ChemBioChem最新文献

We designed a minimalistic zinc(II)-binding peptide featuring the Cys₂His₂ zinc-finger motif. To this aim, several tens of thousands of (His/Cys)-X_n-(His/Cys) protein fragments (n=2-20) were first extracted from the 3D protein structures deposited in Protein Data Bank (PDB). Based on geometrical constraints positioning two Cys (C) and two His (H) side chains at the vertices of a tetrahedron, approximately 22 000 sequences of the (H/C)-X_i-(H/C)-X_j-(H/C)-X_k-(H/C) type, satisfying N_{metal-binding H}=N_{metal-binding C}=2, were processed. Several other criteria, such as the secondary structure content and predicted fold stability, were then used to select the best candidates. To prove the viability of the computational design experimentally, three peptides were synthesized and subjected to isothermal calorimetry (ITC) measurements to determine the binding constants with Zn²⁺, including the entropy and enthalpy terms. For the strongest Zn²⁺ ions binding peptide, P1, the dissociation constant was shown to be in the nanomolar range (K_D=~220 nM; corresponding to ΔG_bind=-9.1 kcal mol^-1). In addition, ITC showed that the [P1 : Zn²⁺] complex forms in 1 : 1 stoichiometry and two protons are released upon binding, which suggests that the zinc coordination involves both cysteines. NMR experiments also indicated that the structure of the [P1 : Zn²⁺] complex might be quite similar to the computationally predicted one. In summary, our proof-of-principle study highlights the usefulness of our computational protocol for designing novel metal-binding peptides.

Pyrimidine nucleotides are essential for a wide variety of cellular processes and are synthesized either via a salvage pathway or through de novo biosynthesis. The latter is particularly important in proliferating cells, such as infectious diseases and cancer cells. Aspartate transcarbamoylase (ATCase) catalyzes the first committed and rate-limiting step in the de novo pyrimidine biosynthesis pathway, making it an attractive therapeutic target for various diseases. This review summarizes the development of a series of allosteric ATCase inhibitors, advancing them as potential candidates for malarial, tuberculosis and cancer therapies. Furthermore, it explores the potential for these compounds to be expanded into drugs targeting neglected tropical diseases, antimicrobial-resistant infections caused by the ESKAPE pathogens, and their possible application as herbicides. We identify the likely equivalent allosteric pocket in these systems and perform a structure and sequence-based analysis of the resides comprising it, providing a rationale for continued exploration of this compound series as both specific and broad-range inhibitors. The review concludes by emphasizing the importance of continued research into ATCase inhibitors, given their potential broad applicability in treating diverse diseases to enhance both human health and agricultural practices.

Delivering cargo into live cells has extensive applications in chemistry, biology, and medicine. Cell-penetrating peptides (CPPs) provide an ideal solution for cellular delivery. Enhancing CPPs with additional functional units can improve delivery efficiency. We investigate the conjugation of boronic acid modules to enhance internalization through interactions with cell surface glycans. The aim of this study is to determine whether adding boronic acid can transform a peptide that typically lacks CPP properties into one that functions as a CPP, enabling the delivery of crucial biological cargo like ubiquitin (Ub). The zinc finger protein in its apo state was selected as a "boronate-enabled" CPP. Results indicate that skeletal point mutations and post-synthetic modifications, combined with conjugated benzoboroxole derivatives, enable the apo-ZFP the ability to transport Ub within A549 cells, confirmed through microscopy and flow cytometry. This effective internalization of cargo offers valuable insights for advancing the development of boronic acid-mediated cell-penetrating peptides.

The human hemoglobin subunit μ (Hb-μ) has been identified as a potential biomarker for α-thalassemia. However, little structural and functional information is available for this subunit. Here, we have overexpressed and purified a double mutant of C49S/C104S Hb-μ and solved its X-ray crystal structure. It adopts a typical protein fold of the globins, similar to that of the α-subunit. The structure also reveals that the protein undergoes self-oxidation of Met62 in the heme distal site, producing the form of sulfoxide (Met-SO). The property and function have also been studied by spectroscopy, which shows that the protein has considerable peroxidase activity due to the presence of a catalytic His-Arg pair in the heme distal site. The structure-function relationship of Hb-μ obtained in this study may provide useful insights into Hb-related diseases.

Phase separation, particularly liquid-liquid phase separation (LLPS), has emerged as a powerful tool in biological research, offering unique advantages for visualizing and analyzing biomolecular interactions. This review highlights recent advances in leveraging LLPS to develop experimental techniques for studying protein-protein interactions (PPIs), protein-RNA interactions, and enzyme activity. The integration of LLPS with advanced techniques has expanded its applications, offering new possibilities for unraveling the complexities of cellular function and disease mechanisms. Looking forward, the development of more versatile, sensitive, and targeted LLPS-based methods is poised to transform molecular biology, providing deeper insights into cellular dynamics and facilitating therapeutic advancements.

Bacterial flagellins are unique for their capacity to activate both the innate and the adaptive immune response through a Toll-like receptor 5 (TLR5) signaling cascade. Used as a carrier protein in conjugate vaccines, it is crucial to preserve their self-adjuvant properties during the conjugation step. Considering the absence of cysteine in the Salmonella enterica flagellin FliC sequence, we have investigated the impact of five mutations (A2 C, K180 C, T240 C, D251 C and S306 C) alone or in combination on TLR5 activation. The FliC mutated at the four positions K180 C, T240 C, D251 C and S306 C displayed much the same activity as native flagellin whether the cysteine residues were free or conjugated. These results pave the way for the preparation of self-adjuvanting conjugate vaccines based on cysteine-mutated FliC as a carrier protein.

Biological membranes achieve selectivity and permeability through protein transporters and channels. The design of artificial compartments with permeable membranes is essential to facilitate substrate and product transfer in enzymatic reactions. In this study, an E. coli outer membrane protein OmpF fused to a modular adaptor was integrated onto a DNA origami skeleton to control the number and polarity of the OmpF trimer. DNA origami skeleton-guided nanoliposomes reconstituted with functional OmpF exhibit pH-responsiveness and size-selective permeability. This approach highlights the potential to construct artificial compartments that incorporate membrane proteins of defined number and polarity, allowing tunable substrate fluxes.

LEA peptides, which are designed based on late embryogenic abundant (LEA) protein sequences, have demonstrated chaperone-like functions, such as improving drought stress tolerance of Escherichia coli (E. coli). Previous studies have focused on the biological functions of linear LEA peptides. However, the function of cyclic LEA peptide still unknown. This study aimed to explore the cyclic LEA peptides' bio function like enhance the drought stress tolerance of E. coli by cyclizing the LEA peptide using SICLOPPS (Split Intein Circular Ligation of Peptides and Proteins). The results indicated that cyclization significantly improved the function and extended the potential applications. At the same time, we found that peptides containing numerous lysine residues exhibited reduced performance, which may be due to the exteins' residues affecting the SICLOPPS efficiency.

Homothymidine DNA oligonucleotides bearing a 3'-terminal 6-phenyl-9H-carbazole C-nucleoside, mercurated at position 1, 8 or both, were synthesized and tested for their potential to form triple helices with homoadenine ⋅ homothymine duplexes. The monomercurated triplex-forming oligonucleotides favored hybridization with fully matched double helices and in some cases considerable increase of the melting temperature could be attributed to Hoogsteen-type Hg(II)-mediated interaction with the homoadenine strand. The dimercurated one, on the other hand, favored hybridization with double helices placing a homo mispair opposite to the carbazole residue, suggesting that simultaneous coordination of each of the two Hg(II) ions to a different strand is only possible in the absence of competition from Watson-Crick base pairing.

Currently, mankind is fiercely struggling with cancer. Recently, we have been winning the battle against cancer through precision medicine and accompanying diagnostic methods, and we are raising many hopes with blockbuster drugs. It would be even better if we could read the cancer nucleotide sequence, identify them in advance, and suggest treatments simultaneously. However, this may be an impossible dream because it takes a lot of time and effort to diagnose and ensure all the long gene sequences of cancer at once. Thus, victory will be even closer if a rapid and accurate diagnosis of the cancer-specific gene biomarkers that will soon be imprinted can be made. With the advent of nanotechnology, a new short cancer diagnostic toolkit has been proposed to achieve the goal. This review presents a small diagnostic device that detects certain cancers' genetic fragments (simply 'Gizmo'). The development of numerous diagnostic methods has focused on (1) directly detecting pre-selectively targeted genes using novel diagnostic systems, and (2) indirectly detecting substantial improvements in diagnostic sensitivity only through detection signal amplification without existing gene amplification steps. Our fight against cancer is not a dream, but the result of success, and it is assumed that victory will accelerate as soon as possible.