Single Residue Research Articles

Vimentin single cysteine residue acts as a tunable sensor for network organization and as a key for actin remodeling in response to oxidants and electrophiles

Cysteine residues can undergo multiple posttranslational modifications with diverse functional consequences, potentially behaving as tunable sensors. The intermediate filament protein vimentin has important implications in pathophysiology, including cancer progression, infection, and fibrosis, and maintains a close interplay with other cytoskeletal structures, such as actin filaments and microtubules. We previously showed that the single vimentin cysteine, C328, is a key target for oxidants and electrophiles. Here, we demonstrate that structurally diverse cysteine-reactive agents, including electrophilic mediators, oxidants and drug-related compounds, disrupt the vimentin network eliciting morphologically distinct reorganizations. As most of these agents display broad reactivity, we pinpointed the importance of C328 by confirming that local perturbations introduced through mutagenesis provoke structure-dependent vimentin rearrangements. Thus, GFP-vimentin wild type (wt) forms squiggles and short filaments in vimentin-deficient cells, the C328F, C328W, and C328H mutants generate diverse filamentous assemblies, and the C328A and C328D constructs fail to elongate yielding dots. Remarkably, vimentin C328H structures resemble the wt, but are strongly resistant to electrophile-elicited disruption. Therefore, the C328H mutant allows elucidating whether cysteine-dependent vimentin reorganization influences other cellular responses to reactive agents. Electrophiles such as 1,4-dinitro-1H-imidazole and 4-hydroxynonenal induce robust actin stress fibers in cells expressing vimentin wt. Strikingly, under these conditions, vimentin C328H expression blunts electrophile-elicited stress fiber formation, apparently acting upstream of RhoA. Analysis of additional vimentin C328 mutants shows that electrophile-sensitive and assembly-defective vimentin variants permit induction of stress fibers by reactive species, whereas electrophile-resistant filamentous vimentin structures prevent it. Together, our results suggest that vimentin acts as a break for actin stress fibers formation, which would be released by C328-aided disruption, thus allowing full actin remodeling in response to oxidants and electrophiles. These observations postulate C328 as a “sensor” transducing structurally diverse modifications into fine-tuned vimentin network rearrangements, and a gatekeeper for certain electrophiles in the interplay with actin.

Enhancement of the thermal stability of G-quadruplex structures by urea

The solute urea has been used extensively as a denaturant in protein folding studies; double-stranded nucleic acid structures are also destabilized by urea, but comparatively less than proteins. In previous research, the solute has been shown to strongly destabilize folded G-quadruplex DNA structures. This contribution demonstrates the stabilizing effect of urea on the G-quadruplex formed by the oligodeoxyribonucleotide (ODN), G3T (d[5′-GGGTGGGTGGGTGGG-3′]), and related sequences in the presence of sodium or potassium cations. Stabilization is observed up to 7 M urea, which was the highest concentration we investigated. The folded structure of G3T has three G-tetrads and three loops that consist of single thymine residues. ODNs related to G3T, in which the thymine residues in the loop are substituted by adenosine residues, also exhibit enhanced stability in the presence of molar concentrations of urea. The circular dichroism (CD) spectra of these ODNs in the presence of urea are consistent with that of a G-quadruplex. As the urea concentration increases, the spectral intensities of the peaks and troughs change, while their positions change very little. The heat-induced transition from the folded to unfolded state, Tm, was measured by monitoring the change in the UV absorption as a function of temperature. G-quadruplex structures with loops containing single bases exhibited large increases in Tm with increasing urea concentrations. These data imply that the loop region play a significant role in the thermal stability of tetra-helical DNA structures in the presence of the solute urea.

Mutation in the Common Docking Domain Affects MAP Kinase ERK2 Catalysis and Stability.

The extracellular-signal-regulated kinase 2 (ERK2), a mitogen-activated protein kinase (MAPK) located downstream of the Ras-Raf-MEK-ERK signal transduction cascade, is involved in the regulation of a large variety of cellular processes. The ERK2, activated by phosphorylation, is the principal effector of a central signaling cascade that converts extracellular stimuli into cells. Deregulation of the ERK2 signaling pathway is related to many human diseases, including cancer. This study reports a comprehensive biophysical analysis of structural, function, and stability data of pure, recombinant human non-phosphorylated (NP-) and phosphorylated (P-) ERK2 wild-type and missense variants in the common docking site (CD-site) found in cancer tissues. Because the CD-site is involved in interaction with protein substrates and regulators, a biophysical characterization of missense variants adds information about the impact of point mutations on the ERK2 structure-function relationship. Most of the P-ERK2 variants in the CD-site display a reduced catalytic efficiency, and for the P-ERK2 D321E, D321N, D321V and E322K, changes in thermodynamic stability are observed. The thermal stability of NP-ERK2 and P-ERK2 D321E, D321G, and E322K is decreased with respect to the wild-type. In general, a single residue mutation in the CD-site may lead to structural local changes that reflects in alterations in the global ERK2 stability and catalysis.

Implications of specific lysine residues within ataxin-3 for the molecular pathogenesis of Machado-Joseph disease.

Lysine residues are one of the main sites for posttranslational modifications of proteins, and lysine ubiquitination of the Machado-Joseph disease protein ataxin-3 is implicated in its cellular function and polyglutamine expansion-dependent toxicity. Despite previously undertaken efforts, the individual roles of specific lysine residues of the ataxin-3 sequence are not entirely understood and demand further analysis. By retaining single lysine residues of otherwise lysine-free wild-type and polyglutamine-expanded ataxin-3, we assessed the effects of a site-limited modifiability on ataxin-3 protein levels, aggregation propensity, localization, and stability. We confirmed earlier findings that levels of lysine-free ataxin-3 are reduced due to its decreased stability, which led to a diminished load of SDS-insoluble species of its polyglutamine-expanded form. The isolated presence of several single lysine residues within the N-terminus of polyglutamine-expanded ataxin-3 significantly restored its aggregate levels, with highest fold changes induced by the presence of lysine 8 or lysine 85, respectively. Ataxin-3 lacking all lysine residues presented a slightly increased nuclear localization, which was counteracted by the reintroduction of lysine 85, whereas presence of either lysine 8 or lysine 85 led to a significantly higher ataxin-3 stability. Moreover, lysine-free ataxin-3 showed increased toxicity and binding to K48-linked polyubiquitin chains, whereas the reintroduction of lysine 85, located between the ubiquitin-binding sites 1 and 2 of ataxin-3, normalized its binding affinity. Overall, our data highlight the relevance of lysine residues 8 and 85 of ataxin-3 and encourage further analyses, to evaluate the potential of modulating posttranslational modifications of these sites for influencing pathophysiological characteristics of the Machado-Joseph disease protein.

Identification of three elevenin receptors and roles of elevenin disulfide bond and residues in receptor activation in Aplysia californica

Neuropeptides are ubiquitous intercellular signaling molecules in the CNS and play diverse roles in modulating physiological functions by acting on specific G-protein coupled receptors (GPCRs). Among them, the elevenin signaling system is now believed to be present primarily in protostomes. Although elevenin was first identified from the L11 neuron of the abdominal ganglion in mollusc Aplysia californica, no receptors have been described in Aplysia, nor in any other molluscs. Here, using two elevenin receptors in annelid Platynereis dumerilii, we found three putative elevenin GPCRs in Aplysia. We cloned the three receptors and tentatively named them apElevR1, apElevR2, and apElevR3. Using an inositol monophosphate (IP1) accumulation assay, we demonstrated that Aplysia elevenin with the disulfide bond activated the three putative receptors with low EC50 values (ranging from 1.2 to 25 nM), supporting that they are true receptors for elevenin. In contrast, elevenin without the disulfide bond could not activate the receptors, indicating that the disulfide bond is required for receptor activity. Using alanine substitution of individual conserved residues other than the two cysteines, we showed that these residues appear to be critical to receptor activity, and the three different receptors had different sensitivities to the single residue substitution. Finally, we examined the roles of those residues outside the disulfide bond ring by removing these residues and found that they also appeared to be important to receptor activity. Thus, our study provides an important basis for further study of the functions of elevenin and its receptors in Aplysia and other molluscs.

Insights into Polyprotein Processing and RNA-Protein Interactions in Foot-and-Mouth Disease Virus Genome Replication.

Foot-and-mouth disease virus (FMDV) is a picornavirus, which infects cloven-hoofed animals to cause foot-and-mouth disease (FMD). The positive-sense RNA genome contains a single open reading frame, which is translated as a polyprotein that is cleaved by viral proteases to produce the viral structural and nonstructural proteins. Initial processing occurs at three main junctions to generate four primary precursors; Lpro and P1, P2, and P3 (also termed 1ABCD, 2BC, and 3AB1,2,3CD). The 2BC and 3AB1,2,3CD precursors undergo subsequent proteolysis to generate the proteins required for viral replication, including the enzymes 2C, 3Cpro, and 3Dpol. These precursors can be processed through both cis and trans (i.e., intra- and intermolecular proteolysis) pathways, which are thought to be important for controlling virus replication. Our previous studies suggested that a single residue in the 3B3-3C junction has an important role in controlling 3AB1,2,3CD processing. Here, we use in vitro based assays to show that a single amino acid substitution at the 3B3-3C boundary increases the rate of proteolysis to generate a novel 2C-containing precursor. Complementation assays showed that while this amino acid substitution enhanced production of some nonenzymatic nonstructural proteins, those with enzymatic functions were inhibited. Interestingly, replication could only be supported by complementation with mutations in cis acting RNA elements, providing genetic evidence for a functional interaction between replication enzymes and RNA elements. IMPORTANCE Foot-and-mouth disease virus (FMDV) is responsible for foot-and-mouth disease (FMD), an important disease of farmed animals, which is endemic in many parts of the world and can results in major economic losses. Replication of the virus occurs within membrane-associated compartments in infected cells and requires highly coordinated processing events to produce an array of nonstructural proteins. These are initially produced as a polyprotein that undergoes proteolysis likely through both cis and trans alternative pathways (i.e., intra- and intermolecular proteolysis). The role of alternative processing pathways may help coordination of viral replication by providing temporal control of protein production and here we analyze the consequences of amino acid substitutions that change these pathways in FMDV. Our data suggest that correct processing is required to produce key enzymes for replication in an environment in which they can interact with essential viral RNA elements. These data further the understanding of RNA genome replication.

In planta deglycosylation improves the SARS-CoV-2 neutralization activity of recombinant ACE2-Fc.

SARS-CoV-2 infects human cells via binding of the viral spike glycoprotein to its main cellular receptor, angiotensin-converting enzyme 2 (ACE2). The spike protein-ACE2 receptor interaction is therefore a major target for the development of therapeutic or prophylactic drugs to combat coronavirus infections. Various engineered soluble ACE2 variants (decoys) have been designed and shown to exhibit virus neutralization capacity in cell-based assays and in vivo models. Human ACE2 is heavily glycosylated and some of its glycans impair binding to the SARS-CoV-2 spike protein. Therefore, glycan-engineered recombinant soluble ACE2 variants might display enhanced virus-neutralization potencies. Here, we transiently co-expressed the extracellular domain of ACE2 fused to human Fc (ACE2-Fc) with a bacterial endoglycosidase in Nicotiana benthamiana to produce ACE2-Fc decorated with N-glycans consisting of single GlcNAc residues. The endoglycosidase was targeted to the Golgi apparatus with the intention to avoid any interference of glycan removal with concomitant ACE2-Fc protein folding and quality control in the endoplasmic reticulum. The in vivo deglycosylated ACE2-Fc carrying single GlcNAc residues displayed increased affinity to the receptor-binding domain (RBD) of SARS-CoV-2 as well as improved virus neutralization activity and thus is a promising drug candidate to block coronavirus infection.

Human versus mouse Beta 2 microglobulin alters TCR antigen recognition kinetics with mouse MHCI

Abstract There are three isoforms of Beta2 microglobulin (B2m) identified in mice and they differ by a single amino acid residue. To study how B2m can modify the peptide binding pocket of MHCI we turned to the comparison of mouse (mB2m) and human (hB2m). It is known that hB2m forms more stable with mouse MHCI heavy chain as compared to mb2m and hB2m is often the choice for most pMHCI tetramer production. Analysis of mB2m and hB2m tetramer (GP33 41M) following LCMV infection revealed no difference in percentage of tetramer positive cells but an increase was observed in staining intensity (MFI) of hB2m. Using steered molecular dynamics we found that there were allosteric effects with mB2m displaying more movement within the binding pocket than hB2m. Consistent with this we saw increased 2D affinity for antigen MHC D band K bhB2m complexes for OT1 and P14 TCRs, respectively. We also measured the force effects on bond lifetime between TCR and pMHC using biomembrane force probe (BFP) and DNA tension sensor. For OTI, we found that mB2m complexes had a longer bond lifetime measured by both methods. We observed signaling and functional effects consistent with the 2D affinity and bond lifetime measurements in mouse verses human B2m. For example, mB2m leads to increased levels of activated LCK (pY394/505) in OT1 T cells. Therefore, alterations in B2m effect the distal peptide binding domain of MHCI and suggests dynamic allostery may influence antigen recognition by TCR. 5T32NS115664-03 5R01AI147641-03

Selective Attachment of Polyethylene Glycol to Hemerythrin for Potential Use in Blood Substitutes.

Due to its ability to reversibly bind O2, alongside a relatively low redox reactivity and a limited cytotoxicity, the oxygen-carrying protein hemerythrin has been considered as an alternative to hemoglobin in preparing blood substitutes. In order to increase the hydrodynamic volume and lower antigenicity, two site-directed variants, H82C and K92C, were engineered that contained a single cysteine residue on the surface of each hemerythrin octamer for the specific attachment of polyethylene glycol (PEG). A sulfhydryl-reactive PEGylation reagent with a 51.9 Å spacer arm was used for selective cysteine derivatization. The mutants were characterized by UV-vis spectroscopy, size-exclusion chromatography, oxygen affinity, and autooxidation rate measurements. The H82C variant showed altered oligomeric behavior compared to the wild-type and was unstable in the met form. The PEGylated K92C variant is reasonably stable, displays an oxygen affinity similar to that of the wild-type, and shows an increased rate of autoxidation; the latter disadvantage may be counteracted by further chemical modifications.

BICD2 phosphorylation regulates dynein function and centrosome separation in G2 and M

The activity of dynein is regulated by a number of adaptors that mediate its interaction with dynactin, effectively activating the motor complex while also connecting it to different cargos. The regulation of adaptors is consequently central to dynein physiology but remains largely unexplored. We now describe that one of the best-known dynein adaptors, BICD2, is effectively activated through phosphorylation. In G2, phosphorylation of BICD2 by CDK1 promotes its interaction with PLK1. In turn, PLK1 phosphorylation of a single residue in the N-terminus of BICD2 results in a structural change that facilitates the interaction with dynein and dynactin, allowing the formation of active motor complexes. Moreover, modified BICD2 preferentially interacts with the nucleoporin RanBP2 once RanBP2 has been phosphorylated by CDK1. BICD2 phosphorylation is central for dynein recruitment to the nuclear envelope, centrosome tethering to the nucleus and centrosome separation in the G2 and M phases of the cell cycle. This work reveals adaptor activation through phosphorylation as crucial for the spatiotemporal regulation of dynein activity.

Specific Residues in the C-Terminal Domain of the Human Metapneumovirus Phosphoprotein Are Indispensable for Formation of Viral Replication Centers and Regulation of the Function of the Viral Polymerase Complex.

Human metapneumovirus (HMPV) is a negative-strand RNA virus that frequently causes respiratory tract infections in infants, the elderly, and the immunocompromised. A hallmark of HMPV infection is the formation of membraneless, liquid-like replication and transcription centers in the cytosol termed inclusion bodies (IBs). The HMPV phosphoprotein (P) and nucleoprotein (N) are the minimal viral proteins necessary to form IB-like structures, and both proteins are required for the viral polymerase to synthesize RNA during infection. HMPV P is a homotetramer with regions of intrinsic disorder and has several known and predicted phosphorylation sites of unknown function. In this study, we found that the P C-terminal intrinsically disordered domain (CTD) must be present to facilitate IB formation with HMPV N, while either the N-terminal intrinsically disordered domain or the central oligomerization domain was dispensable. Alanine substitution at a single tyrosine residue within the CTD abrogated IB formation and reduced coimmunoprecipitation with HMPV N. Mutations to C-terminal phosphorylation sites revealed a potential role for phosphorylation in regulating RNA synthesis and P binding partners within IBs. Phosphorylation mutations which reduced RNA synthesis in a reporter assay produced comparable results in a recombinant viral rescue system, measured as an inability to produce infectious viral particles with genomes containing these single P mutations. This work highlights the critical role HMPV P plays in facilitating a key step of the viral life cycle and reveals the potential role for phosphorylation in regulating the function of this significant viral protein. IMPORTANCE Human metapneumovirus (HMPV) infects global populations, with severe respiratory tract infections occurring in infants, the elderly, and the immunocompromised. There are currently no FDA-approved therapeutics available to prevent or treat HMPV infection. Therefore, understanding how HMPV replicates is vital for the identification of novel targets for therapeutic development. During HMPV infection, viral RNA synthesis proteins localize to membraneless structures called inclusion bodies (IBs), which are sites of genome replication and transcription. The HMPV phosphoprotein (P) is necessary for IBs to form and for the virus to synthesize RNA, but it is not known how this protein contributes to IB formation or if it is capable of regulating viral replication. We show that the C-terminal domain of P is the location of a molecular interaction driving IB formation and contains potential phosphorylation sites where amino acid charge regulates the function of the viral polymerase complex.

FcγRIIA-specific DARPins as novel tools in blood cell analysis and platelet aggregation

Fc receptors are involved in a variety of physiologically and disease-relevant responses. Among them, FcγRIIA (CD32a) is known for its activating functions in pathogen recognition and platelet biology, and, as potential marker of T lymphocytes latently infected with HIV-1. The latter has not been without controversy due to technical challenges complicated by T-B cell conjugates and trogocytosis as well as a lack of antibodies distinguishing between the closely related isoforms of FcγRII. To generate high-affinity binders specific for FcγRIIA, libraries of designed ankyrin repeat proteins (DARPins) were screened for binding to its extracellular domains by ribosomal display. Counterselection against FcγRIIB eliminated binders cross-reacting with both isoforms. The identified DARPins bound FcγRIIA with no detectable binding for FcγRIIB. Their affinities for FcγRIIA were in the low nanomolar range and could be enhanced by cleavage of the His-tag and dimerization. Interestingly, complex formation between DARPin and FcγRIIA followed a two-state reaction model, and discrimination from FcγRIIB was based on a single amino acid residue. In flow cytometry, DARPin F11 detected FcγRIIA+ cells even when they made up less than 1% of the cell population. Image stream analysis of primary human blood cells confirmed that F11 caused dim but reliable cell surface staining of a small subpopulation of T lymphocytes. When incubated with platelets, F11 inhibited their aggregation equally efficient as antibodies unable to discriminate between both FcγRII isoforms. The selected DARPins are unique novel tools for platelet aggregation studies as well as the role of FcγRIIA for the latent HIV-1 reservoir.

A heme pocket aromatic quadrupole modulates gas binding to cytochrome c′-β: Implications for NO sensors

The structural basis by which gas-binding heme proteins control their interactions with NO, CO, and O2 is fundamental to enzymology, biotechnology, and human health. Cytochromes c' (cyts c') are a group of putative NO-binding heme proteins that fall into two families: the well-characterized four alpha helix bundle fold (cyts c'-α) and an unrelated family with a large beta-sheet fold (cyts c'-β) resembling that of cytochromes P460. A recent structure of cyt c'-β from Methylococcus capsulatus Bath revealed two heme pocket phenylalanine residues (Phe 32 and Phe 61) positioned near the distal gas-binding site. This feature, dubbed the "Phe cap," is highly conserved within the sequences of other cyts c'-β but is absent in their close homologs, the hydroxylamine-oxidizing cytochromes P460, although some do contain a single Phe residue. Here, we report an integrated structural, spectroscopic, and kinetic characterization of cyt c'-β from Methylococcus capsulatus Bath complexes with diatomic gases, focusing on the interaction of the Phe cap with NO and CO. Significantly, crystallographic and resonance Raman data show that orientation of the electron-rich aromatic ring face of Phe 32 toward distally bound NO or CO is associated with weakened backbonding and higher off rates. Moreover, we propose that an aromatic quadrupole also contributes to the unusually weak backbonding reported for some heme-based gas sensors, including the mammalian NO sensor, soluble guanylate cyclase. Collectively, this study sheds light on the influence of highly conserved distal Phe residues on heme-gas complexes of cytochrome c'-β, including the potential for aromatic quadrupoles to modulate NO and CO binding in other heme proteins.

An optimized messenger RNA vaccine candidate protects non-human primates from Zika virus infection

Zika virus (ZIKV), an arbovirus transmitted by mosquitoes, was identified as a cause of congenital disease during a major outbreak in the Americas in 2016. Vaccine design strategies relied on limited available isolate sequence information due to the rapid response necessary. The first-generation ZIKV mRNA vaccine, mRNA-1325, was initially generated and, as additional strain sequences became available, a second mRNA vaccine, mRNA-1893, was developed. Herein, we compared the immune responses following mRNA-1325 and mRNA-1893 vaccination and reported that mRNA-1893 generated comparable neutralizing antibody titers to mRNA-1325 at 1/20th of the dose and provided complete protection from ZIKV challenge in non-human primates. In-depth characterization of these vaccines indicated that the observed immunologic differences could be attributed to a single amino acid residue difference that compromised mRNA-1325 virus-like particle formation.

Origin of the kinetic HDAC2-selectivity of an HDAC inhibitor.

A newly synthesized small molecule, KTT-1, exhibits kinetically selective inhibition of histone deacetylase 2, HDAC2, over its homologous enzyme, HDAC1. KTT-1 is hard to be released from the HDAC2/KTT-1 complex, compared to the HDAC1/KTT-1 complex and the residence time of KTT-1 in HDAC2 is longer than that in HDAC1. To explore the physical origin of this kinetic selectivity, we performed replica-exchange umbrella sampling molecular dynamics simulations for formation of both complexes. The calculated potentials of mean force suggest that KTT-1 is stably bound to HDAC2 and that it is easily disassociated from HDAC1. In the direct vicinity of the KTT-1 binding site in both enzymes, there exists a conserved loop consisting of four consecutive glycine residues (Gly304-307 for HDAC2; Gly299-302 for HDA1). The difference between the two enzymes comes from a single un-conserved residue behind this loop, namely, Ala268 in HDAC2 and Ser263 in HDAC1. The Ala268 contributes to the tight binding of KTT-1 to HDAC2 by the linear orientation of Ala268, Gly306, and one carbon atom in KTT-1. On the other hand, Ser263 cannot stabilize the binding of KTT-1 to HDAC1, because it is relatively further away from the glycine loop and because the directions of the two forces are not in line.

A Single Residue within the MCR-1 Protein Confers Anticipatory Resilience.

The envelope stress response (ESR) of Gram-negative enteric bacteria senses fluctuations in nutrient availability and environmental changes to avert damage and promote survival. It has a protective role toward antimicrobials, but direct interactions between ESR components and antibiotic resistance genes have not been demonstrated. Here, we report interactions between a central regulator of ESR viz., the two-component signal transduction system CpxRA (conjugative pilus expression), and the recently described mobile colistin resistance protein (MCR-1). Purified MCR-1 is specifically cleaved within its highly conserved periplasmic bridge element, which links its N-terminal transmembrane domain with the C-terminal active-site periplasmic domain, by the CpxRA-regulated serine endoprotease DegP. Recombinant strains harboring cleavage site mutations in MCR-1 are either protease resistant or degradation susceptible, with widely differing consequences for colistin resistance. Transfer of the gene encoding a degradation-susceptible mutant to strains that lack either DegP or its regulator CpxRA restores expression and colistin resistance. MCR-1 production in Escherichia coli imposes growth restriction in strains lacking either DegP or CpxRA, effects that are reversed by transactive expression of DegP. Excipient allosteric activation of the DegP protease specifically inhibits growth of isolates carrying mcr-1 plasmids. As CpxRA directly senses acidification, growth of strains at moderately low pH dramatically increases both MCR-1-dependent phosphoethanolamine (PEA) modification of lipid A and colistin resistance levels. Strains expressing MCR-1 are also more resistant to antimicrobial peptides and bile acids. Thus, a single residue external to its active site induces ESR activity to confer resilience in MCR-1-expressing strains to commonly encountered environmental stimuli, such as changes in acidity and antimicrobial peptides. Targeted activation of the nonessential protease DegP can lead to the elimination of transferable colistin resistance in Gram-negative bacteria. IMPORTANCE The global presence of transferable mcr genes in a wide range of Gram-negative bacteria from clinical, veterinary, food, and aquaculture environments is disconcerting. Its success as a transmissible resistance factor remains enigmatic, because its expression imposes fitness costs and imparts only moderate levels of colistin resistance. Here, we show that MCR-1 triggers regulatory components of the envelope stress response, a system that senses fluctuations in nutrient availability and environmental changes, to promote bacterial survival in low pH environments. We identify a single residue within a highly conserved structural element of mcr-1 distal to its catalytic site that modulates resistance activity and triggers the ESR. Using mutational analysis, quantitative lipid A profiling and biochemical assays, we determined that growth in low pH environments dramatically increases colistin resistance levels and promotes resistance to bile acids and antimicrobial peptides. We exploited these findings to develop a targeted approach that eliminates mcr-1 and its plasmid carriers.

Identifying Key Residues in Lysine Decarboxylase for Soluble Expression Using Consensus Design Soluble Mutant Screening (ConsenSing)

Although recent advances in deep learning approaches for protein engineering have enabled quick prediction of hot spot residues improving protein solubility, the predictions do not always correspond to an actual increase in solubility under experimental conditions. Therefore, developing methods that rapidly confirm the linkage between computational predictions and empirical results is essential to the success of improving protein solubility of target proteins. Here, we present a simple hybrid approach to computationally predict hot spots possibly improving protein solubility by sequence-based analysis and empirically explore valuable mutants using split GFP as a reporter system. Our approach, Consensus design Soluble Mutant Screening (ConsenSing), utilizes consensus sequence prediction to find hot spots for improvement of protein solubility and constructs a mutant library using Darwin assembly to cover all possible mutations in one pot but still keeps the library as compact as possible. This approach allowed us to identify multiple mutants of Escherichia coli lysine decarboxylase, LdcC, with substantial increases in soluble expression. Further investigation led us to pinpoint a single critical residue for the soluble expression of LdcC and unveiled its mechanism for such improvement. Our approach demonstrated that following a protein's natural evolutionary path provides insights to improve protein solubility and/or increase protein expression by a single residue mutation, which can significantly change the profile of protein solubility.

Arabidopsis COPT1 copper transporter uses a single histidine to regulate transport activity and protein stability

Copper acquisition and subsequent delivery to target proteins are essential for many biological processes. However, the cellular levels of this trace element must be controlled because of its potential toxicity. The COPT1 protein rich in potential metal-binding amino acids functions in high affinity copper uptake at the plasma membrane of Arabidopsis cells. The functional role of these putative metal-binding residues is largely unknown. Through truncations and site-directed mutagenesis, we identified His43, a single residue within the extracellular N-terminal domain as absolutely critical for copper uptake of COPT1. Substitution of this residue with leucine, methionine or cysteine almost inactivated transport function of COPT1, implying that His43 fails to serves as a copper ligand in the regulation of COPT1 activity. Deletion of all extracellular N-terminal metal-binding residues completely blocked copper-stimulated degradation but did not alter the subcellular distribution and multimerization of COPT1. Although mutation of His43 to alanine and serine retained the transporter activity in yeast cells, the mutant protein was unstable and degraded in the proteasome in Arabidopsis cells. Our results demonstrate a pivotal role for the extracellular residue His43 in high affinity copper transport activity, and suggest common molecular mechanisms for regulating both metal transport and protein stability of COPT1.

Determination of multi-class pesticide residues in food commodities from Gujarat, India and evaluation of acute and chronic health risk.

An increasing concern for food safety has drawn attention to the overuse of pesticides, which pose a risk to public health. The present study determined 61 pesticide residues in 120 samples of cauliflower, green chili, cucumber, grapes, bananas and mangoes samples, and these were collected from markets in Ahmedabad, Gandhinagar, Surendranagar, Anand and Sabarkantha districts of Gujarat state, India. The samples were extracted and analyzed using ultra-high performance liquid chromatography-time of flight mass spectrometry (UHPLC-q-TOF-MS) and Gas chromatography-tandem mass spectrometry (GC-MS/MS). In addition, the health risk assessment associated with pesticide residues were evaluated by calculating the Hazard Quotient (HQ) and Hazard Index (HI), which indicate a value of less than 1 is safe for consumption. Out of 61 pesticide residues, 29 residues were detected in 107 samples; 68 samples showed multiple residues, and 39 samples found a single residue. Pesticides such as dimethoate, λ-cyhalothrin, fenvalerate, bifenthrin, and cyfluthrin were frequently detected in samples. HI in adults and adolescents confirmed a value less than 1 in cauliflower, cucumber, grapes and mango samples and greater than 1 in green chili and banana samples, respectively. The overall results depicted that, no considerable risk was observed in the selected food commodities. However, green chili and banana samples were found to exhibit marginal risk to human health. As a result, proper application, implementation of control plans, and continuous monitoring are required to prevent the risk and safeguard human health.

Abstract 3731: The polyamine-hypusine circuit controls an oncogenic translational program essential for malignant transformation in MYC-driven lymphoma

Abstract The MYC oncoprotein is activated in a broad spectrum of human malignancies and transcriptionally reprograms the genome to drive cancer cell growth. Given these functions, it is unclear if targeting a single effector of MYC will have therapeutic benefit. MYC activates the polyamine-hypusine circuit, which post-translationally modifies a single lysine residue (Lys-50) of eukaryotic translation initiation factor eIF5A in a process coined hypusination via two enzymes,deoxyhypusine synthase (DHPS) and deoxyhypusine hydroxylase (DOHH). The roles of this circuit in cancer are unclear. Here we report essential intrinsic roles for hypusinated eIF5A in the development and maintenance of MYC-driven lymphoma, where loss of eIF5A hypusination completely abolishes malignant transformation of MYC-overexpressing B cells in transgenic mice predestined to develop lymphoma. Mechanistically, integrating RNA-seq, Ribo-seq and proteomic analyses revealed that efficient translation of select targets is dependent upon eIF5A hypusination, including regulators of G1-to-S phase cell cycle progression and DNA replication. Thus, this circuit controls MYC’s proliferative response at several levels and is activated in many tumor types. These findings suggest the hypusine circuit as a therapeutic target for a broad spectrum of malignancies. Citation Format: Shima Nakanishi, Jiannong Li, Anders E. Berglund, Youngchul Kim, Yonghong Zhang, Ling Zhang, Chunying Yang, Raghavendra G. Mirmira, John L. Cleveland. The polyamine-hypusine circuit controls an oncogenic translational program essential for malignant transformation in MYC-driven lymphoma. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3731.