FEBS Letters

Mycobacterium tuberculosis (M. tb) has a complex cell wall, largely composed of mycolic acids and long-chain fatty acids that play a crucial role in maintaining its integrity and permeability. This complex lipid structure has a role in abrogating the process of phagosome-lysosome fusion and infection establishment. The M. tb desaturase A1 (DesA1) catalyzes the introduction of position-specific double bonds, a key step in the biosynthesis of a diverse range of mycolic acids. We have previously demonstrated that M. tb DesA1 is a Ca2+-binding protein, belonging to the extended {beta}{gamma}-crystallin superfamily. Using a combination of biophysical and genetic approaches, we investigated the structural and functional significance of Ca2+ binding on DesA1 activity. A protein unfolding assay of the protein in the presence and absence of Ca2+ shows that Ca2+ binding imparts structural stability to DesA1. To identify the role of Ca2+, we introduced mutations at key residues in the identified Ca2+-binding motif of DesA1 and generated F303A, E304Q, and F303A-E304Q variants of DesA1. We identified F303 as a hot point which disables the protein for Ca2+ binding. Two other mutations E304Q and F303A-E304Q showed reduced Ca2+ binding. Complementation of a conditionally complemented desA1 deletion mutant strain of Mycobacterium smegmatis with these mutants, either failed to complement its growth phenotype or led to a compromise in complementation. In addition, the F303A and F303A-E304Q complements exhibit increased sensitivity to isoniazid, a first-line anti-tubercular drug, pointing to a cell wall permeability defect in these strains. Our findings highlight the critical importance of Ca2+ in the functioning of DesA1 and its implicit role in the maintenance of mycobacterial cellular integrity.

Translation is a crucial regulatory mechanism involved in several diseases, including cancer, where pro-inflammatory conditions within the microenvironment have been shown to modulate the translation of specific mRNAs. In the present study, we focused on the regulation of insulin growth factor-like family member 1 (IGFL1) in MCF7 breast cancer cells in response to pro-inflammatory IL-1{beta} and observed an induction of both transcription and translation. We characterized the 3 untranslated region as regulatory hub for the post-transcriptional regulation and identified a distinct G-rich region to confer the IL-1{beta}-dependent translational increase. Our study therefore provides new insights into the translation regulation of IGFL1 in the context of an inflammatory tumor microenvironment.

C9ORF72 is involved in multiple neuronal functions and a major factor in Amyotrophic Lateral Sclerosis, a fatal neurodegenerative disease. Pathogenicity is implemented through an intronic repeat expansion in the genes mRNA leader that produces long, repetitive RNA and dipeptides when translated through a non-canonical mechanism. In contrast, despite the presence of ribosome-occupied, regulatory elements in the mRNA leader, nothing is known about C9ORF72 translation regulation under normal conditions. Surprisingly, when analyzing a series of mutants of the C9ORF72 mRNA leader, we found that translation of C9ORF72 is tightly regulated through a multi-layer system. First, non-canonical, multi-initiation upstream open reading frames (nc-uORFs) in all three frames, a start-stop element in frame 2, and a canonical uORF in frame 1work together to keep baseline C9ORF72 translation very low. Second, a strong secondary structure enhances these repressive elements, mainly the two nc-uORFs in frames 0 and 2. Finally, as initiation at the nc-uORFs reduces initiation at the downstream start-stop, the nc-uORFs effectively dampen the start-stops repressive function, forming a feedforward loop. We hypothesize that this buffered repressor system has likely evolved to ensure reliable, noise-insensitive expression of this critical regulator of neuronal function.

Purple non-sulfur bacteria (PNSB) are metabolically versatile microorganisms that inhabit diverse environments by taking advantage of a remarkably efficient photosynthetic machinery. In this work, we describe the creation of a workflow for experimental studies that aim to compare the energy transfer mechanisms occurring within structural variants of the light-harvesting complex 2 (LH2) of PNSB. Through the creation of a library of LH2 variants using site-directed mutagenesis, we engineered proteins with different spectral properties to be expressed in a LH2-defficient mutant of the model PNSB Rhodobacter sphaeroides. We validated this approach by reproducing a previously described mutant exhibitng a blue-shift, in addition to identifying a novel mutant exhibiting a red-shift of the B850 absorption peak. We characterised the fluorescence lifetime of the purified LH2 spectral variants in vitro, and performed a bacterial growth assay to assess the fitness of the LH2 variants in vivo under oversaturating light conditions. Our results suggest that the LH2s variants expressed by PNSB in nature reflect the intricate tunning of their quantum properties not towards the fastest energy transfer but towards the optimum light-harvesting efficiency which is defined by diverse environmental factors. Statement of significanceIn this work we report a platform for the systematic investigation of the mutational landscape of light harvesting complexes forming part of the photosynthetic machinery of purple non-sulfur bacteria. By conducting directed evolution of selected residues of the light-harvesting antenna (LH2) of Rhodobacter sphaeroides we identified a novel mutant with distinct and red-shifted spectral properties. We characterised the energy transfer dynamics of this and a previous characterised mutant and demonstrated that the new variant confers a phenotypic growth advantage when cultured with high-intensity light. Our findings offer new insights into the mechanisms of light capture and energy transfer also bridging the in vitro observations with quantifiable fitness advantages under the conditions tested.

Synonymous codon substitution, a gene engineering approach in synthetic biology, has been effective in improving the codon composition of recombinant genes of interest based on various criteria without altering the amino acid sequence. The SARS-CoV-2 virus nucleocapsid (N) protein is a stable, conserved and highly immunogenic that is less prone to mutation during infection, making it a key antigen in in vitro diagnosis, vaccine development, immunological and structural studies. While reports have focused on applying optimized N protein for different applications, the basic parameters used by different optimization tools for choosing the best approach for the N gene synonymous codon substitution are often neglected. Here, we analyzed the influence of different synonymous codon substitution strategies on SARS-CoV-2 N-protein expression in E. coli. Using different codon optimization (CO) and harmonization (CH) tools, we predicted and compared how parameters such as GC content, Codon Adaptation Index, codon quality and number of rare codons present in these sequences affect the N-protein expression. Our results also show that Minimum Free Energy (MFE) and RNA structure of N-term and C-tail of the N-protein coding sequence influence protein folding. We then predicted that the SR-rich region of the N-protein may contribute to slowing down the elongation rate during translation. This work presents a fundamental analysis of how different optimization tools affect SARS-CoV-2 N-protein expression and folding and suggests a basic approach to choosing the best strategy for optimal expression and folding of the protein for further studies. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=60 SRC="FIGDIR/small/622014v3_ufig1.gif" ALT="Figure 1"> View larger version (15K): org.highwire.dtl.DTLVardef@1fbd7f8org.highwire.dtl.DTLVardef@11fe81borg.highwire.dtl.DTLVardef@1bf787borg.highwire.dtl.DTLVardef@17edc57_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsCodon substitution affects SARS-CoV-2 nucleocapsid (N) protein expression. SARS-CoV-2 N-protein expression varies with different codon optimization tools. RNA structures of N- and C-term impact RNA stability, protein expression and folding. SR-rich region of the N-protein may slow down elongation rate during translation.

Introns are widely used in the assembly of genetic constructions expressing transgenic proteins in eukaryotic cells for the enhancement of this expression. However, the choice of introns that can be applied for such purposes is limited by the excessively large size of the majority of natural introns (several thousand nucleotides) and therefore they cannot be cloned in a genetic construction. With the help of site-directed mutagenesis we have generated a library of short (99 nucleotides long) introns. The efficiency of these introns in the enhancement of gene expression was analyzed. As a result, a set of 12 introns was selected. The generated intros can be used for genetic constructions with high expression level of recombinant proteins.

Inteins are special proteins that auto-catalytically carry out a protein splicing reaction. Due to their ability to post-translationally modify target proteins in vitro and in vivo, they are used in different applications, ranging from protein purification to the construction of Boolean logic gates. So far inteins have been found to be either encoded by a single gene (contiguous inteins) or by two separate ones (split inteins). Previously, it has been shown that the contiguous Ssp and Rma DnaB inteins and the split Npu DnaE intein could be artificially split in three fragments and retain functionality. Here we report the identification of novel split sites within the N-terminal fragments of the Npu DnaE and gp41-1 split inteins that lead to synthetic functional three-piece versions of these inteins. These variants contribute to the toolkit of three-piece inteins that could be used in biotechnological applications based on highly-fragmented inteins.

ADP-ribosyl-transferases (ADP-ribose polymerases) PARP1 and PARP2 are critical players in DNA damage response in the nucleus. Being activated by a genotoxic stress, these enzymes utilize NAD+ to attach ADP-ribose chains to wide variety of proteins; ribosomal proteins (RPs) have been identified among the major targets of the modification in different cell lines. However, little remained known concerning the peculiarities of the reaction of RPs ADP-ribosylation itself. Here, we study ADP-ribosylation of human RPs within the large (60S) and small (40S) ribosomal subunits and those isolated from the subunits, with PARP1 and PARP2 in vitro using radioactively labeled NAD+. We fail to detect the modification of ribosome-bound RPs but observed ADP-ribosylation of certain ribosome-free RPs when we use total protein isolated from the subunits. RPs from the 60S subunit were globally more modified than those from the 40S subunit, and ADP-ribosylation of several 60S RPs (but not 40S) was considerably enhanced in the presence of histone PARylation factor 1 (HPF1). With all kind RPs, HPF1 switches the modification preferentially to their serine/tyrosine residues. Major targets of the 60S RPs ADP-ribosylation were identified as RPL4 (uL4), RPL6 (eL6) and RPL13A/RPL15 (uL13/eL15). The modification levels of particular RPs differently depend on the concentration of total RP; the most selective HPF1-dependent ADP-ribosylation occurs in RPL6 (eL6). When present simultaneously with histones, RPs win linker histone H1 in the competition for both PARPs; in contrast, core histones strongly compete with RPs for ADP-ribosylation. Possible functional assignments of ADP-ribosylation of RPs are discussed. Bullet points- Free human ribosomal proteins are PARylated by PARP1 and PARP2; - PARylation of ribosomal 60S proteins but not 40S ones is mostly HPF1-dependent; - RPL4, RPL6 and RPL13A/RPL15 are the major targets of PARylation among 60S RPs; - Linker histone H1 is a poor competitor to ribosomal proteins for PARPs; - Core histones strongly competes with ribosomal proteins for PARPs.

Aminoacyl-tRNA synthetases (aaRS) are the main enzymes of protein biosynthesis. Human glycyl-tRNA synthetase, in addition to the main function of amino acid transfer to the corresponding tRNA molecules, is also involved in the initiation of IRES I translation. All members of the enterovirus genus have this type of IRES. It is also known that the presence of point mutations in aaRS leads to the occurrence of diseases in which peripheral nerves are affected. One such disorder of the nervous system is the incurable neurodegenerative disorder Charcot-Marie-Tooth (CMT). The most studied enzyme whose mutations cause CMT is glycyl-tRNA synthetase (GlyRS). In this work, we tested the ability of various mutant forms of glycyl-tRNA synthetase associated with Charcot-Marie-Tooth syndrome to form a stable complex with IRES I. It turned out that neither catalytic activity nor the ability to form a dimer are necessary for the interaction of GlyRS with IRES.

Triosephosphate isomerase (TpiA) is widely regarded as an example of an optimally evolved enzyme due to its essential role in biological systems, its structural conservation, and its near-perfect kinetic parameters. In this study, we investigated the structural robustness of the archetypal TpiA variant from Escherichia coli using an in vitro 5- amino acid linker scanning method. The resulting library was introduced into a tpiA mutant strain for functional complementation. From this library, 15 TpiA variants that were phenotypically indistinguishable from the wild-type enzyme were selected for further analysis. Although all variants retained enzymatic activities within the wild-type range, several insertions were found in highly structured protein domains where the linker was expected to cause significant structural perturbations. Despite these potentially disruptive additions, the enzymes maintained their activity even when expressed in a dnaK mutant, suggesting that chaperones did not compensate for structural abnormalities in vivo. Additionally, when these mutant TpiA variants were produced using the PURE in vitro transcription/translation system, they exhibited enzymatic activity comparable to, and in some cases exceeding, that of the non-mutated enzyme. AlphaFold2 revealed that insertions reconstructed the local architecture of the nearby amino acid sequences. The evolutionary implications of this remarkable structural resilience are discussed.

The epidemiological importance of bacteria from the genus Mycobacterium is indisputable and the necessity to find new molecules that can inhibit their growth is urgent. The shikimate pathway, required for the synthesis of important metabolites in bacteria, represents a target for inhibitors of Mycobacterium tuberculosis growth. The aroA-encoded 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) enzyme catalyzes the sixth step of the shikimate pathway. In this study, we combined gene knockout, gene knockdown and kinetic assays to evaluate aroA gene essentiality and the vulnerability of its protein product, EPSPS synthase from Mycobacterium smegmatis (MsEPSPS), under different nutritional conditions. We demonstrate by an allelic exchange-based gene knockout approach the essentiality of MsEPSPS under rich and poor nutritional conditions. By performing gene complementation experiments with wild-type (WT) and point mutant versions of aroA gene, together with kinetic assays using WT and mutant recombinant proteins, we show that aroA gene essentiality depends on MsEPSPS activity. To evaluate MsEPSPS vulnerability, we performed gene knockdown experiments using the Clustered Regularly Interspaced Short Palindromic Repeats interference (CRISPRi) system. The experiments were performed in both rich and defined (poor) media, using three different repression forces for aroA gene. We only observed growth impairment when bacteria were grown in defined medium without supplementation of aromatic amino acids, thereby indicating that MsEPSPS vulnerability depends on the environment conditions. ImportanceWe evaluated both gene essentiality and target vulnerability of the enzyme that catalyzes the sixth step of the shikimate pathway, the aroA-encoded 5-enolpyruvylshikimate-3-phosphate synthase from Mycobacterium smegmatis (MsEPSPS). Combining gene knockout experiments and kinetic assays, we established a causal link between aroA gene essentiality and the biological function of EPSPS protein, which we advocate is an indispensable step for target validation. Moreover, we characterized MsEPSPS vulnerability under different nutritional conditions and found it is a vulnerable target only when M. smegmatis is grown under poor nutritional conditions without supplementation with aromatic amino acids. Based on our findings, we suggest that gene essentiality information should be obtained from gene knockout experiments and not knockdown approaches, as even low levels of a protein after gene silencing can lead to a different growth phenotype when compared to that under its complete absence, as was the case with aroA and MsEPSPS in our study.

For co-transformation of two plasmids, both have to possess different antibiotic selection markers. If that is not the case, normally the gene of interest (GOI) is subcloned into another vector. Here we introduce a fast and easy method to exchange the antibiotic resistance cassette (ARC) in only two PCR steps. Method SummaryTo shuttle the antibiotic resistance cassette (ARC) from one vector to another, one can amplify the ARC of interest and use the resulting PCR-product as a primer pair for the next amplification step. Simply remove parental DNA template by DpnI digestion, transform PCR product directly in E. coli cells, select transformants on an appropriate agar plate and isolate target vector by plasmid preparation.

The Ton-B dependent outer membrane (OM) transporters are responsible for active iron (Fe) import in Synechocystis sp. strain PCC 6803 (S. 6803 or WT) under Fe depletion. However, the mechanism of Fe acquisition under Fe supplemented conditions remains uncharacterised. In the present study, functional role of OMP Slr1908 in S. 6803 was addressed by insertional mutagenesis. The{Delta} slr1908 cells exhibited slower growth in the first week in comparison to the WT and displayed an absorption and 77K fluorescence spectrum typical of Fe deficiency. Indeed, the mutant had [~] 80% less Fe as confirmed by atomic absorption spectroscopy and 55Fe-radiotracer uptake. The iron deficiency was paralleled with low Mn content. The mutant had low SOD content as well as activity, less cytochromes, less chlorophyll content, less Fv/ Fm, lower ETRII and high oxidative stress in comparison to the WT at the end of first week. Interestingly, the mutant showed transcriptional upregulation of iron stress induced protein isiA and isiB signifying intracellular Fe deficiency. Upregulation of OMP Slr0042 was also observed at RNA and protein level. The results indicate that Slr1908 is a major Fe uptake OMP in S. 6803 the deletion of which leads to initial slow growth that gets partially offset by induction of other Fe importing OMPs.

Phosphorylation is fundamental to modulate protein function and stabilty. There have detected about 300000 site-specic Phosphorylation sites in over 20000 human proteins. However, only 5% of the sites have been experimenrally characterized. In this work, we investigated a phosphorylation event in AGT, an important enzyme due to its detoxifying role of glyoxylate and hundreds of mutations cause a rare disease (primary hyperoxaluria type I or PH1). We analyzed the effect of phosphomimetic mutations Ser81 on the WT proten, the common polymorpshim LM, and the most common disease-associted variants (LM-G170R and LM-I244T). Using biochemical, biophysical and cell biology approaches, we show that phosphorylation at S81 dramatically affects PLP/PMP binding pose and disrupts enzyme activity, without pertubing its subcellular location to peroxisomes. This reversible phenotype is similar to the irreversible effects of some PH1-causing mutaions. Thus, we provide evidence for a novel regulatory mechanism for PH1, in health and disease.

The chemosensory pathway HtChe2 from the marine bacterium Halomonas titanicae KHS3 controls the activity of a diguanylate cyclase. Constitutive activation of the pathway results in colony morphology alterations and increased ability to form biofilm. Such characteristics resemble the behaviour of the Wsp pathway of Pseudomonas. In this work we investigate the specificity of Htc10, the only chemoreceptor coded within the HtChe2 gene cluster. Thermal shift analyses performed with the Htc10 ligand-binding domain led to the identification of purine derivatives as ligands. This ligand-binding domain was crystallized in the presence of guanine or hypoxanthine and its structure was solved by X-ray protein crystallography. The sensor domain adopts a double-cache folding, with ligands bound to the membrane-distal pocket. A high-resolution structure of the occupied guanine-binding pocket allowed the identification of the involved residues. These residues were validated by site directed mutagenesis and thermal shift or isothermal calorimetry analyses of the protein variants. The dissociation constants for guanine or hypoxanthine of the intact domain were in the low micromolar range. To our knowledge, this is the first description of binding specificity for a chemoreceptor that controls the activity of an associated diguanylate cyclase, and opens the way for dynamic studies of the signalling behaviour of this kind of sensory complex. A comparison between Htc10 and the functionally equivalent WspA receptor from Pseudomonas revealed no significant sequence similarities. In contrast, highly conserved Htc10-like receptors were found in distant bacteria carrying HtChe2-like clusters.

Group A streptococcus (GAS), Streptococcus pyogenes manifests plethora of diseases through its explicit virulence factors. Among these, the recently deciphered MSCRAMMs, Streptococcal collagen-like (Scls) adhesins are most studied proteins in context of their biophysically stable collagenous-sequence (Gly-X-Y) despite the difference from analogous mammalian-collagen. Based on recent evidence on collagen-mimetic Scls, we elucidated biomaterial-potential of the unmodified, recombinant Scl1 (rScl1). Initially, rScl1 trimeric- assembly yielded its stability in silico than the monomeric-unit. Thereby, rScl1 matrix characterization was confirmed in vitro. rScl1 exhibited high A549 and HepG2 cell- viability--rScl1 dose incremented to 20.0 {micro}g/ml at time points up to 24 hr, and on 24 hr stored-dishes--deliberating it non-cytotoxic. Imploring cell-adhesion potential, we observed increased cell-counts tangential to rScl1-gradient. This affirmative prelude on rScl1 as a supporting-matrix cued its synergy to collagen; we discerned it through rScl1-augmented, full-thickness diabetic wound-closure in vivo and as a first, we studied > 18-month rabbit alloxan-models. We have ascertained re-epithelialization with higher type III collagen in absence of inflammation evidenced morphometrically and histologically. Finally, we correlated our observations through atomistic-evaluation of rScl1-2{beta}1-integrin interaction, surprisingly, with augmented binding-energy compared to collagen. Hence, connoting recombinant-streptococcal collagen as an alternate; with further characterization, rScl1 can potentiate important revelations conceding homogeneous and safe, bio-available, biomaterial.

Spiroplasma is a cell-wall less, helical bacterium possessing a cytoskeletal ribbon consisting Fibril and 5 MreB paralogs. In absence of any information regarding the filament interface of Fibril, a cytoskeleton protein of novel fold, multiple short constructs of Fibril were designed to identify minimal domains required for polymerization. Purification trials of these short constructs resulted in insoluble protein products. In a parallel approach, we incorporated EGFP at the loop regions aimed to hinder polymerization and obtain non-polymerising constructs. We were able to identify probable interfilament/intrafilament interface residues based on the fluorescence screen. We show that EGFP insertions at many of the positions are tolerated as Fibril filaments were readily observed for these constructs in transmission electron microscopy. Both the approaches suggest that Fibril requires N and C-terminal domains for its polymerization. Earlier studies have shown that FibrilWT interacts with MreB5. Our Fibril-EGFP constructs and MreB5 showed interaction similar to the wild-type. Co-sedimentation assay and visualization of Fibril-EGFP proteins proves that the fluorescent constructs are folded and functional and possess structural and biochemical properties similar to the wild type protein.

The MYC associated factor X (MAX) is the heterodimeric partner of the MYC paralogs (MYC, MYCN and MYCL). When deregulated, high level of the MYC paralogs contribute to all aspects of tumorigenesis and tumor growth. MAX can also heterodimerize with the MXD proteins, MNT and MGA. Heterodimerization and sequence specific DNA binding to the E-Box sequences at gene promoters is controlled by their heterodimerization with the MAX b-HLH-LZ. As a heterodimer with MAX, MYC proteins activate genes involved in cell metabolism, growth and proliferation whereas MXD proteins, MNT and MGA repress them. MAX can also bind to the E-Bos sequence as a homodimer. Being devoid of a transactivation domain it can act as an antagonist of the MYC/MAX heterodimers. Variants of MAX have been reported to be linked to cancer. These variants are either not expressed, inactivated or lead to missense mutations. This has led to the notion that MAX may have a tumor suppressor role. Here, we characterize three of those variants with missense mutations in the basic region, i.e. E32K, R35P and R35C. We analyzed their heterodimerization with the b-HLH-LZ of MYC and their DNA binding properties as homo-and heterodimers. The R35C variant b-HLH-LZ was found to have a markedly increased affinity for the b-HLH-LZ of MYC. We also observed that all three b-HLH-LZ variants have a lower affinity as homodimers for the E-Box than the WT. This was shown to lead to a preferential binding of all the heterodimeric b-LHLH-LZ to the E-Box. This effect is exacerbated in the case of the R35C variant. We argue that this preferential binding of MYC as heterodimers with these variants to E-Box sequences could contribute to tumorigenesis. Hence, our results suggest that, mechanistically, the MAX homodimer bound to the E-Box could act as a tumor suppressor. MATERIALS AND METHODSO_ST_ABSMolecular modelingC_ST_ABSThe open source version 1.7.6.0 of Pymol was used for modeling and molecular rendering [1]. The crystal structure of the MAX homodimer bound to the E-Box (1HLO [2]) was used as a template for the generation of the models. The variants were generated using the mutagenesis function in the wizard. The conformation of the K32 side chain was manually set in order to avoid introducing steric clashes with DNA. Protein expression and purificationThe cDNA, coding for the MAX b-HLH-LZ (Max* hereafter, residues 22-103, UniProt entry P61244-1) to which are added the GSGC residues in c-terminal, inserted in the pET3a vector was already available in the laboratory [3] and was used as a template to generate the plasmids with inserts coding for each of the mutants (E32K, R35C and R35P) through quick-change PCR with Q5 DNA polymerase and DpnI from New England Biolabs. The primers used were purchased from IDT DNA, their sequences are listed in Table S1. Sequence for each construct was confirmed by Sanger sequencing at the Plateforme de sequencage SANGER - Centre de recherche du CHU de Quebec - Universite Laval. The primary structure for the basic region of each construct is given in Fig. 2A. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=137 SRC="FIGDIR/small/715400v1_fig2.gif" ALT="Figure 2"> View larger version (41K): org.highwire.dtl.DTLVardef@1b05d5eorg.highwire.dtl.DTLVardef@1c1d692org.highwire.dtl.DTLVardef@ee469dorg.highwire.dtl.DTLVardef@15e0ba4_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOFigure 2.C_FLOATNO Structure schematics, specific and non-specific interactions dictating specificity and stability of binding of the basic region of MAX to the canonical (CACGTG) E-Box. A. Primary structure for the basic region of MAX and each of the variants. Positions making the most important contacts with the E-box are indicated by black arrows. Positions for the variants studied here are colored according to the Zappo colour scheme, following their physico-chemical properties: red for negative, blue for positive, magenta for proline and yellow for cysteine. B. The side chain (carboxylate) of E32 receives H-Bonds from the CA nucleobases in the leading strand (white carbon atoms). R35 and R36 make a salt bridges with phosphate groups while and the guanidino moiety of R36 makes a specific H-Bond with the nucleobase of the G in the strand of the reverse complement (cyan carbon atoms). C. The R35C mutation removes one non-specific salt-bridge at the interface of the complex. D. The aliphatic portion of the K side chain in the E32K variant is unable to accept the H-Bonds from the CA nucleobases and leads to the stabilisation of the complex and the helical structure of the basic region. E. In addition to removing a salt-bride, the Pro residue in the R35P kinks the path of the basic region, prevents the establishment of the specific H-Bonds mandatory for recognition of the E-Box and leads to unfolding of the helical state. C_FIG The MYC b-HLH-LZ (Myc*), the Max*WT b-HLH-LZ and its variants were expressed and purified as previously described [3,4] After lyophilisation, the b-HLH-LZs were kept at -20{degrees}C and solubilised in Myc buffer (50 mM NaCl, 50 mM NaH2PO4 pH 5.5) for Myc* or PBS for Max* at a final concentration of 1 mM before use. Circular dichroismAll circular dichroism (CD) measurements were performed on a Jasco J-810 spectropolarimeter equipped with a Peltier-type thermostat. The instrument was routinely calibrated using an aqueous solution of d-10-(+)-camphorsulfonic acid at 290.5 nm. Samples were prepared as follows: Max* (either WT or a variant) was diluted in 100 {micro}l 2X CD buffer (40 mM KCl, 11.4 mM K2HPO4, 28.6 mM KH2PO4, pH 6.8) and the volume adjusted to 106 {micro}l with PBS. 10 {micro}l TCEP 16 mM were added, and the volume further adjusted to 192 {micro}l with ddH2O before samples were incubated overnight at room temperature. After reduction, Myc* was added and the volume adjusted to 198 {micro}l with Myc buffer (Na2HPO4 0.95 mM, NaH2PO4 49.05 mM, 50 mM NaCl, pH 5.5). The DNA complexes were prepared as follows. After a 10 minutes incubation of the protein samples at room temperature, 0, 1 or 2 {micro}l of 2 mM of specific or non-specific DNA duplexes in 10 mM Tris pH 8.0 were added and the volume adjusted to 200 {micro}l with 10 mM Tris pH 8.0. The strands of the specific probe were: 5-ATT ACC CAC GTG TCC T*AC-3 and 5-GTA GGA CAC GTG GGT* AAT-3 (with the E-box sequence underlined) and the non-specific probe: 5-ATT ACC TCC GGA TCC T*AC-3 and 5-GTA GGA TCC GGA GGT* AAT-3 (Integrated DNA Technologies). Samples were further incubated for 10 minutes at room temperature and transferred to a 1 mm path length quartz cuvette. All spectra were recorded from 250 to 195 nm at 0.1 nm intervals by accumulating 10 spectra at 25 {degrees}C. Thermal denaturations were recorded at 222 nm from 5 to 95 {degrees}C at a heating rate of 1 {degrees}C/min. CD signal for spectra and thermal denaturations was corrected by substracting the signal from corresponding spectra or thermal denaturation either for buffer alone or the appropriate DNA duplex. CD signal was then converted to mean residue ellipticity using the following formula [5]: [{theta}] = {delta} {middle dot} MRW/(10{middle dot}c l) where [{theta}] is the mean residue ellipticity in deg {middle dot} cm2 dmol-1, {delta} is the CD signal in millidegrees, MRW is the mean residue weight, c is the concentration in mg/ml and l is the pathlength in mm. For the heterodimers, the concentration used was the sum of Max* and Myc* and the MRW was determined using a weighted average.

Tardigrades are microscopic animals that are capable of tolerating extreme environments by entering a desiccated ametabolic state known as anhydrobiosis. While antioxidative stress genes, antiapoptotic pathways and tardigrade-specific intrinsically disordered proteins have been implicated in the anhydrobiotic machinery, conservation of these mechanisms is not universal within the phylum Tardigrada, suggesting the existence of overlooked components. Here, we show that a novel Mn-dependent peroxidase is an important factor in tardigrade anhydrobiosis. Through comparative time-series transcriptome analysis of Ramazzottius varieornatus specimens exposed to desiccation or ultraviolet light, we first identified several novel gene families without similarity to existing sequences that are induced rapidly after stress exposure. Among these, a single gene family with multiple orthologs that is highly conserved within the phylum Tardigrada and enhances oxidative stress tolerance when expressed in human cells was identified. Crystallographic study of this protein suggested Zn or Mn binding at the active site, and we further confirmed that this protein has Mn-dependent peroxidase activity in vitro. Our results demonstrated novel mechanisms for coping with oxidative stress that may be a fundamental mechanism of anhydrobiosis in tardigrades. Furthermore, localization of these sets of proteins in the Golgi apparatus suggests an indispensable role of the Golgi stress response in desiccation tolerance.

Plants have evolved sophisticated mechanisms to adapt to temperature fluctuations, including transcriptional, post-transcriptional, and post-translational processes. Recent discoveries highlight RNA ThermoSwitches, cis-acting elements in several plant mRNAs that regulate protein synthesis based on temperature changes. These mechanisms, first identified in Arabidopsis thaliana, offer a promising tool for biotechnology by enabling temperature-sensitive control of protein expression. This study demonstrates, for the first time, the feasibility the application of plant RNA ThermoSwitches in Agrobacterium-mediated transient expression systems, presenting a novel method for controlled gene expression in plants. This system is particularly advantageous due to its homogeneous nature and independence from chemical inducers or suppressors.