SV-AUC as a stability-indicating method for the characterization of mRNA-LNPs.

During the SARS-CoV2 pandemic mRNA vaccines in the form of lipid nanoparticles (LNPs) containing the mRNA, have set the stage for a new area of vaccines. Analytical methods to quantify changes in size and structure of LNPs are crucial, as changes in these parameters could have implications for potency. We investigated the application of sedimentation velocity analytical ultracentrifugation (SV-AUC) as quantitative stability-indicating method to detect structural changes of mRNA-LNP vaccines upon relevant stress factors (freeze/thaw, heat and mechanical stress), in comparison to qualitative dynamic light scattering (DLS) analysis. DLS was capable to qualitatively determine size and homogeneity of mRNA-LNPs with sufficient precision. Stress factors, in particular freeze/thaw and mechanical stress, led to increased particle size and content of larger species in DLS and SV-AUC. Changes upon heat stress at 50 °C were only detected as increased flotation rates by SV-AUC. In addition, SV-AUC was able to observe changes in particle density, which cannot be detected by DLS. In conclusion, SV-AUC can be used as a highly valuable quantitative stability-indicating method for characterization of LNPs.

Binding of the HSF-1 DNA-binding domain to multimeric C. elegans consensus HSEs is guided by cooperative interactions.

The protein HSF-1 is the controlling transcription factor of the heat-shock response (HSR). Its binding to the heat-shock elements (HSEs) induces the strong upregulation of conserved heat-shock proteins, including Hsp70s, Hsp40s and small HSPs. Next to these commonly known HSPs, more than 4000 other HSEs are found in the promoter regions of C. elegans genes. In microarray experiments, few of the HSE-containing genes are specifically upregulated during the heat-shock response. Most of the 4000 HSE-containing genes instead are unaffected by elevated temperatures and coexpress with genes unrelated to the HSR. This is also the case for several genes related to the HSP chaperone system, like dnj-12, dnj-13, and hsp-1. Interestingly, several promoters of the dedicated HSR-genes, like F44E5.4p, hsp-16.48p or hsp-16.2p, contain extended HSEs in their promoter region, composed of four or five HSE-elements instead of the common trimeric HSEs. We here aim at understanding how HSF-1 interacts with the different promoter regions. To this end we purify the nematode HSF-1 DBD and investigate the interaction with DNA sequences containing these regions. EMSA assays suggest that the HSF-1 DBD interacts with most of these HSE-containing dsDNAs, but with different characteristics. We employ sedimentation analytical ultracentrifugation (SV-AUC) to determine stoichiometry, affinity, and cooperativity of HSF-1 DBD binding to these HSEs. Interestingly, most HSEs show cooperative binding of the HSF-1 DBD with up to five DBDs being bound. In most cases binding to the HSEs of inducible promoters is stronger, even though the consensus scores are not always higher. The observed high affinity of HSF-1 DBD to the non-inducible HSEs of dnj-12, suggests that constitutive expression may be supported from some promoter regions, a fact that is evident for this transcription factor, that is essential also under non-stress conditions.

Nematode CDC-37 and DNJ-13 form complexes and can interact with HSP-90.

The molecular chaperones Hsc70 and Hsp90 are required for proteostasis control and specific folding of client proteins in eukaryotic and prokaryotic organisms. Especially in eukaryotes these ATP-driven molecular chaperones are interacting with cofactors that specify the client spectrum and coordinate the ATPase cycles. Here we find that a Hsc70-cofactor of the Hsp40 family from nematodes, DNJ-13, directly interacts with the kinase-specific Hsp90-cofactor CDC-37. The interaction is specific for DNJ-13, while DNJ-12 another DnaJ-like protein of C. elegans, does not bind to CDC-37 in a similar manner. Analytical ultracentrifugation is employed to show that one CDC-37 molecule binds to a dimeric DNJ-13 protein with low micromolar affinity. We perform cross-linking studies with mass spectrometry to identify the interaction site and obtain specific cross-links connecting the N-terminal J-domain of DNJ-13 with the N-terminal domain of CDC-37. Further AUC experiments reveal that both, the N-terminal part of CDC-37 and the C-terminal domain of CDC-37, are required for efficient interaction. Furthermore, the presence of DNJ-13 strengthens the complex formation between CDC-37 and HSP-90 and modulates the nucleotide-dependent effects. These findings on the interaction between Hsp40 proteins and Hsp90-cofactors provide evidence for a more intricate interaction between the two chaperone systems during client processing.

hsp-90 and unc-45 depletion induce characteristic transcriptional signatures in coexpression cliques of C. elegans.

Nematode development is characterized by progression through several larval stages. Thousands of genes were found in large scale RNAi-experiments to block this development at certain steps, two of which target the molecular chaperone HSP-90 and its cofactor UNC-45. Aiming to define the cause of arrest, we here investigate the status of nematodes after treatment with RNAi against hsp-90 and unc-45 by employing an in-depth transcriptional analysis of the arrested larvae. To identify misregulated transcriptional units, we calculate and validate genome-wide coexpression cliques covering the entire nematode genome. We define 307 coexpression cliques and more than half of these can be related to organismal functions by GO-term enrichment, phenotype enrichment or tissue enrichment analysis. Importantly, hsp-90 and unc-45 RNAi induce or repress many of these cliques in a coordinated manner, and then several specifically regulated cliques are observed. To map the developmental state of the arrested nematodes we define the expression behaviour of each of the cliques during development from embryo to adult nematode. hsp-90 RNAi can be seen to arrest development close to the L4 larval stage with further deviations in daf-16 regulated genes. unc-45 RNAi instead leads to arrested development at young adult stage prior to the programmatic downregulation of sperm-cell specific genes. In both cases processes can be defined to be misregulated upon depletion of the respective chaperone. With most of the defined gene cliques showing concerted behaviour at some stage of development from embryo to late adult, the "clique map" together with the clique-specific GO-terms, tissue and phenotype assignments will be a valuable tool in understanding concerted responses on the genome-wide level in Caenorhabditis elegans.

HSP-90/kinase complexes are stabilized by the large PPIase FKB-6.

Protein kinases are important regulators in cellular signal transduction. As one major type of Hsp90 client, protein kinases rely on the ATP-dependent molecular chaperone Hsp90, which maintains their structure and supports their activation. Depending on client type, Hsp90 interacts with different cofactors. Here we report that besides the kinase-specific cofactor Cdc37 large PPIases of the Fkbp-type strongly bind to kinase•Hsp90•Cdc37 complexes. We evaluate the nucleotide regulation of these assemblies and identify prominent interaction sites in this quaternary complex. The synergistic interaction between the participating proteins and the conserved nature of the interaction suggests functions of the large PPIases Fkbp51/Fkbp52 and their nematode homolog FKB-6 as contributing factors to the kinase cycle of the Hsp90 machinery.

head-bent resistant Hsc70 variants show reduced Hsp40 affinity and altered protein folding activity.

The molecular chaperone Hsc70 performs essential tasks by folding proteins. Hsc70 is driven by the hydrolysis of ATP and tuned by the association with various co-chaperones. One such cofactor is the nematode nucleotide exchange factor UNC-23, whose mutation disrupts muscle attachment and induces a severe head-bent phenotype in C.elegans. Interestingly, four mutations in Hsc70 can suppress this phenotype, but the molecular mechanism underlying this suppression is unknown. Here we characterize these four suppressor variants, Hsc70 D233N, S321F, A379V and D384N. In vitro only Hsc70 S321F shows reduced stability and altered nucleotide interaction, but all mutations affect the ATPase stimulation. In particular, Hsc70 D233N and Hsc70 A379V show strongly reduced interactions with DNJ-12 and DNJ-13. Nucleotide exchange factor binding instead is barely influenced in Hsc70 D233N, A379V and D384N and their chaperone activity is preserved. Molecular dynamics simulations suggest that effects in Hsc70 S321F and Hsc70 A379V originate from steric clashes in the vicinity of the mutation site, while D233N disrupts a salt bridge that contributes to Hsc70's nucleotide-induced conformational changes. In summary, the analyzed mutants show altered ATPase and refolding activity caused by changes in Hsp40 binding.

Genome-wide analysis of yeast expression data based on a priori generated co-regulation cliques.

DNA microarrays are highly sensitive tools to evaluate the gene expression status of organismic samples and standardized array formats exist for many different sample types. Differential expression studies usually utilize the strongest upor downregulated genes to generate networks visualizing the relationships among these genes. To include all yeast genes in one analysis and to get broader information on all cellular responses, we test a priori input of predefined genome-wide expression cliques and subsequent statistical analysis of the expression data. To this end, we generate a set of 72 co-regulation cliques using the information from 3196 microarray experiments. The obtained cliques performed highly significant in gene ontology and transcription factor enrichment analyses. We then tested the clique set on individual microarray experiments reporting on responses to pheromone, glycerol versus glucose based growth and the cellular response to heat. In all cases a highly significant determination of affected expression cliques was possible based on their average expression differences, the positions of their genes within hit rankings (UpRegScore) or the enrichment of the Top200 hits in certain cliques. The 72 cliques were finally used to compare experiments, which reported on the transcriptional response to polyglutamine proteins of different lengths. Using the predefined clique set it is possible to identify with high sensitivity and good significance sample and condition specific changes to gene expression. We thus conclude that an analysis, starting with these 72 preformed expression cliques, can complement traditional microarray analyses by visualizing the entire response on a static genome-wide gene set.

Activity of a Holin-Endolysin System in the Insecticidal Pathogenicity Island of Yersinia enterocolitica.

Yersinia enterocolitica is a pathogen that causes gastroenteritis in humans. Because of its low-temperature-dependent insecticidal activity, it can oscillate between invertebrates and mammals as host organisms. The insecticidal activity of strain W22703 is associated with a pathogenicity island of 19 kb (Tc-PAI Ye ), which carries regulators and genes encoding the toxin complex (Tc). The island also harbors four phage-related and highly conserved genes of unknown functions, which are polycistronically transcribed. Two open reading frames showed significant homologies to holins and endolysins and exhibited lytic activity in Escherichia coli cells upon overexpression. When a set of Yersinia strains was tested in an equivalent manner, highly diverse susceptibilities to lysis were observed, and some strains were resistant to lysis. If cell lysis occurred (as demonstrated by membrane staining), it was more pronounced when two accessory elements of the cassette coding for an i-spanin and an o-spanin were included in the overexpression construct. The pore-forming function of the putative holin, HolY, was demonstrated by complementation of the lysis defect of a phage λ S holin mutant. In experiments performed with membrane preparations, ElyY exhibited high specificity for W22703 peptidoglycan, with a cleavage activity resembling that of lysozyme. Although the functionality of the lysis cassette from Tc-PAI Ye was demonstrated in this study, its biological role remains to be elucidated.IMPORTANCE The knowledge of how pathogens survive in the environment is pivotal for our understanding of bacterial virulence. The insecticidal and nematocidal activity of Yersinia spp., by which the bacteria gain access to nutrients and thus improve their environmental fitness, is conferred by the toxin complex (Tc) encoded on a highly conserved pathogenicity island termed Tc-PAI Ye While the regulators and the toxin subunits of the island had been characterized in some detail, the role of phage-related genes within the island remained to be elucidated. Here, we demonstrate that this cassette encodes a holin, an endolysin, and two spanins that, at least upon overexpression, lyse Yersinia strains.

Metabolic engineering to expand the substrate spectrum of Pseudomonas putida toward sucrose.

Sucrose is an important disaccharide used as a substrate in many industrial applications. It is a major component of molasses, a cheap by-product of the sugar industry. Unfortunately, not all industrially relevant organisms, among them Pseudomonas putida, are capable of metabolizing sucrose. We chose a metabolic engineering approach to circumvent this blockage and equip P. putida with the activities necessary to consume sucrose. Therefore, we constructed a pair of broad-host range mini-transposons (pSST - sucrose splitting transposon), carrying either cscA, encoding an invertase able to split sucrose into glucose and fructose, or additionally cscB, encoding a sucrose permease. Introduction of cscA was sufficient to convey sucrose consumption and the additional presence of cscB had no further effect, though the sucrose permease was built and localized to the membrane. Sucrose was split extracellularly by the activity of the invertase CscA leaking out of the cell. The transposons were also used to confer sucrose consumption to Cupriavidus necator. Interestingly, in this strain, CscB acted as a glucose transporter, such that C. necator also gained the ability to grow on glucose. Thus, the pSST transposons are functional tools to extend the substrate spectrum of Gram-negative bacterial strains toward sucrose.