with K143. Here, additional amino acids involved in glucose modulation were identified using a structure-based approach of site-directed mutagenesis followed by whole-cell patch-clamp analysis. We identified two additional lysine residues in the α1 GlyR extracellular domain, K16 and K281, that were involved in glucose modulation. Mutation of either residue to alanine abolished glucose potentiation. Residue K281 is located in the same pocket as K143 and could thus contribute to glucose binding. The double mutant K143A-K281A showed a 6-fold increase of EC50, while EC50 of both single mutants K143A and K281A was only slightly increased (1.7- and 1.3-fold, respectively). K16 is located at an analgesic binding site that is distant from the agonist or glucose sites, and the K16A mutation may generate a receptor species that is not potentiated. GlyR position α1-S267 is close to the postulated glucose binding site and known for interactions with ethanol and anesthetics. In the presence of glucose, GlyR α1 mutants S267A, S267I, and S267R showed potentiation, no effect, and reduction of current responses, respectively. This pattern follows that of ethanol modulation and suggests that the interaction sites of glucose and ethanol are identical or located close to each other. Our results support the presence of a distinct binding site for glucose on the glycine receptor, overlapping with the ivermectin/ethanol binding pocket near the transmembrane region and the TM2-3 loop.

Chromatographic separation of hemoglobin variants using robust molecularly imprinted polymers

Devising a robust, efficient and cost effective hemoglobin (Hb) purification strategy is one of the key challenges in the development of Hb-based blood substitutes. The aim of this study was to use molecularly imprinted polymers (MIPs) as a novel and efficient chromatographic resin to selectively recognize and purify different Hb variants. The results showed that the Hb-MIP material developed here could selectively recognize and purify various Hb directly from either crude E. coli extracts or human body fluids, such as blood plasma and cerebrospinal fluid (CSF), in one-step. The dynamic binding capacity at 10% breakthrough was around 7.4 mg mL−1resin for adult Hb (HbA) and fetal Hb (HbF). This chromatographic material also allowed identification of changes related to amino acid substitutions on the Hb protein surface. For instance, when an additional lysine residue was introduced, the HbA αY42K mutant eluted later in an Hb-MIP column than wildtype HbA. Additional negative charges on the protein surface, such as aspartate, mitigated the interaction between the protein and imprinted polymers, and therefore an αA19D-αA12D HbF mutant eluted earlier, at −2.7 column volumes compared to wildtype HbF.

Functional expression of monomeric streptavidin and fusion proteins in Escherichia coli: applications in flow cytometry and ELISA.

Monomeric streptavidin (mSA) offers a combination of structural and binding properties that are useful in many applications, including a small size and monovalent biotin binding. Because mSA contains a structurally important disulfidebond, the molecule does not fold correctly when expressed inside the cell. We show that mSA can be expressed in a functional form in Escherichia coli by fusing the OmpA signal sequence at the amino terminus. Expressed mSA is exported to the periplasm, from which the molecule leaks to the medium under vigorous shaking. Purified mSA can be conjugated with FITC and used to label microbeads and yeast cells for analysis by flow cytometry, further expanding the scope of mSA-based applications. Some applications require recombinant fusion of mSA with another protein. mSA fused to EGFP cannot be secreted to the medium but was successfully expressed in an engineered cell line that supports oxidative folding in the cytoplasm. Purified mSA-EGFP and mSA-mCherry bound biotin with high affinity and were successfully used in conventional flow cytometry and imaging flow cytometry. Finally, we demonstrate the use of mSA in ELISA, in which horseradish peroxidase-conjugated mSA and biotinylated secondary antibody are used together to detect primary antibody captured on an ELISA plate. Engineering mSA to introduce additional lysine residues can increase the reporter signal above that of wild-type streptavidin. Together, these examples establish mSA as a convenient reagent with a potentially unique role in biotechnology.

Mutation of M13 Bacteriophage Major Coat Protein for Increased Conjugation to Exogenous Compounds

Over the past ten years there has been increasing interest in the conjugation of exogenous compounds to the surface of the M13 bacteriophage. M13 offers a convenient scaffold for the development of nanoassemblies with useful functions, such as highly specific drug delivery and pathogen detection. However, the progress of these technologies has been hindered by the limited efficiency of conjugation to the bacteriophage. Here we generate a mutant version of M13 with an additional lysine residue expressed on the outer surface of the M13 major coat protein, pVIII. We show that this mutation is accommodated by the bacteriophage and that up to an additional 520 exogenous groups can be attached to the bacteriophage surface via amine-directed conjugation. These results could aid the development of high payload drug delivery nanoassemblies and pathogen detection systems with increased sensitivity.

Peptide ligands for targeting the extracellular domain of EGFR: Comparison between linear and cyclic peptides.

Colorectal cancer (CRC) is the third most common solid internal malignancy among cancers. Early detection of cancer is key to increasing the survival rate of colorectal cancer patients. Overexpression of the EGFR protein is associated with CRC. We have designed a series of peptides that are highly specific for the extracellular domain of EGFR, based on our earlier studies on linear peptides. The previously reported linear peptide LARLLT, known to bind to EGFR, was modified with the goals of increasing its stability and its specificity toward EGFR. Peptide modifications, including D-amino acid substitution, cyclization, and chain reversal, were investigated. In addition, to facilitate labeling of the peptide with a fluorescent dye, an additional lysine residue was introduced onto the linear (KLARLLT) and cyclic peptides cyclo(KLARLLT) (Cyclo.L1). The lysine residue was also converted into an azide group in both a linear and reversed cyclic peptide sequences cyclo(K(N3)larllt) (Cyclo.L1.1) to allow for subsequent "click" conjugation. The cyclic peptides showed enhanced binding to EGFR by SPR. NMR and molecular modeling studies suggest that the peptides acquire a β-turn structure in solution. In vitro stability studies in human serum show that the cyclic peptide is more stable than the linear peptide.

Acid-labile pHPMA modification of four-arm oligoaminoamide pDNA polyplexes balances shielding and gene transfer activity in vitro and in vivo

We report novel pH-reversibly surface-shielded polyplexes with enhanced gene transfer activity upon systemic administration. A four-arm-structured sequence-defined cationic oligomer KK[HK[(H-Sph-K)3HC]2]2 was designed and synthesized on solid-phase, containing additional lysine residues not only for improved pDNA polyplex stability, but also providing attachment points for subsequent polyplex functionalization with amine-reactive shielding polymers. Herein, the surface of polyplexes was shielded with hydrophilic polymers, monovalent PEG or monovalent and multivalent pHPMA, optionally attached to the polyplex via the acid-labile linker AzMMMan. Overall, surface modification with PEG or pHPMA resulted in a decrease in the zeta potential of polyplexes, consistent with the degree of surface shielding. At pH 6.0, only polyplexes modified via the acid-labile linkage showed an increase in zeta potential, consistent with a “deshielding” in acidic environment, expected as beneficial for endosomal escape. Shielding was more efficient for multivalent pHPMA (20kDa, 30kDa) as compared to monovalent pHPMA (10kDa, 20kDa, 30kDa) or PEG (5kDa). In vitro transfection studies revealed higher gene expression by the polyplexes with the acid-labile shield as compared to their irreversibly shielded counterparts. Intravenous administration of AzMMMan-pHPMA modified polyplexes in an in vivo tumor mouse model mediated enhanced gene expression in the subcutaneous tumor and reduced undesirable expression in the liver.

Importance of the N-terminal proline for the promiscuous activity of 4-oxalocrotonate tautomerase (4-OT)

Michael addition of aldehydes to nitroolefins is an effective method to obtain useful chiral ?-nitroaldehydes. ?-Nitroaldehydes are precursors for chiral ?-aminobytiric acid analogues which have numerous pharmacological activities and are used for treatment of neurological disorders. A whole-cell system based on recombinantly expressed 4-oxalocrotonate tautomerase (4-OT) has been developed and shown to be an effective biocatalyst for Michael addition of branched aldehydes to ?-nitrostyrenes. The aim of this study was to investigate the influence of the substitution of the N-terminal proline with lysine and arginine both containing the reactive ?-amino group, on the Michael addition catalyzed by 4-OT. Firstly, the effect of these mutations was examined by in silico analysis, followed by generation of three terminal ly-sine mutants. The generated mutants, 4-OT_K, 4-OT_PK and 4-OT_KK were tested for the ability to utilise ?-nitrostyrene (1), (E)-2-(thiophene-2-yl)nitroethen (2) and p-chloro-trans-?-nitrostyrene (3) as Michael acceptors with isobutanal as the donor. For comparison, the lithium salt of lysine was used in the same organocatalytic reactions. In general, the introduction of lysine had a negative effect on Michael additions based on overall product yields. However, additional lysine residues at the N-terminus of the protein resulted in structural changes that enhanced the activity towards 2 and 3. Therefore, the N-terminal proline is important for the 4-OT catalysed Michael-additons, but it is not essential.

A porcine circovirus-2 mutant isolated in Brazil contains low-frequency substitutions in regions of immunoprotective epitopes in the capsid protein.

Porcine circovirus-2 (PCV2) is the etiologic agent of several diseases in pigs, including multi-systemic wasting syndrome (PMWS). In this work, a new mutant PCV2b was isolated from PMWS-affected pigs on a Brazilian farm. Its genome showed high sequence similarity (>99% identity) to those from a group of emerging mutants isolated from cases of PMWS outbreaks in vaccinated pigs in China, the USA and South Korea. Here, we show that these isolates share a combination of low-frequency substitutions (single amino acid polymorphisms with a frequency of ≤25%) in the viral capsid protein, mainly in regions of immunoprotective epitopes, and an additional lysine residue at position 234. These isolates were phylogenetically grouped in the PCV2b clade, reinforcing the idea of the emergence of a new group of mutants PCV2b associated with outbreaks worldwide. The identification of these polymorphisms in the viral capsid highlights the importance of considering these isolates for the development of more-effective vaccines.

SIRT1 mediates FOXA2 breakdown by deacetylation in a nutrient-dependent manner.

The Forkhead transcription factor FOXA2 plays a fundamental role in controlling metabolic homeostasis in the liver during fasting. The precise molecular regulation of FOXA2 in response to nutrients is not fully understood. Here, we studied whether FOXA2 could be controlled at a post-translational level by acetylation. By means of LC-MS/MS analyses, we identified five acetylated residues in FOXA2. Sirtuin family member SIRT1 was found to interact with and deacetylate FOXA2, the latter process being dependent on the NAD+-binding catalytic site of SIRT1. Deacetylation by SIRT1 reduced protein stability of FOXA2 by targeting it towards proteasomal degradation, and inhibited transcription from the FOXA2-driven G6pase and CPT1a promoters. While mutation of the five identified acetylated residues weakly affected protein acetylation and stability, mutation of at least seven additional lysine residues was required to abolish acetylation and reduce protein levels of FOXA2. The importance of acetylation of FOXA2 became apparent upon changes in nutrient levels. The interaction of FOXA2 and SIRT1 was strongly reduced upon nutrient withdrawal in cell culture, while enhanced Foxa2 acetylation levels were observed in murine liver in vivo after starvation for 36 hours. Collectively, this study demonstrates that SIRT1 controls the acetylation level of FOXA2 in a nutrient-dependent manner and in times of nutrient shortage the interaction between SIRT1 and FOXA2 is reduced. As a result, FOXA2 is protected from degradation by enhanced acetylation, hence enabling the FOXA2 transcriptional program to be executed to maintain metabolic homeostasis.

β-Lactam Antibiotics Form Distinct Haptenic Structures on Albumin and Activate Drug-Specific T-Lymphocyte Responses in Multiallergic Patients with Cystic Fibrosis

β-Lactam antibiotics provide the cornerstone of treatment for respiratory exacerbations in patients with cystic fibrosis. Unfortunately, approximately 20% of patients develop multiple nonimmediate allergic reactions that restrict therapeutic options. The purpose of this study was to explore the chemical and immunological basis of multiple β-lactam allergy through the analysis of human serum albumin (HSA) covalent binding profiles and T-cell responses against 3 commonly prescribed drugs; piperacillin, meropenem, and aztreonam. The chemical structures of the drug haptens were defined by mass spectrometry. Peripheral blood mononuclear cells (PBMC) were isolated from 4 patients with multiple allergic reactions and cultured with piperacillin, meropenem, and aztreonam. PBMC responses were characterized using the lymphocyte transformation test and IFN-γ /IL-13 ELIspot. T-cell clones were generated from drug-stimulated T-cell lines and characterized in terms of phenotype, function, and cross-reactivity. Piperacillin, meropenem, and aztreonam formed complex and structurally distinct haptenic structures with lysine residues on HSA. Each drug modified Lys190 and at least 6 additional lysine residues in a time- and concentration-dependent manner. PBMC proliferative responses and cytokine release were detected with cells from the allergic patients, but not tolerant controls, following exposure to the drugs. 122 CD4+, CD8+, or CD4+CD8+ T-cell clones isolated from the allergic patients were found to proliferate and release cytokines following stimulation with piperacillin, meropenem, or aztreonam. Cross-reactivity with the different drugs was not observed. In conclusion, our data show that piperacillin-, meropenem-, and aztreonam-specific T-cell responses are readily detectable in allergic patients with cystic fibrosis, which indicates that multiple β-lactam allergies are instigated through priming of naïve T-cells against the different drug antigens. Characterization of complex haptenic structures on distinct HSA lysine residues provides a chemical basis for the drug-specific T-cell response.

Tailor-made design of penicillin G acylase surface enables its site-directed immobilization and stabilization onto commercial mono-functional epoxy supports

Four different mutants of penicillin G acylase from E. coli (PGA) were site-direct immobilized and rigidification on Eupergit C. The resulting immobilized preparations were more stable against both temperature and organic solvents than those obtained by using a more complex tailor-made disulfide-epoxy supports. The region around the position β380 was the most interesting one to achieve the highest PGA stabilization values (this derivative was 150-fold more stable than the corresponding soluble enzyme). Such region was further enriched in 4 additional lysine residues, and the resulting immobilized derivative was 1500-fold more stable than the same protein variant uni-punctually immobilized through position β380. This stabilization factor was the highest reported for PGA by immobilization on Eupergit C as commercial epoxy-acrylic immobilization carrier.

LOX Out, Histones: A New Enzyme Is Nipping at Your Tails

In the current issue of Molecular Cell, Herranz et al. (2012) demonstrate that LOXL2 deaminates trimethylated histone 3 lysine 4 (H3K4me3), which uncovers a new chromatin modification and a new enzymatic mechanism with the potential to regulate additional lysine residues.

Role of Ubiquitin in Parainfluenza Virus 5 Particle Formation

Ubiquitin is important for the budding of many retroviruses and other enveloped viruses, but the precise role of ubiquitin in virus budding remains unclear. Here, we characterized the ubiquitination of the matrix (M) protein of a paramyxovirus, parainfluenza virus 5 (PIV5). The PIV5 M protein (but not the PIV5 nucleocapsid protein) was found to be targeted for monoubiquitination in transfected mammalian cells. Major sites of ubiquitin attachment identified by mass spectrometry analysis were lysine residues at amino acid positions 79/80, 130, and 247. The cumulative mutation of lysine residues 79, 80, and 130 to arginines led to an altered pattern of M protein ubiquitination and impaired viruslike particle (VLP) production. However, the cumulative mutation of lysine residues 79, 80, 130, and 247 to arginines restored M protein ubiquitination and VLP production, suggesting that ubiquitin is attached to alternative sites on the M protein when the primary ones have been removed. Additional lysine residues were targeted for mutagenesis based on the UbiPred algorithm. An M protein with seven lysine residues changed to arginines exhibited altered ubiquitination and poor VLP production. A recombinant virus encoding an M protein with seven lysines mutated was generated, and this virus exhibited a 6-fold-reduced maximum titer, with the defect being attributed mainly to the budding of noninfectious particles. The recombinant virus was assembly deficient, as judged by the redistribution of viral M and hemagglutinin-neuraminidase proteins in infected cells. Similar assembly defects were observed for the wild-type (wt) virus after treatment with a proteasome inhibitor. Collectively, these findings suggest that the monoubiquitination of the PIV5 M protein is important for proper virus assembly and for the budding of infectious particles.

The Role of Protein Context in Disease-Related Huntingtin Protein/Lipid Interface Interactions

Huntington's disease (HD) is a neurodegenerative disorder caused by an expansion of a poly-glutamine (poly-Q) region near the N-terminus of the huntingtin (htt) protein. Expansion of the poly-Q region above 35-40 repeats results in the disease that is characterized by inclusion body aggregates of mutated protein. The poly-Q region of htt is flanked by a 17 amino acid N-terminal sequence (N17) and a region of proline repeats (P11). As the cell membrane has been proposed to play a role in mediating htt aggregation, Langmuir trough techniques were used to investigate the effects of flanking regions on the surface activity and insertion of htt peptide constructs into a lipid monolayer. Four peptide constructs were tested: N17-Q35-KK, N17-Q35-P11-KK, KK-Q35-KK and KK-Q35-P11-KK, where the additional lysine residues were attached to improve solubility. Surface activity was measured to determine the affinity of each peptide for the air-water interface. The constructs containing the N-terminal sequence had higher surface activities compared to those without. Inclusion of the polyproline region with the N-terminal sequence contributed to the highest surface activity. To determine peptide association with a cell membrane, insertion of each peptide into a monolayer of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) held at a constant surface pressure was also investigated. Peptides with the flanking N-terminus showed appreciable monolayer insertion. The polyproline region only mediated and increased the degree of insertion when the N-terminus was also present. Rates of insertion of the constructs containing the N-terminus were comparable, while those without this region were markedly slower. Experiments were repeated using monolayers of total brain extract to better model a physiological environment with similar results. The amino acid environment surrounding the poly-Q region dramatically affects peptide association with the cell membrane and this may potentially mediate the aggregation process.

Differential N termini in epithelial Na+ channel δ-subunit isoforms modulate channel trafficking to the membrane

The epithelial Na(+) channel (ENaC) is a heteromultimeric ion channel that plays a key role in Na(+) reabsorption across tight epithelia. The canonical ENaC is formed by three analogous subunits, α, β, and γ. A fourth ENaC subunit, named δ, is expressed in the nervous system of primates, where its role is unknown. The human δ-ENaC gene generates at least two splice isoforms, δ(1) and δ(2) , differing in the N-terminal sequence. Neurons in diverse areas of the human and monkey brain differentially express either δ(1) or δ(2) , with few cells coexpressing both isoforms, which suggests that they may play specific physiological roles. Here we show that heterologous expression of δ(1) in Xenopus oocytes and HEK293 cells produces higher current levels than δ(2) . Patch-clamp experiments showed no differences in single channel current magnitude and open probability between isoforms. Steady-state plasma membrane abundance accounts for the dissimilarity in macroscopic current levels. Differential trafficking between isoforms is independent of β- and γ-subunits, PY-motif-mediated endocytosis, or the presence of additional lysine residues in δ(2)-N terminus. Analysis of δ(2)-N terminus identified two sequences that independently reduce channel abundance in the plasma membrane. The δ(1) higher abundance is consistent with an increased insertion rate into the membrane, since endocytosis rates of both isoforms are indistinguishable. Finally, we conclude that δ-ENaC undergoes dynamin-independent endocytosis as opposed to αβγ-channels.

Comparison of Tax-1 and Tax-2B post-translational modifications using specific lysine mutants in relation to activation of NF-κB and intracellular localization

Post-translational modifications of HTLV-1 and HTLV-2 Tax-1 and Tax-2 proteins have been shown to play a critical role in their cellular localization, transactivation and protein interactions. Five of ten lysine residues were found to be major targets for Tax-1 modifications: Lys189(K4); Lys197(K5), Lys263(K6), Lys280(K7) and Lys284(K8), are essential for ubiquitination, while sumoylation takes place on Lys280 (K7) and Lys284(K8). Tax-2 contains four additional lysine residues, namely at position Lys100(K2i), Lys149(K3i), Lys185(K3ii), and Lys356(K10i). Very few studies have been so far performed on Tax-2 lysine mutants. We have previously demonstrated that Tax-2B is ubiquitinated and sumoylated similarly to Tax-1. To identify the Tax-2 lysine residues which are directly involved in post-translational modifications, we have constructed a series of Tax-2B mutants with substitutions of lysine (K) residues by arginines (R) and analyzed them for NF-kB and CREB/ATF transactivation, intracellular distribution and extent of ubiquitination and sumoylation. We have found that Tax-2 K7-8R mutant, contrary to its Tax-1 homologue, is only partially affected in its capacity to transactivate NF-κB pathway, is regularly sumoylated and presents formation of nuclear bodies by confocal analysis. However, Tax-2 mutants with extended (K3ii-8R) and/or total (K1-10iR) mutation rate were severely affected for NF-kB transactivation and sumoylation. By comparing Tax-2 WT with mutants K7-8R and K3ii-8R, we observed that the reduction of NF-κB activity is correlated to a parallel decrease in sumoylation. These results suggest that the target for Tax-2 ubiquitination and sumoylation differs from that described for Tax-1.