DNA-binding properties of the MADS-domain transcription factor SEPALLATA3 and mutant variants characterized by SELEX-seq

Key message We studied the DNA-binding profile of the MADS-domain transcription factor SEPALLATA3 and mutant variants by SELEX-seq. DNA-binding characteristics of SEPALLATA3 mutant proteins lead us to propose a novel DNA-binding mode. Abstract MIKC-type MADS-domain proteins, which function as essential transcription factors in plant development, bind as dimers to a 10-base-pair AT-rich motif termed CArG-box. However, this consensus motif cannot fully explain how the abundant family members in flowering plants can bind different target genes in specific ways. The aim of this study was to better understand the DNA-binding specificity of MADS-domain transcription factors. Also, we wanted to understand the role of a highly conserved arginine residue for binding specificity of the MADS-domain transcription factor family. Here, we studied the DNA-binding profile of the floral homeotic MADS-domain protein SEPALLATA3 by performing SELEX followed by high-throughput sequencing (SELEX-seq). We found a diverse set of bound sequences and could estimate the in vitro binding affinities of SEPALLATA3 to a huge number of different sequences. We found evidence for the preference of AT-rich motifs as flanking sequences. Whereas different CArG-boxes can act as SEPALLATA3 binding sites, our findings suggest that the preferred flanking motifs are almost always the same and thus mostly independent of the identity of the central CArG-box motif. Analysis of SEPALLATA3 proteins with a single amino acid substitution at position 3 of the DNA-binding MADS-domain further revealed that the conserved arginine residue, which has been shown to be involved in a shape readout mechanism, is especially important for the recognition of nucleotides at positions 3 and 8 of the CArG-box motif. This leads us to propose a novel DNA-binding mode for SEPALLATA3, which is different from that of other MADS-domain proteins known.

Effect of Glyoxal Modification on a Critical Arginine Residue (Arg-31α) of Hemoglobin: Physiological Implications of Advanced Glycated end Product an in vitro Study

Background: Non-enzymatic protein glycation is involved in structure and stability changes that impair protein functionality, resulting in several human diseases, such as diabetes and amyloidotic neuropathies (Alzheimer’s disease, Parkinson’s disease and Andrade’s syndrome). Glyoxal, an endogenous reactive oxoaldehyde, increases in diabetes and reacts with several proteins to form advanced glycation end products through Maillard-like reaction. Objective: Human hemoglobin, the most abundant protein in blood cells is subjected to nonenzymatic modification by reactive oxoaldehydes in diabetic condition. In the present study, the effect of a low concentration of glyoxal (5 μM) on hemoglobin (10 μM) has been investigated following a period of 30 days incubation in vitro. Methods: Different techniques, mostly biophysical and spectroscopic (e.g. circular dichroism, differential scanning calorimetric study, dynamic light scattering, mass spectrometry, etc.) were used to study glyoxal-induced changes of hemoglobin. Results: Glyoxal-treated hemoglobin exhibits decreased absorbance around 280 nm, decreased fluorescence and reduced surface hydrophobicity compared to normal hemoglobin. Glyoxal treatment enhances the stability of hemoglobin and lowers its susceptibility to thermal aggregation compared to control hemoglobin as seen by different studies. Finally, peptide mass fingerprinting study showed glyoxal to modify an arginine residue of α-chain of hemoglobin (Arg-31α) to hydroimidazolone. Conclusion: Increased level of glyoxal in diabetes mellitus as well as its high reactivity may cause modifications of the heme protein. Thus, considering the significance of glyoxal-induced protein modification under physiological conditions, the observation appears clinically relevant in terms of understanding hydroimidazolone-mediated protein modification under in vivo conditions.

Synthesis and Biological Evaluation of Iodinated Fidaxomicin Antibiotics

<div><div><div><p>Fidaxomicin (1, tiacumicin B, lipiarmycin A3) is a marketed antibiotic that is used in the treatment of C. difficile infections. Based on the analysis of a cryo- EM structure of fidaxomicin binding to its target enzyme (RNA-polymerase), a cation-p interaction of the aromatic moiety with an arginine residue was identified. Therefore, the variation of the substituents and concurrently changing the electronic properties of the aryl moiety represents an interesting strategy in search for new fidaxomicin analogs. Herein, we report the first semisynthetic access to new fidaxomicin analogs with varying halogen substituents via a Pd-catalyzed hydrodechlorination reaction. Subsequent iodination gave access to the first iodo-fidaxomicin derivatives, which matched or improved antibacterial properties compared to fidaxomicin against Mycobacterium tuberculosis and Staphylococcus aureus ATCC 29213.</p></div></div></div>

Synthesis and Biological Evaluation of Iodinated Fidaxomicin Antibiotics

<div><div><div><p>Fidaxomicin (1, tiacumicin B, lipiarmycin A3) is a marketed antibiotic that is used in the treatment of C. difficile infections. Based on the analysis of a cryo- EM structure of fidaxomicin binding to its target enzyme (RNA-polymerase), a cation-p interaction of the aromatic moiety with an arginine residue was identified. Therefore, the variation of the substituents and concurrently changing the electronic properties of the aryl moiety represents an interesting strategy in search for new fidaxomicin analogs. Herein, we report the first semisynthetic access to new fidaxomicin analogs with varying halogen substituents via a Pd-catalyzed hydrodechlorination reaction. Subsequent iodination gave access to the first iodo-fidaxomicin derivatives, which matched or improved antibacterial properties compared to fidaxomicin against Mycobacterium tuberculosis and Staphylococcus aureus ATCC 29213.</p></div></div></div>