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Understanding how complex biological systems behave over time is essential for predicting their future states, especially when studying Alzheimer's disease. Mathematical modeling of such systems offers a valuable approach to exploring not only theoretical aspects of network behavior but also their biological and medical implications. This study investigates how reducing the complexity of the amyloid-beta signaling pathway influences its dynamic behavior. We apply Boolean modeling and network simulations in BooleSim using data from the SIGNOR database under different initial conditions that reflect healthy and disease-related cellular states. Complexity reduction involved removing non-essential interactions and simplifying regulatory motifs. Our findings show that while simplification can shorten the time to reach steady states, it does not eliminate important regulatory pathways unless critical nodes are removed. Importantly, pro-disease nodes such as BAX and GSK3ß retained their functional significance even in simplified models, confirming their central role in Alzheimer's pathology and supporting their relevance as potential therapeutic targets, consistent with current Alzheimer's drug development strategies. This work illustrates how simplified Boolean modeling can provide a practical framework for analyzing neurodegenerative systems while preserving essential biological insights.

This study uses high-resolution cryo-electron microscopy (cryo-EM) to reveal the precise binding site of the antibiotic GE81112 on the bacterial ribosome's 30S subunit. GE81112 targets the initiation phase of bacterial protein synthesis, specifically interacting within the mRNA channel, distant from the initiation factor and initiator tRNA-binding sites. This indicates that GE81112 acts allosterically, disrupting and preventing conformational rearrangements in IF3 and the proper positioning of the initiator tRNA, stalling the ribosome in an unlocked pre-initiation complex. The findings identify key ribosomal interactions, including conserved nucleotides in helices 23, 24, and 45, and protein S11, highlighting GE81112's unique binding mode among initiation inhibitors. This structural characterization enhances our understanding of antibiotic interference with translation initiation and provides insights to support rational design strategies for improved GE81112 derivatives.

The World Health Organization has identified antimicrobial resistance as a substantial threat to human health. Because of the emergence of pathogenic bacteria resistant to multiple antibiotics worldwide, there is a need to identify the mode of action of antibiotics and to unravel the basic mechanisms responsible for drug resistance. Antibiotic producers’ microorganisms can protect themselves from the toxic effect of the drug using different strategies; one of the most common involves the modification of the antibiotic’s target site. In this work, we report a detailed analysis of the molecular mechanism, based on protein modification, devised by the soil microorganism Streptomyces sp. strain AM-2504 to protect itself from the activity of the peptide antibiotic dityromycin. Furthermore, we demonstrate that this mechanism can be reproduced in E. coli , thereby eliciting antibiotic resistance in this human commensal bacterium. Dityromycin is a peptide antibiotic isolated from the culture broth of the soil microorganism Streptomyces sp. strain AM-2504. Recent structural studies have shown that dityromycin targets the ribosomal protein S12 in the 30S ribosomal subunit, inhibiting translocation. Herein, by using in vitro protein synthesis assays, we identified the resistance mechanism of the producer strain to the secondary metabolite dityromycin. The results show that the self-resistance mechanism of the Streptomyces sp. strain AM-2504 is due to a specific modification of the ribosome. In particular, two amino acid substitutions, located in a highly conserved region of the S12 protein corresponding to the binding site of the antibiotic, were found. These mutations cause a substantial loss of affinity of the dityromycin for the 30S ribosomal subunit, protecting the producer strain from the toxic effect of the antibiotic. In addition to providing a detailed description of the first mechanism of self-resistance based on a mutated ribosomal protein, this work demonstrates that the molecular determinants of the dityromycin resistance identified in Streptomyces can be transferred to Escherichia coli ribosomes, where they can trigger the same antibiotic resistance mechanism found in the producer strain. IMPORTANCE The World Health Organization has identified antimicrobial resistance as a substantial threat to human health. Because of the emergence of pathogenic bacteria resistant to multiple antibiotics worldwide, there is a need to identify the mode of action of antibiotics and to unravel the basic mechanisms responsible for drug resistance. Antibiotic producers’ microorganisms can protect themselves from the toxic effect of the drug using different strategies; one of the most common involves the modification of the antibiotic’s target site. In this work, we report a detailed analysis of the molecular mechanism, based on protein modification, devised by the soil microorganism Streptomyces sp. strain AM-2504 to protect itself from the activity of the peptide antibiotic dityromycin. Furthermore, we demonstrate that this mechanism can be reproduced in E. coli , thereby eliciting antibiotic resistance in this human commensal bacterium.

Ribosome biogenesis is an energetically expensive and complex cellular process that involves the coordinated folding of the ribosomal RNA and dozens of ribosomal proteins. It proceeds along multiple parallel pathways and is guided by trans-acting factors called ribosome assembly factors. Although this process has been studied for decades, there are still many open questions regarding the role of the ribosome assembly factors in directing the folding of ribosome biogenesis intermediates. RimP is one of the early acting factors and guides the assembly of the small 30S ribosomal subunit by facilitating the binding of ribosomal proteins uS5 and uS12. Here we report the virtually complete 1H, 15N, and 13C chemical shift assignment of RimP from Escherichia coli. The NMR chemical shift data, deposited in the BMRB data bank under Accession No. 28014, indicates a widely folded protein composed of three alpha helices and eight beta strands.

RbfA (ribosome binding factor A; 15.2 kDa) is a protein involved in ribosome biogenesis and has been shown to be important for growth at low temperatures and to act as a suppressor for a cold-sensitive mutation (C23U) in the ribosomal RNA of the small 30S ribosomal subunit. The 3D structure of isolated RbfA has been determined from several organisms showing that RbfA has type-II KH-domain fold topology similar to the KH domain of another assembly factor, Era, whose overexpression can compensate for the deletion of rbfA, suppressing both the cold sensitivity and abnormal accumulation of 17S rRNA in rbfA knockout stains. Interestingly, a RbfAΔ25 variant used in previous NMR studies, truncated at the C-terminal domain to remove 25 unstructured residues causing aggregation at room temperature, was biologically active in the sense that it could complement a knock-out of wildtype RbfA, although it did not act as a suppressor for a 16S cold-sensitive mutation (C23U), nor did it interact stably with the 30S subunit. To complement this work, we report the 1H, 13C, and 15 N backbone and sidechain NMR resonance assignments of full length RbfA from Escherichia coli measured under physiological conditions (pH 7.6). This construct contains seven additional C-terminal residues from the cloning (i.e. one alanine and six residues from the HRV 3C cleavage site) and no aggregation issues were observed over a 1-week period at 293 K. The assignment data has been deposited in the BMRB data bank under Accession No. 27857.

The ribosome is a major target for antibiotics owing to its essential cellular role in protein synthesis. Structural analysis of ribosome-antibiotic complexes provides insight into the molecular basis for their inhibitory action and highlights possible avenues to improve their potential or overcome existing resistance mechanisms. Here we use X-ray crystallography and pre-steady state kinetics to detail the inhibitory mechanism of the antimicrobial on the large ribosomal subunit.

The initiation phase is the rate-limiting step of protein synthesis (translation) and is finely regulated, making it an important drug target. In bacteria, initiation is guided by three initiation factors and involves positioning the start site on the messenger RNA within the P-site on the small ribosomal subunit (30S), where it is decoded by the initiator tRNA. This process can be efficiently inhibited by GE81112, a natural hydrophilic, noncyclic, nonribosomal tetrapeptide. It is found in nature in three structural variants (A, B and B1 with molecular masses of 643-658 Da). Previous biochemical and structural characterisation of GE81112 indicates that the primary mechanism of action of this antibiotic is to (1) prevent the initiator tRNA from binding correctly to the P-site and (2) block conformational rearrangements in initiation factor IF3, resulting in an 30S pre/C state. In this study, using cryoEM, we have determined the binding site of GE81112 in initiation complexes (3.2-3.7Å) and on empty ribosomes (2.09 Å). This binding site is within the mRNA channel (E-site) but remote from the binding site of the initiation factors and initiator tRNA. This suggests that it acts allosterically to prevent the initiator tRNA from being locked into place. The binding mode is consistent with previous biochemical studies and recent work identifying the key pharmacophores of GE81112.

Antibiotic resistance is a growing threat to human health requiring the discovery or development of new anti-infectives. As such, KBP-7072 is a novel tetracycline derivative that exhibits broad-spectrum activity against Gram-positive and -negative bacterial strains. To determine the mechanism of action of KBP-7072 and understand how its unique C9 extension can be used to combat the growing problem of antibiotic resistance we determined the structure of KBP-7072 bound to the bacterial 30S ribosomal subunit, the inhibitory target of typical tetracyclines. We show that KBP-7072 binds to the primary tetracycline binding site on the 30S ribosomal subunit consistent with it acting as a protein synthesis inhibitor that blocks A-site occupation. Moreover, the unique chemical nature of KBP-7072s C9 extension leads to a distinctive interaction pattern with the 30S subunit that distinguishes KBP-7072 from the third-generation tetracycline, Tigecycline, and thus expands the interaction potential of the primary tetracycline binding pocket.

While a structural description of the molecular mechanisms guiding ribosome assembly in eukaryotic systems is emerging, bacteria employ an unrelated core set of assembly factors for which high-resolution structural information is still missing. To address this, we used single-particle cryo-EM to visualize the effects of bacterial ribosome assembly factors RimP, RbfA, RsmA, and RsgA on the conformational landscape of the 30S ribosomal subunit and obtained eight snapshots representing late steps in the folding of the decoding center. Analysis of these structures identifies a conserved secondary structure switch in the 16S rRNA central to decoding site maturation, and suggests both a sequential order of action and molecular mechanisms for the assembly factors in coordinating and controlling this switch. Structural and mechanistic parallels between bacterial and eukaryotic systems indicate common folding features inherent to all ribosomes. Competing Interest Statement The authors have declared no competing interest. Footnotes * Editorial changes * Abbreviations used cryo-EM cryo-electron microscopy