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Background: Vascular adhesion protein-1 (VAP-1) is an adhesion molecule and primary amine oxidase, and Gallium-68-labeled 1,4,7,10-tetraazacyclododecane-N,N′,N″,N‴-tetra-acetic acid conjugated sialic acid-binding immunoglobulin-like lectin 9 motif containing peptide ([68Ga]Ga-DOTA-Siglec-9) is a positron emission tomography (PET) tracer targeting VAP-1. We evaluated the feasibility of PET imaging with [68Ga]Ga-DOTA-Siglec-9 for the detection of myocardial lesions in rats with autoimmune myocarditis. Methods: Rats (n = 9) were immunized twice with porcine cardiac myosin in complete Freund's adjuvant. Control rats (n = 6) were injected with Freund's adjuvant alone. On day 21, in vivo PET/computed tomography (CT) imaging with [68Ga]Ga-DOTA-Siglec-9 was performed, followed by ex vivo autoradiography, histology, and immunohistochemistry of tissue sections. In addition, myocardial samples from three patients with cardiac sarcoidosis were studied. Results: [68Ga]Ga-DOTA-Siglec-9 PET/CT images of immunized rats showed higher uptake in myocardial lesions than in myocardium outside lesions (SUVmean, 0.5 ± 0.1 vs 0.3 ± 0.1; P =.003) or control rats (SUVmean, 0.2 ± 0.03; P <.0001), which was confirmed by ex vivo autoradiography of tissue sections. Immunohistochemistry showed VAP-1-positive staining in lesions of rats with myocarditis and in patients with cardiac sarcoidosis. Conclusion: VAP-1-targeted [68Ga]Ga-DOTA-Siglec-9 PET is a potential novel technique for the detection of myocardial lesions. [ABSTRACT FROM AUTHOR]

The three-dimensional structure of proteins determines their function in vital biological processes. Thus, when the structure is known, the molecular mechanism of protein function can be understood in more detail and obtained information utilized in biotechnological, diagnostics, and therapeutic applications. Over the past five years, machine learning (ML)-based modeling has pushed protein structure prediction to the next level with AlphaFold in the front line, predicting the structure for hundreds of millions of proteins. Further advances recently report promising ML-based approaches for solving remaining challenges by incorporating functionally important metals, co-factors, post-translational modifications, structural dynamics, and interdomain and multimer interactions in the structure prediction process. [ABSTRACT FROM AUTHOR]

Correct timing of the induction of photoperiodic reproductive diapause has been found to play an important role in the life cycle of several northern insect species. However, even when the environmental conditions are favourable for diapause, the switch to diapause can only take place when the females are in a proper developmental and physiological stage, referred to as the sensitive period (SP) for diapause. We have previously shown that in a northern fly species, Drosophila montana, the developmental pathway of the ovaries (direct maturation vs. diapause) is determined by photoperiodic cues that the females receive after eclosion. Here, we have studied the effects of temperature on the duration of the sensitive period, and on the number of short day cycles that the females have to experience before half of them will enter diapause (RDN=required day number). Ovarian development rate of the females was first traced under long and short day conditions in 16 and 19°C, and then reciprocal transfers were done between the diapause-inducing short day conditions, and the vitellogenesis-inducing long day conditions to determine the females’ SP and RDN. Close to 100% of the females of all study strains entered reproductive diapause under short day conditions in 16°C, and the same occurred also in 19°C in strains from the more northern univoltine population. The sensitive period for diapause induction was affected by temperature, as it was shorter in higher temperature (circa 8days in 16°C and 4–5days in 19°C), and was restricted by the faster development rate of the ovaries. D. montana females had to experience approximately three short day cycles during the sensitive period, before half of them entered diapause, which also explains the decrease in the number of diapausing females at higher temperatures. This system clearly differs from that of the more southern Drosophila species, e.g. D. melanogaster, where the females’ developmental pathway is determined already during the first day after eclosion. [ABSTRACT FROM AUTHOR]

Abstract: Couch potato (cpo) has previously been connected to reproductive diapause in several insect species including Drosophila melanogaster, where it has been suggested to provide a link between the insulin signalling pathway and the hormonal control of diapause. In the first part of the study we sequenced nearly 3.6kb of this gene in a northern Drosophila species (Drosophila montana) with a robust photoperiodically determined diapause and found several types of polymorphisms along the sequenced area. We also found variation among five Drosophila virilis group species in the length of the 5th exon of cpo and in the site of the stop codon at the end of this exon. The second part of the study was targeted on a deletion of six amino acids located in the last section of exon 5, which in D. melanogaster, is translated only in one short transcript lacking the following exons. The studied deletion appeared to be extremely rare in the wild D. montana population where it was found, but its frequency rapidly increased during laboratory culture. qPCR analyses showed the expression level of the deletion allele to be significantly downregulated in both the diapausing and non-diapausing females compared to the wild type allele. At the phenotypic level, the deletion and the decreased expression of cpo transcript involving it did not have direct effect on the incidence of female reproductive diapause, but it was associated with a reduction in development time under diapause-inducing conditions. This suggests that while the cpo transcript containing the prolonged version of the 5th exon with a stop codon is clearly associated with fly development time, the exons with RNA domains included in other transcripts of the gene may be more directly related to diapause regulation. [Copyright &y& Elsevier]

Purpose: To evaluate the possible selection bias related to the differential participation of mobile phone users and non-users in a Finnish case–control study on mobile phone use and brain tumors. Methods: Mobile phone use was investigated among 777 controls and 726 cases participating in the full personal interview (full participants), and 321 controls and 103 cases giving only a brief phone interview (incomplete participants). To assess selection bias, the Mantel-Haenszel estimate of odds ratio was calculated for three different groups: full study participants, incomplete participants, and a combined group consisting of both full and incomplete participants. Results: Among controls, 83% of the full participants and 73% of the incomplete participants had regularly used a mobile phone. Among cases, the figures were 76% and 64%, respectively. The odds ratio for brain tumor based on the combined group of full and incomplete participants was slightly closer to unity than that based only on the full participants. Conclusions: Selection bias tends to distort the effect estimates below unity, while analyses based on more comprehensive material gave results close to unity. [Copyright &y& Elsevier]

Dysregulation of the developmentally important Notch signaling pathway is implicated in several types of cancer, including breast cancer. However, the specific roles and regulation of the four different Notch receptors have remained elusive. We have previously reported that the oncogenic PIM kinases phosphorylate Notch1 and Notch3. Phosphorylation of Notch1 within the second nuclear localization sequence of its intracellular domain (ICD) enhances its transcriptional activity and tumorigenicity. In this study, we analyzed Notch3 phosphorylation and its functional impact. Unexpectedly, we observed that the PIM target sites are not conserved between Notch1 and Notch3. Notch3 ICD (N3ICD) is phosphorylated within a domain, which is essential for formation of a transcriptionally active complex with the DNA-binding protein CSL. Through molecular modeling, X-ray crystallography, and isothermal titration calorimetry, we demonstrate that phosphorylation of N3ICD sterically hinders its interaction with CSL and thereby inhibits its CSL-dependent transcriptional activity. Surprisingly however, phosphorylated N3ICD still maintains tumorigenic potential in breast cancer cells under estrogenic conditions, which support PIM expression. Taken together, our data indicate that PIM kinases modulate the signaling output of different Notch paralogs by targeting distinct protein domains and thereby promote breast cancer tumorigenesis via both CSL-dependent and CSL-independent mechanisms. [ABSTRACT FROM AUTHOR]

Collagens are the most abundant extracellular matrix proteins in vertebrates and have a characteristic triple-helix structure. Hydroxylation of proline residues is critical for helix stability, and diminished prolyl hydroxylase activity causes wide-spread defects in connective tissues. Still, the role of proline hydroxylation in the binding of collagen receptors such as integrins is unclear. Here, we isolated skin collagen from genetically modified mice having reduced prolyl 4-hydroxylase activity. At room temperature, the reduced proline hydroxylation did not affect interactions with the recombinant integrin α2I domain, but at 37 °C, collagen hydroxylation correlated with the avidity of α2I domain binding. Of note, LC-MS/MS analysis of isolated skin collagens revealed no major changes in the hydroxyproline content of the main integrin-binding sites. Thus, the disrupted α2I domain binding at physiological temperatures was most likely due to structural destabilization of the collagenous helix. Integrin α2I binding to the triple-helical GFPGER motif was slightly weaker than to GFOGER (O = hydroxyproline). This phenomenon was more prominent when α1 integrin was tested. Integrin α1β1 expressed on CHO cells and recombinant α1I domain showed remarkably slower binding velocity and weaker avidity to GFPGER when compared with GFOGER. Structural modeling revealed the critical interaction between Arg-218 in α1I and the hydroxyproline residue in the integrin-binding motif. The role of Arg-218 was further validated by testing a variant R218D α1I domain in solid-phase binding assays. Thus, our results show that the lack of proline hydroxylation in collagen can affect integrin binding by a direct mechanism and via structural destabilization of the triple helix. [ABSTRACT FROM AUTHOR]

The intracellular polyamine contents are regulated not only by polyamine biosynthesis and transport but also by polyamine degradation catalyzed by copper-dependent amine oxidase (DAO) and FAD-dependent polyamine oxidase (PAO). The genome sequence of Synechocystis sp. PCC 6803 reveals the presence of at least one putative polyamine oxidase gene, slr5093 . The open reading frame of slr5093 encoding Synechocystis polyamine oxidase (SynPAO, E.C. 1.5.3.17) was expressed in Escherichia coli . The purified recombinant enzyme had the characteristic absorption spectrum of a flavoprotein with absorbance peaks at 380 and 450 nm. The optimum pH and temperature for the oxidation of both spermidine and spermine are 8.5 and 30 °C, respectively. The enzyme catalyzed the conversion of spermine and spermidine to spermidine and putrescine, respectively, with higher catalytic efficiency when spermine served as substrate. These results suggest that SynPAO is a polyamine oxidase involved in a polyamine back-conversion pathway. Based on the structural analysis, Gln94, Tyr403 and Thr440 in SynPAO are predicted to be important residues in the active site. [ABSTRACT FROM AUTHOR]

Previous reports have connected non-symbiotic and truncated hemoglobins (Hbs) to metabolism of nitric oxide (NO), an important signalling molecule involved in wood formation. We have studied the capability of poplar ( Populus tremula × tremuloides ) Hbs PttHb1 and PttTrHb proteins alone or with a flavin-protein reductase to relieve NO cytotoxicity in living cells. Complementation tests in a Hb-deficient, NO-sensitive yeast ( Saccharomyces cerevisiae ) Δ yhb1 mutant showed that neither PttHb1 nor PttTrHb alone protected cells against NO. To study the ability of Hbs to interact with a reductase, ferredoxin NADP + oxidoreductase PtthFNR was characterized by sequencing and proteomics. To date, by far the greatest number of the known dual-targeted plant proteins are directed to chloroplasts and mitochondria. We discovered a novel variant of hFNR that lacks the plastid presequence and resides in cytosol. The coexpression of PttHb1 and PtthFNR partially restored NO resistance of the yeast Δ yhb1 mutant, whereas PttTrHb coexpressed with PtthFNR failed to rescue growth. YFP fusion proteins confirmed the interaction between PttHb1 and PtthFNR in plant cells. The structural modelling results indicate that PttHb1 and PtthFNR are able to interact as NO dioxygenase. This is the first report on dual targeting of central plant enzyme FNR to plastids and cytosol. [ABSTRACT FROM AUTHOR]

Stem cell renewal and differentiation are regulated by interactions with the niche. Although multiple cell populations have been identified in distinct anatomical compartments, little is known about niche-specific molecular factors. Using skin as a model system and combining single-cell RNA-seq data analysis, immunofluorescence, and transgenic mouse models, we show that the transmembrane protein embigin is specifically expressed in the sebaceous gland and that the number of embigin-expressing cells is negatively regulated by Wnt. The loss of embigin promotes exit from the progenitor compartment and progression toward differentiation, and also compromises lipid metabolism. Embigin modulates sebaceous niche architecture by affecting extracellular matrix organization and basolateral targeting of monocarboxylate transport. We discover through ligand screening that embigin is a direct fibronectin receptor, binding to the N-terminal fibronectin domain without impairing integrin function. Our results solve the long-standing question of how embigin regulates cell adhesion and demonstrate a mechanism that couples adhesion and metabolism. [Display omitted] • Embigin (EMB) marks Wnt-regulated, lipid-producing, sebaceous gland cells • EMB supports adhesion to ECM via direct binding to the N-terminal part of fibronectin • EMB deletion drives sebocyte progenitors from basal layer to suprabasal differentiation • EMB regulates stiffness, ECM composition, and MCT1 localization in sebaceous glands Extracellular matrix (ECM) interaction is an important regulator of epithelial stem cells and progenitors. Sipilä et al. report a mechanism by which embigin directly binds to fibronectin and mediates progenitor cell anchoring to the ECM, regulating the differentiation and metabolism of sebocytes as well as the stiffness of the sebaceous gland microenvironment. [ABSTRACT FROM AUTHOR]

Abstract: The non-specific lipid transfer proteins (nsLTPs) are characterized by a compact structure with a central hydrophobic cavity very suitable for binding hydrophobic ligands, such as lipids. The nsLTPs are encoded by large gene families in all land plant lineages, but seem to be absent from green algae. The nsLTPs are classified to different types based on molecular weight, sequence similarity, intron position or spacing between the cysteine residues. The Type G nsLTPs (LTPGs) have a GPI-anchor in the C-terminal region which may attach the protein to the exterior side of the plasma membrane. Here, we present the first characterization of nsLTPs from an early diverged plant, the moss Physcomitrella patens. Moss LTPGs were heterologously produced and purified from Pichia pastoris. The purified moss LTPGs were found to be extremely heat stable and showed a binding preference for unsaturated fatty acids. Structural modeling implied that high alanine content could be important for the heat stability. Lipid profiling revealed that cutin monomers, such as C16 and C18 mono- and di-hydroxylated fatty acids, could be identified in P. patens. Expression of a moss LTPG-YFP fusion revealed localization to the plasma membrane. The expressions of many of the moss LTPGs were found to be upregulated during drought and cold treatments. [Copyright &y& Elsevier]

We have analyzed the structure and function of the integrin α1I domain harboring a gain-of-function mutation E317A. To promote protein crystallization, a double variant with an additional C139S mutation was used. In cell adhesion assays, the E317A mutation promoted binding to collagen. Similarly, the double mutation C139S/E317A increased adhesion compared with C139S alone. Furthermore, soluble α1I C139S/E317A was a higher avidity collagen binder than α1I C139S, indicating that the double variant represents an activated form. The crystal structure of the activated variant of α1I was solved at 1.9 Å resolution. The E317A mutation results in the unwinding of the αC helix, but the metal ion has moved toward loop 1, instead of loop 2 in the open α2I. Furthermore, unlike in the closed αI domains, the metal ion is pentacoordinated and, thus, prepared for ligand binding. Helix 7, which has moved downward in the open α2I structure, has not changed its position in the activated α1I variant. During the integrin activation, Glu335 on helix 7 binds to the metal ion at the metal ion-dependent adhesion site (MIDAS) of the β1 subunit. Interestingly, in our cell adhesion assays E317A could activate collagen binding even after mutating Glu335. This indicates that the stabilization of helix 7 into its downward position is not required if the α1 MIDAS is already open. To conclude, the activated α1I domain represents a novel conformation of the αI domain, mimicking the structural state where the Arg287-Glu317 ion pair has just broken during the integrin activation. [ABSTRACT FROM AUTHOR]

Yersinia enterocolitica (Ye) is a Gram-negative bacterium; Ye serotype O:3 expresses lipopolysaccharide (LPS) with a hexasaccharide branch known as the outer core (OC). The OC is important for the resistance of the bacterium to cationic antimicrobial peptides and also functions as a receptor for bacteriophage φbR1-37 and enterocoliticin. The biosynthesis of the OC hexasaccharide is directed by the OC gene cluster that contains nine genes (wzx, wbcKLMNOPQ, and gne). In this study, we inactivated the six OC genes predicted to encode glycosyltransferases (GTase) one by one by nonpolar mutations to assign functions to their gene products. The mutants expressed no OC or truncated OC oligosaccharides of different lengths. The truncated OC oligosaccharides revealed that the minimum structural requirements for the interactions of OC with bacteriophage φR1-37, enterocoliticin, and OC-specific monoclonal antibody 2B5 were different. Furthermore, using chemical and structural analyses of the mutant LPSs, we could assign specific functions to all six GTases and also revealed the exact order in which the transferases build the hexasaccharide. Comparative modeling of the catalytic sites of glucosyltransferases WbcK and WbcL followed by site-directed mutagenesis allowed us to identify Asp-182 and Glu-181, respectively, as catalytic base residues of these two GTases. In general, conclusive evidence for specific GTase functions have been rare due to difficulties in accessibility of the appropriate donors and acceptors; however, in this work we were able to utilize the structural analysis of LPS to get direct experimental evidence for five different GTase specificities. [ABSTRACT FROM AUTHOR]

Members of the vascular endothelial growth factor (VEGF) family play a pivotal role in angiogenesis and lymphangiogenesis. They are potential therapeutics to induce blood vessel formation in myocardium and skeletal muscle, when normal blood flow is compromised. Most members of the VEGF/platelet derived growth factor protein superfamily exist as covalently bound antiparallel dimers. However, the mature form of VEGF-D (VEGF-DΔnΔC) is predominantly a non-covalent dimer even though the cysteine residues (Cys-44 and Cys-53) forming theintersubunit disulfide bridges in the other members of the VEGF family are also conserved in VEGF-D. Moreover, VEGF-D bears an additional cysteine residue (Cys-25) at the subunit interface. Guided by our model of VEGF-DΔnΔC, the cysteines at the subunit interface were mutated to study the effect of these residues on the structural and functional properties of VEGF-DΔnΔC. The conserved cysteines Cys-44 and Cys-53 were found to be essential for the function of VEGF-DΔnΔC. More impor- tantly, the substitution of the Cys-25 at the dimer interface by various amino acids improved the activity of the recombinant VEGF-DΔnΔC and increased the dimer to monomer ratio. Specifically, substitutions to hydrophobic amino acids lie, Leu, and Val, equivalent to those found in other VEGFs, most favorably affected the activity of the recombinant VEGF-DΔnΔC. The increased activity of these mutants was mainly due to stabilization of the protein. This study enables us to better understand the structural determinants controlling the biological activity of VEGF-D. The novel variants of VEGF-DΔnΔC described here are potential agents for therapeutic applications, where induction of vascular formation is required. [ABSTRACT FROM AUTHOR]

Abstract: Caspase 8 (CASP8) is a key regulator of apoptosis or programmed cell death, and hence a defence against cancer. The CASP8 polymorphism D302H has recently been shown to influence the risk of breast cancer. We tested the hypothesis that the CASP8 polymorphism D302H may influence risk of meningioma through analysis of five independent series of case patients and controls (n =631 and 637, respectively). Carrier status for 302H was not associated with a statistically significantly increased risk (OR=1.16; 95% CI: 0.87–1.53; P =0.31) making it unlikely that this variant contributes to the inherited risk of meningioma. [Copyright &y& Elsevier]

The molecular mechanisms by which the AP-1 transcription factor c-Jun exerts its biological functions are not clearly understood. In addition to its well established role in transcriptional regulation of gene expression, several reports have suggested that c-Jun may also regulate cell behavior by non-transcriptional mechanisms. Here, we report that small interfering RNA-mediated depletion of c-Jun from mammalian cells results in inhibition of 28 S and 18 S rRNA accumulation. Moreover, we show that c-Jun depletion results in partial translocation of RNA helicase DDX21, implicated in rRNA processing, from the nucleolus to the nucleoplasm. We demonstrate that DDX21 translocation is rescued by exogenous c-Jun expression and that c-Jun depletion inhibits rRNA binding of DDX21. Furthermore, the direct interaction between c-Jun and DDX21 regulates nucleolar localization of DDX21. These results demonstrate that in addition to its transcriptional effects, c-Jun regulates rRNA processing and nucleolar compartmentalization of the rRNA processing protein DDX21. Thus, our results demonstrate a nucleolar mechanism through which c-Jun can regulate cell behavior. Moreover, these results suggest that the phenotypes observed previously in c-Jun-depleted mouse models and cell lines could be partly due to the effects of c-Jun on rRNA processing. [ABSTRACT FROM AUTHOR]

Integrin α1β1 is one of four collagen-binding integrins in humans. Collagens bind to the αI domain and in the case of α2I collagen binding is competitively inhibited by peptides containing the RKKH sequence and derived from the metalloproteinase jararhagin of snake venom from Bothrops jararaca. In α2I, these peptides bind near the metal ion-dependent adhesion site (MIDAS), where a collagen (I)-like peptide is known to bind; magnesium is required for binding. Published structures of the ligandbound "open" conformation of α2I differs significantly from the "closed" conformation seen in the structure of apo-α2I near MIDAS. Here we show that two peptides, CTRKKHDC and CARKKHDC, derived from jararhagin also bind to α1I and competitively inhibit collagen I binding. Furthermore, calorimetric and fluorimetric measurements show that the structure of the complex of α1I with Mg2+ and CTRKKHDC differs from structure in the absence of peptide. A comparison of the x-ray structure of apo-α1I ("closed" conformation) and a model structure of the α1I ("open" conformation) based on the closely related structure of α2I reveals that the binding site is partially blocked to ligands by Glu255 and Tyr285 in the "closed" structure, whereas in the "open" structure helix C is unwound and these residues are shifted, and the "RKKH" peptides fit well when docked. The "open" conformation of α2I resulting from binding a collagen (I)-like peptide leads to exposure of hydrophobic surface, also seen in the model of α1I and shown experimentally for α1I using a fluorescent hydrophobic probe. [ABSTRACT FROM AUTHOR]