Substrate-binding Domain Research Articles

A novel and unique ATP hydrolysis to AMP by a human Hsp70 Binding immunoglobin protein (BiP).

Hsp70s are ubiquitous and highly conserved molecular chaperones. They play crucial roles in maintaining cellular protein homeostasis. It is well established that Hsp70s use the energy of ATP hydrolysis to ADP to power the chaperone activity regardless of the cellular locations and isoforms. Binding immunoglobin protein (BiP), the major member of Hsp70s in the endoplasmic reticulum, is essential for protein folding and quality control. Unexpectedly, our structural analysis of BiP demonstrated a novel ATP hydrolysis to AMP during crystallization under the acidic conditions. Our biochemical studies confirmed this newly discovered ATP to AMP hydrolysis in solutions. Unlike the canonical ATP to ADP hydrolysis observed for Hsp70s, this ATP hydrolysis to AMP depends on the substrate-binding domain of BiP and is inhibited by the binding of a peptide substrate. Intriguingly, this ATP to AMP hydrolysis is unique to BiP, not shared by two representative Hsp70 proteins from the cytosol. Taken together, this novel and unique ATP to AMP hydrolysis may provide a potentially new direction for understanding the activity and cellular function of BiP.

Structural and mechanistic basis of the high catalytic activity of monooxygenase Tet(X4) on tigecycline

BackgroundTigecycline is a tetracycline derivative that constitutes one of the last-resort antibiotics used clinically to treat infections caused by both multiple drug-resistant (MDR) Gram-negative and Gram-positive bacteria. Resistance to this drug is often caused by chromosome-encoding mechanisms including over-expression of efflux pumps and ribosome protection. However, a number of variants of the flavin adenine dinucleotide (FAD)-dependent monooxygenase TetX, such as Tet(X4), emerged in recent years as conferring resistance to tigecycline in strains of Enterobacteriaceae, Acinetobacter sp., Pseudomonas sp., and Empedobacter sp. To date, mechanistic details underlying the improvement of catalytic activities of new TetX enzymes are not available.ResultsIn this study, we found that Tet(X4) exhibited higher affinity and catalytic efficiency toward tigecycline when compared to Tet(X2), resulting in the expression of phenotypic tigecycline resistance in E. coli strains bearing the tet(X4) gene. Comparison between the structures of Tet(X4) and Tet(X4)-tigecycline complex and those of Tet(X2) showed that they shared an identical FAD-binding site and that the FAD and tigecycline adopted similar conformation in the catalytic pocket. Although the amino acid changes in Tet(X4) are not pivotal residues for FAD binding and substrate recognition, such substitutions caused the refolding of several alpha helixes and beta sheets in the secondary structure of the substrate-binding domain of Tet(X4), resulting in the formation of a larger number of loops in the structure. These changes in turn render the substrate-binding domain of Tet(X4) more flexible and efficient in capturing substrate molecules, thereby improving catalytic efficiency.ConclusionsOur works provide a better understanding of the molecular recognition of tigecycline by the TetX enzymes; these findings can help guide the rational design of the next-generation tetracycline antibiotics that can resist inactivation of the TetX variants.

Characterization and functional analysis of RdDFR1 regulation on flower color formation in Rhododendron delavayi

Rhododendron delavayi is a popular ornamental plant with globular flowers noted for their bright red color, but very limited studies have been reported on its flower color formation. In this study, we successfully isolated a novel DFR gene (RdDFR1) from red flowers of Rhododendron delavayi. Multiple sequence alignments revealed that RdDFR1 had the conserved NADP and substrate binding domain, and was classified into Asn-type DFR. Meanwhile, quantitative real-time PCR analysis showed that transcript levels of RdDFR1 matched the accumulation patterns of anthocyanins during flower development, hinting its potential role involved in anthocyanin biosynthesis. Then in vitro enzymatic analysis indicated that recombinant RdDFR1 protein could catalyze the production of leucoanthocyanidins from dihydroquercetin and dihydromyricetin. Furthermore, the in planta assay, using Arabidopsis thaliana dfr mutant (tt3-1) and tobacco, displayed that RdDFR1 transgenes recovered the defective proanthocyanidin and anthocyanin biosynthesis at seed coats, hypocotyl as well as cotyledon, and altered the flowers color of tobacco from pale pink to dark pink which demonstrated its function as dihydroflavonol 4-reductase in vivo. In summary, our findings suggest that RdDFR1 plays a crucial role in the biosynthesis of anthocyanin and will also make a contribution to understand the mechanisms of flower color formation in Rhododendron delavayi.

Structure of Pseudomonas aeruginosa spermidine dehydrogenase: a polyamine oxidase with a novel heme-binding fold.

The opportunistic pathogen Pseudomonas aeruginosa can utilize polyamines (including putrescine, cadaverine, 4-aminobutyrate, spermidine, and spermine) as its sole source of carbon and nitrogen. Spermidine dehydrogenase (SpdH) is a component of one of the two polyamine utilization pathways identified in P. aeruginosa, but little is known about its structure and function. Here, we report the first crystal structure of SpdH from P. aeruginosa to 1.85 Å resolution. The resulting core structure confirms that SpdH belongs to the polyamine oxidase (PAO) family with flavin-binding and substrate-binding domains. A unique N-terminal extension wraps around the flavin-binding domain of SpdH and is required for heme binding, placing a heme cofactor in close proximity to the FAD cofactor. Structural and mutational analysis reveals that residues in the putative active site at the re side of the FAD isoalloxazine ring form part of the catalytic machinery. PaSpdH features an unusual active site and lacks the conserved lysine that forms part of a lysine-water-flavin N5 atom interaction in other PAO enzymes characterized to date. Mutational analysis further confirms that heme is required for catalytic activity. This work provides an important starting point for understanding the role of SpdH, which occurs universally in P. aeruginosa strains, in polyamine metabolism.

Multifaceted N-Degron Recognition and Ubiquitylation by GID/CTLH E3 Ligases

N-degron E3 ubiquitin ligases recognize specific residues at the N-termini of substrates. Although molecular details of N-degron recognition are known for several E3 ligases, the range of N-terminal motifs that can bind a given E3 substrate binding domain remains unclear. Here, we discovered capacity of Gid4 and Gid10 substrate receptor subunits of yeast “GID”/human “CTLH” multiprotein E3 ligases to tightly bind a wide range of N-terminal residues whose recognition is determined in part by the downstream sequence context. Screening of phage displaying peptide libraries with exposed N-termini identified novel consensus motifs with non-Pro N-terminal residues binding Gid4 or Gid10 with high affinity. Structural data reveal that conformations of flexible loops in Gid4 and Gid10 complement sequences and folds of interacting peptides. Together with analysis of endogenous substrate degrons, the data show that degron identity, substrate domains harboring targeted lysines, and varying E3 ligase higher-order assemblies combinatorially determine efficiency of ubiquitylation and degradation.

PDI Cleavage of Disulfide Bonds within the TP Receptor Inhibits Signaling through Gα 13

G-protein coupled receptors (GPCRs) are the most abundant superfamily of cell surface receptors. Approximately 35% of approved drugs target GPCRs and class A GPCRs account for ~85% of this superfamily. Class A GPCRs are characterized in part by two highly conserved disulfides in their extracellular domains that are thought to stabilize protein structure. Whether these disulfides can be enzymatically modified to influence G-protein signaling is not known. We and others have previously shown that disulfide bond modification by thiol isomerases such as protein disulfide isomerase (PDI) represent a previously unrecognized level of control of thrombus formation. We therefore evaluated the ability of recombinant PDI (rPDI) to modulate signaling through modification of the canonical disulfides within platelet GPCRs. Exposure of platelets to rPDI had no effect on stimulation through PAR1, PAR4, or the α 2A-adrenergic receptor. In contrast, rPDI exposure dramatically decreased platelet activation induced by the TP receptor agonists U46619 or arachidonic acid, implicating rPDI-mediated modulation of TP receptor signaling. Consistent with this finding, rPDI blocked U46619-mediated activation of α IIbβ 3, α-granule release, and dense granule release. Conversely, inhibition of endogenous PDI using either inhibitory antibodies or PDI-targeted small molecules enhanced TP receptor-mediated platelet aggregation and granule release, indicating that endogenous platelet PDI influences TP receptor signaling. Inhibition of TP receptor-mediated signaling required PDI active site cysteines since rPDI mutants lacking these cysteines lost inhibitory activity. Evaluation of the inhibitory activity of different PDI fragments showed that the PDI substrate binding domain is also critical for inhibitory activity. The ability to inhibit signaling through the TP receptor was specific for rPDI since incubation with other recombinant thiol isomerases including ERp57, ERp5, and ERp72 had no effect. To determine the specific modifications to TP receptor canonical disulfides induced by PDI, HEK cells were transfected with TP receptor and exposed to rPDI. TP receptor was subsequently immunopreciptated and disulfide-linked peptide analysis was performed using mass spectrometry. Compared to untreated controls, TP receptor exposed to rPDI demonstrated cleavage of Cys11-Cys102 and Cys105-Cys183 bonds and the generation of a new Cys102-Cys183 bond. To determine how modification of the disulfide bonding pattern affected signaling through the TP receptor, we evaluated signaling through specific Gα subunits in platelets. The platelet TP receptor signals through Gα q (which couples to phospholipase and increases calcium flux) and Gα 13 (which couples to RhoA and myosin light chain kinase). PDI-mediated cleavage of the platelet TP receptor resulted in biased signaling, with substantial inhibition of G α13-mediated RhoA-GTP activation and myosin light chain phosphorylation and little effect on Gα q-mediated calcium flux. These results show how PDI can modify platelet signaling and represent the first demonstration that a thiol isomerase can modulate the function of a GPCR via rearrangement of canonical disulfide bonds. DisclosuresNo relevant conflicts of interest to declare.

Physics-based modeling provides predictive understanding of selectively promiscuous substrate binding by Hsp70 chaperones.

To help cells cope with protein misfolding and aggregation, Hsp70 molecular chaperones selectively bind a variety of sequences ("selective promiscuity"). Statistical analyses from substrate-derived peptide arrays reveal that DnaK, the E. coli Hsp70, binds to sequences containing three to five branched hydrophobic residues, although otherwise the specific amino acids can vary considerably. Several high-resolution structures of the substrate -binding domain (SBD) of DnaK bound to peptides reveal a highly conserved configuration of the bound substrate and further suggest that the substrate-binding cleft consists of five largely independent sites for interaction with five consecutive substrate residues. Importantly, both substrate backbone orientations (N- to C- and C- to N-) allow essentially the same backbone hydrogen-bonding and side-chain interactions with the chaperone. In order to rationalize these observations, we performed atomistic molecular dynamics simulations to sample the interactions of all 20 amino acid side chains in each of the five sites of the chaperone in the context of the conserved substrate backbone configurations. The resulting interaction energetics provide the basis set for deriving a predictive model that we call Paladin (Physics-based model of DnaK-Substrate Binding). Trained using available peptide array data, Paladin can distinguish binders and nonbinders of DnaK with accuracy comparable to existing predictors and further predicts the detailed configuration of the bound sequence. Tested using existing DnaK-peptide structures, Paladin correctly predicted the binding register in 10 out of 13 substrate sequences that bind in the N- to C- orientation, and the binding orientation in 16 out of 22 sequences. The physical basis of the Paladin model provides insight into the origins of how Hsp70s bind substrates with a balance of selectivity and promiscuity. The approach described here can be extended to other Hsp70s where extensive peptide array data is not available.

Characterizing the potentially neuronal acetylcholinesterase reactivity toward chiral pyraclofos: Enantioselective insights from spectroscopy, in silico docking, molecular dynamics simulation and per-residue energy decomposition studies

Chiral organophosphorus agents are distributed ubiquitously in the environment, but the neuroactivity of these asymmetric chemicals to humans remains uncertain. This scenario was to explore the stereoselective neurobiological response of human acetylcholinesterase (AChE) to chiral pyraclofos at the enantiomeric scale, and then decipher the microscopic basis of enantioselective neurotoxicity of pyraclofos enantiomers. The results indicated that (R)-/(S)-pyraclofos can form the bioconjugates with AChE with a stoichiometric ratio of 1:1, but the neuronal affinity of (R)-pyraclofos (K = 6.31 × 104 M−1) with AChE was larger than that of (S)-pyraclofos (K = 1.86 × 104 M−1), and significant enantioselectivity was existed in the biochemical reaction. The modes of neurobiological action revealed that pyraclofos enantiomers were situated at the substrate binding domain, and the strength of the overall noncovalent bonds between (S)-pyraclofos and the residues was weaker than that of (R)-pyraclofos, resulting in the high inhibitory effect of (R)-pyraclofos toward the activity of AChE. Dynamic enantioselective biointeractions illustrated that the intervention of inherent conformational flexibility in the AChE-(R)-pyraclofos was greater than that of the AChE-(S)-pyraclofos, which arises from the big spatial displacement and the conformational flip of the binding domain composed of the residues Thr-64～Asn-89, Gly-122～Asp-134, and Thr-436～Tyr-449. Energy decomposition exhibited that the Gibbs free energies of the AChE-(R)-/(S)-pyraclofos were ΔG° = －37.4/－30.2 kJ mol−1, respectively, and the disparity comes from the electrostatic energy during the stereoselective neurochemical reactions. Quantitative conformational analysis further confirmed the atomic-scale computational chemistry conclusions, and the perturbation of (S)-pyraclofos on the AChE's ordered conformation was lower than that of (R)-pyraclofos, which is germane to the interaction energies of the crucial residues, e.g. Tyr-124, Tyr-337, Asp-74, Trp-86, and Tyr-119. Evidently, this attempt will contribute mechanistic information to uncovering the neurobiological effects of chiral organophosphates on the body.

Identification of three gene families coordinating the conversion between fructose-6-phosphate and fructose-1,6-bisphosphate in wheat

Saccharides are a direct energy source for most organisms and the primary components in grains of common wheat (Triticum aestivum L., 2n = 6x = 42, AABBDD). However, genes involved in the metabolism of primary saccharides such as glucose and fructose have not been fully characterized in wheat, which limits our understanding of how these genes influence wheat growth. In this study, genes coding ATP-dependent phosphofructokinase (PFK), fructose-1,6-bisphosphatase (FBP), and pyrophosphate-dependent fructose-6-phosphate 1-phosphotransferase (PFP), which participate in the conversion between fructose 6-phosphate (F-6-P) and fructose 1,6-bisphosphate (F-1,6-P2), were identified at the genome-wide level. A total of 24, 13, and 12 genes were found encoding TaPFK, TaFBP, and TaPFP, respectively. All predicted peptides of these genes exhibited conserved substrate-binding domain, suggesting they are active enzymes in vivo. Transcriptome data ranked the gene levels as follows: TacyFBP-1 > TacpFBP-1 > TaPFPα-2 ≈ TaPFPβ >> TaPFK-1 ≈ TaPFK-5 >> all remaining genes at different developmental stages of wheat. In the three tapfp-a, b, and d knockout lines, there was a decrease in the plant height, anther length, and thousand-grain mass, while the percentage of abnormal pollen increased compared to that of wild type cv. Huapei3 (HP3). During germination, tapfpβ-a exhibited a lower germination rate, shorter coleoptile and primary root length, and higher fructose content than HP3, tapfpβ-b, and tapfpβ-d lines. Expressions were ranked as follows: TaPFK-5 ≈ TaPFPα-2 >> TaPFPα-1 ≈ TaPFPβ > TacyFBP-1 ≈ TaPFK-7, 9 in HP3. All these genes were downregulated during the 24 - 96 h germinating process in three mutant lines. Collectively, main TaPFK, TaFBP, and TaPFP members cooperated during wheat growth, while TaPFPβ knockout decreased wheat vitality. Results from this study can aid more systematic studies of the physiological and molecular functions of TaPFK, TaFBP, and TaPFP.

Selective promiscuity in the binding of E. coli Hsp70 to an unfolded protein

Heat shock protein 70 (Hsp70) chaperones bind many different sequences and discriminate between incompletely folded and folded clients. Most research into the origins of this "selective promiscuity" has relied on short peptides as substrates to dissect the binding, but much less is known about how Hsp70s bind full-length client proteins. Here, we connect detailed structural analyses of complexes between the Escherichia coli Hsp70 (DnaK) substrate-binding domain (SBD) and peptides encompassing five potential binding sites in the precursor to E. coli alkaline phosphatase (proPhoA) with SBD binding to full-length unfolded proPhoA. Analysis of SBD complexes with proPhoA peptides by a combination of X-ray crystallography, methyl-transverse relaxation optimized spectroscopy (methyl-TROSY), and paramagnetic relaxation enhancement (PRE) NMR and chemical cross-linking experiments provided detailed descriptions of their binding modes. Importantly, many sequences populate multiple SBD binding modes, including both the canonical N to C orientation and a C to N orientation. The favored peptide binding mode optimizes substrate residue side-chain compatibility with the SBD binding pockets independent of backbone orientation. Relating these results to the binding of the SBD to full-length proPhoA, we observe that multiple chaperones may bind to the protein substrate, and the binding sites, well separated in the proPhoA sequence, behave independently. The hierarchy of chaperone binding to sites on the protein was generally consistent with the apparent binding affinities observed for the peptides corresponding to these sites. Functionally, these results reveal that Hsp70s "read" sequences without regard to the backbone direction and that both binding orientations must be considered in current predictive algorithms.

Chloroplast SRP43 autonomously protects chlorophyll biosynthesis proteins against heat shock.

The assembly of light-harvesting chlorophyll-binding proteins (LHCPs) is coordinated with chlorophyll biosynthesis during chloroplast development. The ATP-independent chaperone known as chloroplast signal recognition particle 43 (cpSRP43) mediates post-translational LHCP targeting to the thylakoid membrane and also participates in tetrapyrrole biosynthesis (TBS). How these distinct actions of cpSRP43 are controlled has remained unclear. Here, we demonstrate that cpSRP43 effectively protects several TBS proteins from heat-induced aggregation and enhances their stability during leaf greening and heat shock. While the substrate-binding domain of cpSRP43 is sufficient for chaperoning LHCPs, the stabilization of TBS clients requires the chromodomain 2 of the protein. Strikingly, cpSRP54-which activates cpSRP43's LHCP-targeted function-inhibits the chaperone activity of cpSRP43 towards TBS proteins. High temperature weakens the interaction of cpSRP54 with cpSRP43, thus freeing cpSRP43 to interact with and protect the integrity of TBS proteins. Our data indicate that the temperature sensitivity of the cpSRP43-cpSRP54 complex enables cpSRP43 to serve as an autonomous chaperone for the thermoprotection of TBS proteins.

Conformational equilibria in allosteric control of Hsp70 chaperones

Heat-shock proteins of 70kDa (Hsp70s) are vital for all life and are notably important in protein folding. Hsp70s use ATP binding and hydrolysis at a nucleotide-binding domain (NBD) to control the binding and release of client polypeptides at a substrate-binding domain (SBD); however, the mechanistic basis for this allostery has been elusive. Here, we first characterize biochemical properties of selected domain-interface mutants in bacterial Hsp70 DnaK. We then develop a theoretical model for allosteric equilibria among Hsp70 conformational states to explain the observations: a restraining state, Hsp70R-ATP, restricts ATP hydrolysis and binds peptides poorly, whereas a stimulating state, Hsp70S-ATP, hydrolyzes ATP rapidly and has high intrinsic substrate affinity but rapid binding kinetics. We support this model for allosteric regulation with DnaK structures obtained in the postulated stimulating state S with biochemical tests of the S-state interface and with improved peptide-binding-site definition in an R-state structure.

A novel sulfamethoxazole derivative as an inhibitory agent against HSP70: A combination of computational with in vitro studies

In the current study, a novel derivative of sulfamethoxazole (a sulfonamide containing anti-biotic) named ZM-093 (IUPAC name: (E)-4-((4-(bis(2-hydroxyethyl)amino)phenyl)diazenyl)-N-(5-methylisoxazole-3-yl)benzenesulfonamide) was synthesized via common diazotization-coupling reactions from sulfamethoxazole and subsequently characterized through NMR/FT-IR spectroscopy. After evaluation, the compound was geometrically optimized at the DFT level of theory with BL3YP method and 6/31++G (d,p) basis set and from the optimized structure, several molecular descriptors important in the biological reactivity of the compound, such as global reactivity parameters, molecular electrostatic potential, average local ionization energy, and drug-likeness features of the compound were computationally analyzed. The experimental in vitro investigations of the interaction between ZM-093 and heat shock protein 70 (HSP70), a protein that is highly expressed in several types of cancers, exhibited a significant inhibitory effect against the chaperone activity of HSP70 for the titled compound (P-value < 0.01) and the comparison between the experimental studies with the mentioned computational analysis, as well as molecular docking, illustrated that ZM-093 may inhibit HSP70 through binding to its substrate-binding domain. Finally, by taking all the previous results into account, a new method for assessing the inhibitory activity of ligand to HSP70 is introduced based on protonography, a recently developed method that is dependent on the catalytic activity of carbonic anhydrase on polyacrylamide gel electrophoresis.

USP2 is an SKP2 deubiquitylase that stabilizes both SKP2 and its substrates

The stability of a protein is regulated by a balance between its ubiquitylation and deubiquitylation. S-phase kinase-associated protein 2 (SKP2) is an oncogenic F-box protein that recognizes tumor suppressor substrates for targeted ubiquitylation by the E3 ligase SKP1-Cullin1-F-box and degradation by proteasome. SKP2 is itself ubiquitylated by the E3 ligases APC/CCDH1 and SCFFBXW2, and deubiquitylated by deubiquitylases (DUBs) USP10 and USP13. Given the biological significance of SKP2, it is likely that the other E3s or DUBs may also regulate its stability. Here, we report the identification and characterization of USP2 as a new DUB. We first screened a panel of DUBs and found that both USP2 and USP21 bound to endogenous SKP2, but only USP2 deubiquitylated and stabilized SKP2 protein. USP2 inactivation via siRNA knockdown or small-molecule inhibitor treatment remarkably shortened SKP2 protein half-life by enhancing its ubiquitylation and subsequent degradation. Unexpectedly, USP2-stabilized SKP2 did not destabilize its substrates p21 and p27. Mechanistically, USP2 bound to SKP2 via the leucine-rich repeat substrate-binding domain on SKP2 to disrupt the SKP2-substrate binding, leading to stabilization of both SKP2 and these substrates. Biologically, growth suppression induced by USP2 knockdown or USP2 inhibitor is partially mediated via modulation of SKP2 and its substrates. Our study revealed a new mechanism of the cross-talk among the E3–DUB substrates and its potential implication in targeting the USP2–SKP2 axis for cancer therapy.

Interdomain interactions dictate the function of the Candida albicans Hsp110 protein Msi3

Heat shock proteins of 110 kDa (Hsp110s), a unique class of molecular chaperones, are essential for maintaining protein homeostasis. Hsp110s exhibit a strong chaperone activity preventing protein aggregation (the “holdase” activity) and also function as the major nucleotide-exchange factor (NEF) for Hsp70 chaperones. Hsp110s contain two functional domains: a nucleotide-binding domain (NBD) and substrate-binding domain (SBD). ATP binding is essential for Hsp110 function and results in close contacts between the NBD and SBD. However, the molecular mechanism of this ATP-induced allosteric coupling remains poorly defined. In this study, we carried out biochemical analysis on Msi3, the sole Hsp110 in Candida albicans, to dissect the unique allosteric coupling of Hsp110s using three mutations affecting the domain–domain interface. All the mutations abolished both the in vivo and in vitro functions of Msi3. While the ATP-bound state was disrupted in all mutants, only mutation of the NBD-SBDβ interfaces showed significant ATPase activity, suggesting that the full-length Hsp110s have an ATPase that is mainly suppressed by NBD-SBDβ contacts. Moreover, the high-affinity ATP-binding unexpectedly appears to require these NBD-SBD contacts. Remarkably, the “holdase” activity was largely intact for all mutants tested while NEF activity was mostly compromised, although both activities strictly depended on the ATP-bound state, indicating different requirements for these two activities. Stable peptide substrate binding to Msi3 led to dissociation of the NBD-SBD contacts and compromised interactions with Hsp70. Taken together, our data demonstrate that the exceptionally strong NBD-SBD contacts in Hsp110s dictate the unique allosteric coupling and biochemical activities.

Yeast J-protein Sis1 prevents prion toxicity by moderating depletion of prion protein.

[PSI+] is a prion of Saccharomyces cerevisiae Sup35, an essential ribosome release factor. In [PSI+] cells, most Sup35 is sequestered into insoluble amyloid aggregates. Despite this depletion, [PSI+] prions typically affect viability only modestly, so [PSI+] must balance sequestering Sup35 into prions with keeping enough Sup35 functional for normal growth. Sis1 is an essential J-protein regulator of Hsp70 required for the propagation of amyloid-based yeast prions. C-terminally truncated Sis1 (Sis1JGF) supports cell growth in place of wild-type Sis1. Sis1JGF also supports [PSI+] propagation, yet [PSI+] is highly toxic to cells expressing only Sis1JGF. We searched extensively for factors that mitigate the toxicity and identified only Sis1, suggesting Sis1 is uniquely needed to protect from [PSI+] toxicity. We find the C-terminal substrate-binding domain of Sis1 has a critical and transferable activity needed for the protection. In [PSI+] cells that express Sis1JGF in place of Sis1, Sup35 was less soluble and formed visibly larger prion aggregates. Exogenous expression of a truncated Sup35 that cannot incorporate into prions relieved [PSI+] toxicity. Together our data suggest that Sis1 has separable roles in propagating Sup35 prions and in moderating Sup35 aggregation that are crucial to the balance needed for the propagation of what otherwise would be lethal [PSI+] prions.

'Candidatus Liberibacter asiaticus' Multimeric LotP Mediates Citrus sinensis Defense Response Activation.

‘Candidatus Liberibacter asiaticus’ is known as the most pathogenic organism associated with citrus greening disease. Since its publicized emergence in Florida in 2005, ‘Ca. L. asiaticus’ remains unculturable. Currently, a limited number of potential disease effectors have been identified through in silico analysis. Therefore, these potential effectors remain poorly characterized and do not fully explain the complexity of symptoms observed in citrus trees infected with ‘Ca. L. asiaticus.’ LotP has been identified as a potential effector and have been partially characterized. This protein retains structural homology to the substrate binding domain of the Lon protease. LotP interacts with chaperones like GroEL, Hsp40, DnaJ, and ClpX and may exercise its biological role through interactions with different proteins involved in proteostasis networks. Here, we evaluate the interactome of LotP—revealing a new protein–protein interaction target (Lon-serine protease) and its effect on citrus plant tissue integrity. We found that via protein–protein interactions, LotP can enhance Lon protease activity, increasing the degradation rate of its specific targets. Infiltration of purified LotP strained citrus plant tissue causing photoinhibition and chlorosis after several days. Proteomics analysis of LotP tissues recovering after the infiltration revealed a large abundance of plant proteins associated with the stabilization and processing of mRNA transcripts, a subset of important transcription factors; and pathways associated with innate plant defense were highly expressed. Furthermore, interactions and substrate binding module of LotP suggest potential interactions with plant proteins, most likely proteases.

Structures of the Plasmodium falciparum heat-shock protein 70-x ATPase domain in complex with chemical fragments identify conserved and unique binding sites.

Plasmodium falciparum invades erythrocytes and extensively modifies them in a manner that increases the virulence of this malaria parasite. A single heat-shock 70 kDa-type chaperone, PfHsp70-x, is among the parasite proteins exported to the host cell. PfHsp70-x assists in the formation of a key protein complex that underpins parasite virulence and supports parasite growth during febrile episodes. Previous work resolved the crystallographic structures of the PfHsp70-x ATPase and substrate-binding domains, and showed them to be highly similar to those of their human counterparts. Here, 233 chemical fragments were screened for binding to the PfHsp70-x ATPase domain, resulting in three crystallographic structures of this domain in complex with ligands. Two binding sites were identified, with most ligands binding proximal to the ATPase nucleotide-binding pocket. Although amino acids participating in direct ligand interactions are conserved between the parasite and human erythrocytic chaperones, one nonconserved residue is also present near the ligand. This work suggests that PfHsp70-x features binding sites that may be exploitable by small-molecule ligands towards the specific inhibition of the parasite chaperone.

Hsp70 in Liquid Biopsies-A Tumor-Specific Biomarker for Detection and Response Monitoring in Cancer.

Simple SummaryTumor-specific biomarkers in liquid biopsies provide useful tools for detection of tumors, monitoring of tumor responses and prediction of outcomes. Nearly all malignant solid tumor cells, but not normal cells, present the major stress-inducible Heat shock protein 70 (Hsp70) on their cell surface and actively release it into the blood in small extracellular vesicles. Therefore, vesicular Hsp70 might serve as a biomarker for viable tumor cells. Presently, no validated test system is available that allows the quantification of vesicular Hsp70 in the blood. Based on two Hsp70-specific monoclonal antibodies, we have developed the complete (comp)Hsp70 ELISA that provides a highly sensitive and reliable tool for measuring both, free and vesicular Hsp70 in the circulation of tumor patients. Hsp70 levels in the blood reflect the presence and risk characteristics of tumors and their membrane-Hsp70 status, and might be predictive for therapeutic responses.In contrast to normal cells, tumor cells of multiple entities overexpress the Heat shock protein 70 (Hsp70) not only in the cytosol, but also present it on their plasma membrane in a tumor-specific manner. Furthermore, membrane Hsp70-positive tumor cells actively release Hsp70 in small extracellular vesicles with biophysical characteristics of exosomes. Due to conformational changes of Hsp70 in a lipid environment, most commercially available antibodies fail to detect membrane-bound and vesicular Hsp70. To fill this gap and to assess the role of vesicular Hsp70 in circulation as a potential tumor biomarker, we established the novel complete (comp)Hsp70 sandwich ELISA, using two monoclonal antibodies (mAbs), that is able to recognize both free and lipid-associated Hsp70 on the cell surface of viable tumor cells and on small extracellular vesicles. The epitopes of the mAbs cmHsp70.1 (aa 451–461) and cmHsp70.2 (aa 614–623) that are conserved among different species reside in the substrate-binding domain of Hsp70 with measured affinities of 0.42 nM and 0.44 nM, respectively. Validation of the compHsp70 ELISA revealed a high intra- and inter-assay precision, linearity in a concentration range of 1.56 to 25 ng/mL, high recovery rates of spiked liposomal Hsp70 (>84%), comparable values between human serum and plasma samples and no interference by food intake or age of the donors. Hsp70 concentrations in the circulation of patients with glioblastoma, squamous cell or adeno non-small cell lung carcinoma (NSCLC) at diagnosis were significantly higher than those of healthy donors. Hsp70 concentrations dropped concomitantly with a decrease in viable tumor mass upon irradiation of patients with approximately 20 Gy (range 18–22.5 Gy) and after completion of radiotherapy (60–70 Gy). In summary, the compHsp70 ELISA presented herein provides a sensitive and reliable tool for measuring free and vesicular Hsp70 in liquid biopsies of tumor patients, levels of which can be used as a tumor-specific biomarker, for risk assessment (i.e., differentiation of grade III vs. IV adeno NSCLC) and monitoring of therapeutic outcomes.

Antiviral peptides against the main protease of SARS-CoV-2: A molecular docking and dynamics study.

The recent coronavirus outbreak has changed the world’s economy and health sectors due to the high mortality and transmission rates. Because the development of new effective vaccines or treatments against the virus can take time, an urgent need exists for the rapid development and design of new drug candidates to combat this pathogen. Here, we obtained antiviral peptides obtained from the data repository of antimicrobial peptides (DRAMP) and screened their predicted tertiary structures for the ability to inhibit the main protease of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) using multiple combinatorial docking programs, including PatchDock, FireDock, and ClusPro. The four best peptides, DRAMP00877, DRAMP02333, DRAMP02669, and DRAMP03804, had binding energies of −1125.3, −1084.5, −1005.2, and −924.2 Kcal/mol, respectively, as determined using ClusPro, and binding energies of −55.37, −50.96, −49.25, −54.81 Kcal/mol, respectively, as determined using FireDock, which were better binding energy values than observed for other peptide molecules. These peptides were found to bind with the active cavity of the SARS-CoV-2 main protease; at Glu166, Cys145, Asn142, Phe140, and Met165, in addition to the substrate-binding sites, Domain 2 and Domain 3, whereas fewer interactions were observed with Domain 1. The docking studies were further confirmed by a molecular dynamics simulation study, in which several descriptors, including the root-mean-square difference (RMSD), root-mean-square fluctuation (RMSF), solvent-accessible surface area (SASA), radius of gyration (Rg), and hydrogen bond formation, confirmed the stable nature of the peptide–main protease complexes. Toxicity and allergenicity studies confirmed the non-allergenic nature of the peptides. This present study suggests that these identified antiviral peptide molecules might inhibit the main protease of SARS-CoV-2, although further wet-lab experiments remain necessary to verify these findings.