The formation of superoxide dismutase 1 (SOD1) filaments has been implicated in amyotrophic lateral sclerosis (ALS). Although the disulfide bond formed between Cys57 and Cys146 in the active state has been well studied, the role of the reduced cysteine residues, Cys6 and Cys111, in SOD1 filament formation remains unclear. In this study, we investigated the role of reduced cysteine residues by determining and comparing cryoelectron microscopy (cryo-EM) structures of wild-type (WT) and C6A/C111A SOD1 filaments under thiol-based reducing and metal-depriving conditions, starting with protein samples possessing enzymatic activity. The C6A/C111A mutant SOD1 formed filaments more rapidly than the WT protein. The mutant structure had a unique paired-protofilament arrangement, with a smaller filament core than that of the single-protofilament structure observed in WT SOD1. Although the single-protofilament form developed more slowly, cross-seeding experiments demonstrated the predominance of single-protofilament morphology over paired protofilaments, regardless of the presence of the Cys6 and Cys111 mutations. These findings highlight the importance of the number of amino acid residues within the filament core in determining the energy requirements for assembly. Our study provides insights into ALS pathogenesis by elucidating the initiation and propagation of filament formation, which potentially leads to deleterious amyloid filaments.

The formation of superoxide dismutase 1 (SOD1) filaments has been implicated in amyotrophic lateral sclerosis (ALS). Although the disulfide bond formed between Cys57 and Cys146 in the active state has been well studied, the role of the reduced cysteine residues, Cys6 and Cys111, in SOD1 filament formation remains unclear. In this study, we investigated the role of reduced cysteine residues by determining and comparing cryoelectron microscopy (cryo-EM) structures of wild-type (WT) and C6A/C111A SOD1 filaments under thiol-based reducing and metal-depriving conditions, starting with protein samples possessing enzymatic activity. The C6A/C111A mutant SOD1 formed filaments more rapidly than the WT protein. The mutant structure had a unique paired-protofilament arrangement, with a smaller filament core than that of the single-protofilament structure observed in WT SOD1. Although the single-protofilament form developed more slowly, cross-seeding experiments demonstrated the predominance of single-protofilament morphology over paired protofilaments, regardless of the presence of the Cys6 and Cys111 mutations. These findings highlight the importance of the number of amino acid residues within the filament core in determining the energy requirements for assembly. Our study provides insights into ALS pathogenesis by elucidating the initiation and propagation of filament formation, which potentially leads to deleterious amyloid filaments.

SignificanceYeiE has been identified as a master virulence factor of <i>Cronobacter sakazakii</i>. In this study, we determined the crystal structures of the regulatory domain of YeiE in complex with its physiological ligand sulfite ion (SO₃^2-). The structure provides the basis for the molecular mechanisms for sulfite sensing and the ligand-dependent conformational changes of the regulatory domain. The genes under the control of YeiE in response to sulfite were investigated to reveal the functional roles of YeiE in the sulfite tolerance of the bacteria. We propose the molecular mechanism underlying the ability of gram-negative pathogens to defend against the innate immune response involving sulfite, thus providing a strategy to control the pathogenesis of bacteria.

Pyridoxal 5'-phosphate (PLP)-dependent enzymes participate in various reactions involved in methionine and cysteine metabolism. The representative foodborne pathogen Staphylococcus aureus expresses the PLP-dependent enzyme MccB, which exhibits both cystathionine gamma-lyase (CGL) and cysteine desulfhydrase activities. In this study, we investigated the role of Ser323 in MccB, a conserved residue in many PLP-dependent enzymes in the transsulfuration pathway. Our findings reveal that Ser323 forms a hydrogen bond with the catalytic lysine in the absence of PLP, and upon internal aldimine formation, PLP-bound lysine is repositioned away from Ser323. Substituting Ser323 with alanine abolishes the enzymatic activity, similar to mutations at the catalytic lysine site. Spectroscopic analysis suggests that Ser323 is essential for the rapid formation of the internal aldimine with lysine in wild-type MccB. This study highlights the crucial role of Ser323 in catalysis, with broader implications for other PLP-dependent enzymes, and enhances our understanding of the molecular mechanisms involved in the selective control of foodborne pathogenic bacteria.

<i>Cronobacter sakazakii</i> is a Gram-negative pathogen that causes severe infections such as neonatal meningitis and sepsis. Bacteriophages (phages) rely on tail fibers for host recognition and infection, but the mechanisms of how phages recognize their bacterial hosts are not completely elucidated. In this study, two lytic <i>C. sakazakii</i> phages belonging to the <i>Drexlerviridae</i> family, CRES7 and CRES9, were isolated from sewage in South Korea. The genomes of both phages are almost the same, with only two nucleotide differences in the gene encoding a putative tail fiber, causing two amino acid differences at amino acid residues 400 and 550 of the tail fiber. The predicted structure of the tail fiber revealed that the two amino acid residues are located on the surface of the tail fiber, suggesting that these two amino acid residues may affect receptor binding. These amino acid differences resulted in differential host ranges, adsorption rates, and burst sizes of CRES7 and CRES9; CRES7, which could infect only the <i>C. sakazakii</i> serotype O1 strain, exhibited a higher adsorption rate and larger burst size compared to CRES9, whereas CRES9 could infect both serotypes O1 and O3 strains. These findings provide insights into how the mutations in the tail fiber gene contribute to the fitness of phages within natural environments and help develop phage-based strategies with expanded host range or enhanced specificity for targeted biocontrol of <i>C. sakazakii</i>.IMPORTANCEAccurate recognition and attachment to the bacterial host, mediated by tail fibers, are crucial for successful phage infection. Understanding the mechanisms underlying host specificity of phages is essential for developing targeted biocontrol applications. This study identified specific amino acid residues responsible for host specificity in the tail fibers of two newly isolated <i>Cronobacter sakazakii</i> phages, CRES7 and CRES9. Differences in these residues showed variation in O serotype recognition, leading to differences in host range, adsorption efficiency, and burst size. These findings provide valuable insights into tail fiber-mediated host specificity, facilitating the development of more effective phage-based strategies against <i>C. sakazakii</i>.

<i>Candida auris</i> is a pathogenic fungus associated with high-mortality infections and forms resilient biofilms on various surfaces. In this study, we introduced a novel antifungal strategy against <i>C. auris</i> by integrating an AI-powered protein design tool, ProteinMPNN, with classical molecular dynamics (MD) simulations to design artificial proteins from a miniprotein library. This combined approach accelerated and enhanced the design process, enabling the rapid development of effective miniprotein inhibitors specifically targeting <i>C. auris</i> biofilm formation. The miniproteins developed in this study exhibited potent inhibitory effects on <i>C. auris</i> biofilms, representing a significant advancement in antifungal therapy. Notably, the combined application of these miniproteins enhanced suppression of biofilm formation. These findings highlight not only the strong therapeutic potential of these designed miniproteins but also the power of combining AI-driven protein design with MD simulations to advance biomedical research.

The Bacillus subtilis spore crust is an exceptionally robust proteinaceous layer that protects spores under extreme environmental conditions. Among its key components, CgeA, a glycosylation-associated protein, plays a critical role in modifying crust properties through its glycosylated moiety, enhancing spore dispersal in aqueous environments. In this study, we present the high-resolution cryo-electron microscopy structure of the core region of CgeA at 3.05 Å resolution, revealing a doughnut-like hexameric assembly. The N-terminal regions are disordered, whereas the C-terminal region forms the core of the hexamer. Although the loop containing Thr112 was not resolved in the density map, its location can be inferred from surrounding residues, suggesting that Thr112 is situated on the exposed surface of the hexamer. On the opposite face, a distinct electrostatic pattern is observed, featuring a negatively charged central pore and a positively charged outer surface. Modeling and biochemical studies with the putative glycosyltransferase CgeB provide insights into how the glycosyl group is transferred to Thr112. This study offers a molecular-level understanding of the assembly, glycosylation, and environmental adaptability of the B. subtilis spore crust, with valuable implications for controlling spore formation in industrial applications.

The formation of SOD1 filament is implicated in the pathogenesis of amyotrophic lateral sclerosis (ALS), with numerous genetic variants identified in familial cases. This study reveals a novel cryo-EM structure of the wild-type SOD1 amyloid filament formed under physiological conditions. The difference in filament morphology compared to the previous structure is attributed to the metalation state of the SOD1 protein before filamentation. In contrast to the previous study that used metal-deprived apo- SOD1 protein, we utilized as-isolated wild-type SOD1, maintaining a high proportion of the enzymatically active holo-form. We found the Trp32 residue at the core of the SOD1 filament structure, where the Trp32 residue engages in crucial interactions with adjacent Lys91. The W32A mutation or the addition of Trp32-containing peptides modified the rate of filament formation in holo-SOD1. Additionally, the Cys111 residue located in a confined hydrophobic space within the filament undergoes bulkier modifications that impede filament assembly, emphasizing the role of the Cys111 in filament formation. Moreover, we revealed the cryo-EM structure of ALS-related and cysteine mutant SOD1 protein filaments. Comparative analysis of wild-type and mutant SOD1 structures elucidates the amyloid filament formation mechanism, emphasizing the significance of amino acid residue counts within the filament core for assembly energy requirements. These insights enhance our understanding of ALS pathogenesis by elucidating how genetic variations contribute to the development of potentially harmful filaments.

Ung thư da được phát triển từ các tế bào da bị phân chia mất kiểm soát do ảnh hưởng kéo dài của bức xạ cực tím (UVR). Hiện tại ở Việt Nam chưa có thang đo nào uy tín đánh giá về thói quen tiếp xúc và bảo vệ da dưới tác động của ánh sáng mặt trời. Nghiên cứu của chúng tôi chuẩn hóa phiên bản tiếng Việt và đánh giá độ tin cậy của thang đo chỉ số tiếp xúc và bảo vệ da dưới tác động của ánh sáng mặt trời (Sun exposure and protection index - SEPI), được thực hiện từ tháng 10 năm 2024 đến tháng 12 năm 2024, khảo sát lần thứ nhất được thực hiện trên 47 sinh viên trường Đại học Y Hà Nội. Sau 2 tuần tiến hành khảo sát lần thứ hai. Kết quả được sử dụng để đo lường tính thống nhất nội bộ qua chỉ số Cronbach alpha và độ tin cậy thử nghiệm lại qua chỉ số tương quan nội bộ (Intraclass Correlation - ICC). Phiên bản tiếng Việt của bộ câu hỏi SEPI cho thấy tính thống nhất nội bộ cao với chỉ số Cronbach’s alpha bằng 0.755. Độ tin cậy thử nghiệm lại giữa hai lần trả lời của bộ câu hỏi rất cao với chỉ số ICC là 0.927. Nghiên cứu đã cho thấy thang đo SEPI phiên bản tiếng Việt là một bộ công cụ hợp lý để đánh giá chỉ số tiếp xúc và bảo vệ da dưới ánh sáng mặt trời cho người Việt Nam.