CRISPR metabolic screen identifies ATM and KEAP1 as targetable genetic vulnerabilities in solid tumors.

Cancer treatments targeting DNA repair deficiencies often encounter drug resistance, possibly due to alternative metabolic pathways that counteract the most damaging effects. To identify such alternative pathways, we screened for metabolic pathways exhibiting synthetic lethality with inhibition of the DNA damage response kinase Ataxia-telangiectasia-mutated (ATM) using a metabolism-centered Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 library. Our data revealed Kelch-like ECH-associated protein 1 (KEAP1) as a key factor involved in desensitizing cancer cells to ATM inhibition both in vitro and in vivo. Cells depleted of KEAP1 exhibited an aberrant overexpression of the cystine transporter SLC7A11, robustly accumulated cystine inducing disulfide stress, and became hypersensitive to ATM inhibition. These hallmarks were reversed in a reducing cellular environment indicating that disulfide stress was a crucial factor. In The Cancer Genome Atlas (TCGA) pan-cancer datasets, we found that ATM levels negatively correlated with KEAP1 levels across multiple solid malignancies. Together, our results unveil ATM and KEAP1 as new targetable vulnerabilities in solid tumors.

Structural insight into the subclass B1 metallo-ß-lactamase AFM-1.

The emergence of drug-resistant bacteria, facilitated by metallo-beta-lactamases (MBLs), presents a significant obstacle to the effective use of antibiotics in the management of clinical drug-resistant bacterial infections. AFM-1 is a MBL derived from Alcaligenes faecalis and shares 86% homology with the NDM-1 family. Both AFM-1 and NDM-1 demonstrate the ability to hydrolyze ampicillin and other ß-lactam antibiotics, however, their substrate affinities vary, and the specific reason for this variation remains unknown. We present the high-resolution structure of AFM-1. The active center of AFM-1 binds two zinc ions, and the conformation of the key amino acid residues in the active center is in accordance with that of NDM-1. However, the substrate-binding pocket of AFM-1 is considerably smaller than that of NDM-1. Additionally, the mutation of amino acid residues in the Loop3 region, as compared to NDM-1, results in the formation of a dense hydrophobic patch comprised of hydrophobic amino acid residues in this area, which facilitates substrate binding. Our findings lay the foundation for understanding the molecular mechanism of AFM-1 with a high affinity for substrates and provide a novel theoretical foundation for addressing the issue of drug resistance caused by B1 MBLs.

Computer-assisted semi-rational design enhanced the enzymatic activity and protein stability of Proteinase K in calcium-free conditions.

Wild-type Proteinase K binds to two Ca2+ ions, which play an important role in regulating enzymaticactivity and maintaining protein stability. Therefore, a predetermined concentration of Ca2+ must be added during the use of Proteinase K, which increases its commercial cost. Herein, we addressed this challenge using a computational strategy to engineer a Proteinase K mutant that does not require Ca2+ and exhibits high enzymatic activity and protein stability. In the absence of Ca2+, the best mutant, MT24 (S17W-S176N-D260F), displayed an activity approximately 9.2-fold higher than that of wild-type Proteinase K. It also exhibited excellent protein stability, retaining 56.2 % of its enzymatic activity after storage at 4 °C for 5 days. The residual enzymatic activity was 65-fold higher than that of the wild-type Proteinase K under the same storage conditions. Structural analysis and molecular dynamics simulations suggest that the introduction of new hydrogen bond and π-π stacking at the Ca2+ binding sites due to the mutation may be the reasons for the increased enzymatic activity and stability of MT24.

Structural and mechanistic insights into the complexes formed by Wolbachia cytoplasmic incompatibility factors.

Wolbachia bacteria, inherited through the female germ line, infect a large fraction of arthropod species. Many Wolbachia strains manipulate host reproduction, most commonly through cytoplasmic incompatibility (CI). CI, a conditional male sterility, results when Wolbachia-infected male insects mate with uninfected females; viability is restored if the female is similarly infected (called "rescue"). CI is used to help control mosquito-borne viruses such as dengue and Zika, but its mechanisms remain unknown. The coexpressed CI factors CifA and CifB form stable complexes in vitro, but the timing and function of this interaction in the insect are unresolved. CifA expression in the female germ line is sufficient for rescue. We report high-resolution structures of a CI-factor complex, CinA-CinB, which utilizes a unique binding mode between the CinA rescue factor and the CinB nuclease; the structures were validated by biochemical and yeast growth analyses. Importantly, transgenic expression in Drosophila of a nonbinding CinA mutant, designed based on the CinA-CinB structure, suggests CinA expressed in females must bind CinB imported by sperm in order to rescue embryonic viability. Binding between cognate factors is conserved in an enzymatically distinct CI system, CidA-CidB, suggesting universal features in Wolbachia CI induction and rescue.

Structural basis for GTP-induced dimerization and antiviral function of guanylate-binding proteins.

Guanylate-binding proteins (GBPs) form a family of dynamin-related large GTPases which mediate important innate immune functions. They were proposed to form oligomers upon GTP binding/hydrolysis, but the molecular mechanisms remain elusive. Here, we present crystal structures of C-terminally truncated human GBP5 (hGBP51-486), comprising the large GTPase (LG) and middle (MD) domains, in both its nucleotide-free monomeric and nucleotide-bound dimeric states, together with nucleotide-free full-length human GBP2. Upon GTP-loading, hGBP51-486 forms a closed face-to-face dimer. The MD of hGBP5 undergoes a drastic movement relative to its LG domain and forms extensive interactions with the LG domain and MD of the pairing molecule. Disrupting the MD interface (for hGBP5) or mutating the hinge region (for hGBP2/5) impairs their ability to inhibit HIV-1. Our results point to a GTP-induced dimerization mode that is likely conserved among all GBP members and provide insights into the molecular determinants of their antiviral function.

Computation-Aided Phylogeny-Oriented Engineering of ß-Xylosidase: Modification of "Blades" to Enhance Stability and Activity for the Bioconversion of Hemicellulose to Produce Xylose.

Hemicellulose is a highly abundant, ubiquitous, and renewable natural polysaccharide, widely present in agricultural and forestry residues. The enzymatic hydrolysis of hemicellulose has generally been accomplished using ß-xylosidases, but concomitantly increasing the stability and activity of these enzymes remains challenging. Here, we rationally engineered a ß-xylosidase from Bacillus clausii to enhance its stability by computation-aided design combining ancestral sequence reconstruction and structural analysis. The resulting combinatorial mutant rXYLOM25I/S51L/S79E exhibited highly improved robustness, with a 6.9-fold increase of the half-life at 60 °C, while also exhibiting improved pH stability, catalytic efficiency, and hydrolytic activity. Structural analysis demonstrated that additional interactions among the propeller blades in the catalytic module resulted in a much more compact protein structure and induced the rearrangement of the opposing catalytic pocket to mediate the observed improvement of activity. Our work provides a robust biocatalyst for the hydrolysis of agricultural waste to produce various high-value-added chemicals and biofuels.

[Detection methods for polyethylene terephthalate degrading enzymes: a review].

Polyethylene terephthalate (PET) is one of the most widely used synthetic polyester. It poses serious threat to terrestrial, aquatic ecosystems and human health since it is difficult to be broken down and deposited in the environment. The biodegradation based on enzymatic catalysis offers a sustainable method for recycling PET. A number of PET hydrolases have been discovered in the last 20 years, and protein engineering has increased their degradation capabilities. However, no PET hydrolases that are practical for widespread industrial use have been identified. Screening of PET hydrolase using conventional detection techniques is laborious and inefficient process. Effective detection techniques are required to promote the commercialization of PET hydrolases. Using efficient detection techniques to screen potent industrial enzymes is essential for supporting the widespread industrial implementation of PET hydrolases. To define PET hydrolase, scientists have created a number of analytical techniques recently. The detection techniques that can be used to screen PET hydrolase, including high performance liquid chromatography, ultraviolet absorption spectrometric, and fluorescence activated droplet sorting method, are summarized in this study along with their potential applications.

Poloxamer 407 Induces Hypertriglyceridemia but Decreases Atherosclerosis in Ldlr-/- Mice.

BACKGROUND: Hypertriglyceridemia (HTG) increases the risk for atherosclerotic cardiovascular disease, but underlying mechanisms are incompletely understood. Circulating monocytes play an important role in atherogenesis by infiltrating arterial walls, where they differentiate into macrophages. We tested the hypothesis that HTG is mechanistically linked to atherogenesis by altering the monocyte phenotype and infiltration into atherosclerotic lesions in a model of diet-induced atherogenesis in Ldlr-/- mice. METHODS: HTG was induced in male Ldlr-/- mice, fed a Western, high-fat high-cholesterol diet, by daily injection of poloxamer 407 (P407), a lipoprotein lipase inhibitor, for seven weeks. Atherosclerosis, monocyte phenotypes, and monocyte migration into atherosclerotic lesions were determined by well-validated methods. RESULTS: Compared with the saline control, P407 injection in Ldlr-/- mice rapidly induced profound and persistent HTG, modestly elevated plasma cholesterol levels, and increased levels of triglyceride and cholesterol carried in very-low-density lipoprotein and low-density lipoprotein. Unexpectedly, mice receiving P407 versus saline control showed less atherosclerosis. Following induction of HTG by P407, CD36+ (also CD11c+), but not CD36- (CD11c-), monocytes showed early increases in lipid accumulation, but the number of CD36+ (not CD36-) monocytes was dramatically decreased afterwards in the circulation until the end of the test. Concurrently, CD36+ (CD11c+) monocyte migration into atherosclerotic lesions was also reduced in mice receiving P407 versus controls. CONCLUSIONS: P407 induced severe HTG, but reduced atherosclerosis, in Ldlr-/- mice, possibly because of profound reductions of circulating CD36+ (CD11c+) monocytes, leading to decreased monocyte migration into atherosclerotic lesions.

The Structure of the Porcine Deltacoronavirus Main Protease Reveals a Conserved Target for the Design of Antivirals.

The existing zoonotic coronaviruses (CoVs) and viral genetic variants are important microbiological pathogens that cause severe disease in humans and animals. Currently, no effective broad-spectrum antiviral drugs against existing and emerging CoVs are available. The CoV main protease (Mpro) plays an essential role in viral replication, making it an ideal target for drug development. However, the structure of the Deltacoronavirus Mpro is still unavailable. Porcine deltacoronavirus (PDCoV) is a novel CoV that belongs to the genus Deltacoronavirus and causes atrophic enteritis, severe diarrhea, vomiting and dehydration in pigs. Here, we determined the structure of PDCoV Mpro complexed with a Michael acceptor inhibitor. Structural comparison showed that the backbone of PDCoV Mpro is similar to those of alpha-, beta- and gamma-CoV Mpros. The substrate-binding pocket of Mpro is well conserved in the subfamily Coronavirinae. In addition, we also observed that Mpros from the same genus adopted a similar conformation. Furthermore, the structure of PDCoV Mpro in complex with a Michael acceptor inhibitor revealed the mechanism of its inhibition of PDCoV Mpro. Our results provide a basis for the development of broad-spectrum antivirals against PDCoV and other CoVs.

Biodegradation of highly crystallized poly(ethylene terephthalate) through cell surface codisplay of bacterial PETase and hydrophobin.

The process of recycling poly(ethylene terephthalate) (PET) remains a major challenge due to the enzymatic degradation of high-crystallinity PET (hcPET). Recently, a bacterial PET-degrading enzyme, PETase, was found to have the ability to degrade the hcPET, but with low enzymatic activity. Here we present an engineered whole-cell biocatalyst to simulate both the adsorption and degradation steps in the enzymatic degradation process of PETase to achieve the efficient degradation of hcPET. Our data shows that the adhesive unit hydrophobin and degradation unit PETase are functionally displayed on the surface of yeast cells. The turnover rate of the whole-cell biocatalyst toward hcPET (crystallinity of 45%) dramatically increases approximately 328.8-fold compared with that of purified PETase at 30 °C. In addition, molecular dynamics simulations explain how the enhanced adhesion can promote the enzymatic degradation of PET. This study demonstrates engineering the whole-cell catalyst is an efficient strategy for biodegradation of PET.

Crystal Structures of Wolbachia CidA and CidB Reveal Determinants of Bacteria-induced Cytoplasmic Incompatibility and Rescue.

Cytoplasmic incompatibility (CI) results when Wolbachia bacteria-infected male insects mate with uninfected females, leading to embryonic lethality. "Rescue" of viability occurs if the female harbors the same Wolbachia strain. CI is caused by linked pairs of Wolbachia genes called CI factors (CifA and CifB). The co-evolution of CifA-CifB pairs may account in part for the incompatibility patterns documented in insects infected with different Wolbachia strains, but the molecular mechanisms remain elusive. Here, we use X-ray crystallography and AlphaFold to analyze the CI factors from Wolbachia strain wMel called CidAwMel and CidBwMel. Substituting CidAwMel interface residues with those from CidAwPip (from strain wPip) enables the mutant protein to bind CidBwPip and rescue CidBwPip-induced yeast growth defects, supporting the importance of CifA-CifB interaction in CI rescue. Sequence divergence in CidAwPip and CidBwPip proteins affects their pairwise interactions, which may help explain the complex incompatibility patterns of mosquitoes infected with different wPip strains.

Yeast cell surface display of bacterial PET hydrolase as a sustainable biocatalyst for the degradation of polyethylene terephthalate.

Enzymatic hydrolysis of polyethylene terephthalate (PET) is considered to be an environmentally friendly method for the recycling of plastic waste. Recently, a bacterial enzyme named IsPETase was found in Ideonella sakaiensis with the ability to degrade amorphous PET at ambient temperature suggesting its possible use in recycling of PET. However, applying the purified IsPETase in large-scale PET recycling has limitations, i.e., a complicated production process, high cost of single-use, and instability of the enzyme. Yeast cell surface display has proven to be an effectual alternative for improving enzyme degradation efficiency and realizing industrial applications. This chapter deals with the construction and application of a whole-cell biocatalyst by displaying IsPETase on the surface of yeast (Pichia pastoris) cells.

A microfluidic chip with a serpentine channel enabling high-throughput cell separation using surface acoustic waves.

As an acute inflammatory response, sepsis may cause septic shock and multiple organ failure. Rapid and reliable detection of pathogens from blood samples can promote early diagnosis and treatment of sepsis. However, traditional pathogen detection methods rely on bacterial blood culture, which is complex and time-consuming. Although pre-separation of bacteria from blood can help with the identification of pathogens for diagnosis, the required low-velocity fluid environment of most separation techniques greatly limits the processing capacity for blood samples. Here, we present an acoustofluidic device for high-throughput bacterial separation from human blood cells. Our device utilizes a serpentine microfluidic design and standing surface acoustic waves (SSAWs), and separates bacteria from blood cells effectively based on their size difference. The serpentine microstructure allows the operating distance of the acoustic field to be multiplied in a limited chip size via the "spatial multiplexing" and "pressure node matching" of SSAW field. Microscopic observation and flow cytometry analysis shows that the device is helpful in improving the flow rate (2.6 µL min-1 for blood samples; the corresponding velocity is ∼3 cm s-1) without losing separation purity or cell recovery. The serpentine microfluidic design provides a compatible solution for high-throughput separation, which can synergize with other functional designs to improve device performance. Further, its advantages such as low cost, high biocompatibility, label-free separation and ability to integrate with on-chip biosensors are promising for clinical utility in point-of-care diagnostic platforms.

Dual-functional protein for one-step production of a soluble and targeted fluorescent dye.

Low water solubility and poor selectivity are two fundamental limitations that compromise applications of near-infrared (NIR) fluorescent probes. Methods: Here, a simple strategy that can resolve these problems simultaneously was developed by using a novel hybrid protein named RGD-HFBI that is produced by fusion of hydrophobin HFBI and arginine-glycine-aspartic acid (RGD) peptide. This unique hybrid protein inherits self-assembly and targeting functions from HFBI and RGD peptide respectively. Results: Boron-dipyrromethene (BODIPY) used as a model NIR dye can be efficiently dispersed in the RGD-HFBI solution by simple mixing and sonication for 30 min. The data shows that self-assembled RGD-HFBI forms a protein nanocage by using the BODIPY as the assembly template. Cell uptake assay proves that RGD-HFBI/BODIPY can efficiently stain αvß3 integrin-positive cancer cells. Finally, in vivo affinity tests fully demonstrate that the soluble RGD-HFBI/BODIPY complex selectively targets and labels tumor sites of tumor-bearing mice due to the high selectivity of the RGD peptide. Conclusion: Our one-step strategy using dual-functional RGD-HFBI opens a novel route to generate soluble and targeted NIR fluorescent dyes in a very simple and efficient way and may be developed as a general strategy to broaden their applications.

Effective Bioactivity Retention of Low-Concentration Antibodies on HFBI-Modified Fluorescence ICTS for Sensitive and Rapid Detection of PSA.

Nowadays, increasing analytical sensitivity is still a big challenge in constructing membrane-based fluorescence immunochromatography test strips (FICTS). However, the bioactivity of antibody (Ab) immobilized on the test line (T line) of porous nitrocellulose membrane (PNM), which directly influences the analytical sensitivity, is less studied. In this work, a novel amphiphilic hydrophobin (HFBI) protein was introduced to modify the T line to effectively retain the Abs' bioactivity. The results indicated that HFBI could self-assemble on the PNM and immobilize the Abs in the "stand-up" orientation. Compared with the conventional FICTS, the HFBI-modified FICTS with only 0.2 mg/mL of monoclonal Abs on T line enable more accurate quantitative detection and better sensitivity (0.06 ng/mL for prostate specific antigen), which is more than 2 orders of magnitude lower than that of the conventional FICTS with the same concentration of monoclonal Abs on T line. Furthermore, the accuracy of this HFBI-modified FICTS was investigated by testing 150 clinical serum samples and the detection results were coincident with those by electrochemiluminescence immunoassay. Our results provide a novel and promising strategy of Ab immobilization on FICTS for near-patient and point-of-care application.

Self-assembled hydrophobin for producing water-soluble and membrane permeable fluorescent dye.

Low water solubility and poor membrane permeability are major disadvantages that compromise applications of most fluorescent dyes. To resolve these problems, herein, using Boron-dipyrromethene (BODIPY) as a model fluorescent dye, for the first time, we provide a new strategy for the rapid and efficient production of a water-soluble and membrane-permeable dye by mixing with an amphiphilic protein named hydrophobin. Data shows BODIPY could be effectively solubilized and dispersed in 200 µg/mL hydrophobin by simple mixing and sonication. Subsequent experiments indicated that hydrophobin self-assembled into a protein film on the surface of BODIPY forming stable hydrophobin-BODIPY complexes with a size range of 10-30 nm. Furthermore, we demonstrated hydrophobin-functionalized BODIPY are toxicity free to cells. The hydrophobin-BODIPY complex could pass through both the cell plasma membrane and nuclear membrane efficiently. Our work opens a novel route to modify and functionalize fluorescent dyes and may be developed as a general strategy for broadening their applications.

RNA stability under different combinations of amber force fields and solvation models.

The proper matching of force field and solvent is critical to obtain correct result in molecular dynamics simulation of bio-molecules. This problem has been intensively investigated for protein but not for RNA yet. In this paper, we use standard molecular dynamics and replica exchange molecular dynamics to take a series of tests on the RNA stability under different combinations of Amber force field parameters (ff98, ff99 and ff99bsc0) and the general Born implicit solvent models (igb1, igb2 and igb5). It is found that only ff98 and ff99bsc0 with igb1 can keep the native conformations of RNA hairpin and duplex. Our results suggest that ff98 plus igb1 may be reasonable choice for molecular dynamics simulation of RNA dynamics.