Identification of SARS-CoV-2 PLpro and 3CLpro human proteome substrates using substrate phage display coupled with protein network analysis.

Viral proteases play key roles in viral replication, and they also facilitate immune escape by proteolyzing diverse target proteins. Deep profiling of viral protease substrates in host cells is beneficial for understanding viral pathogenesis and for antiviral drug discovery. Here, we utilized substrate phage display coupled with protein network analysis to identify human proteome substrates of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viral proteases, including papain-like protease (PLpro) and 3C-like protease (3CLpro). We first performed peptide substrates selection of PLpro and 3CLpro, and we then used the top 24 preferred substrate sequences to identify a total of 290 putative protein substrates. Protein network analysis revealed that the top clusters of PLpro and 3CLpro substrate proteins contain ubiquitin-related proteins and cadherin-related proteins, respectively. We verified that cadherin-6 and cadherin-12 are novel substrates of 3CLpro, and CD177 is a novel substrate of PLpro using in vitro cleavage assays. We thus demonstrated that substrate phage display coupled with protein network analysis is a simple and high throughput method to identify human proteome substrates of SARS-CoV-2 viral proteases for further understanding of virus-host interactions.

Visible-Light-Driven, Copper-Catalyzed Decarboxylative C(sp3 )-H Alkylation of Glycine and Peptides.

Despite a well-developed and growing body of work in Cu catalysis, the potential of Cu to serve as a photocatalyst remains underexplored. Reported herein is the first example of visible-light-induced Cu-catalyzed decarboxylative C(sp3 )-H alkylation of glycine for preparing α-alkylated unnatural α-amino acids. It merits mentioning that the mild conditions and the good functional-group tolerance allow the modification of peptides using this method. The mechanistic studies revealed that a radical-radical coupling pathway is involved in the reaction.

A single backbone amide modification method to achieve single site-specific chemical protein conjugation.

Efficient and precise chemical protein modification methods are highly sought after in biotechnology. However, chemically distinguishing a single site within a large protein is challenging. This study introduces a Copper Assisted Sequence-specific Conjugation Tag (CAST) method, enabling rapid (second order rate 8.1 M-1s-1) and site-specific chemical modification of the protein backbone with pinpoint accuracy. The versatility of this method is demonstrated through the preparation of antibody-drug conjugates, showcasing high plasma stability and potent efficacy in both in vitro and in vivo settings. Thus, CAST emerges as an efficient and quantitative approach for attaching payloads to large, native proteins.

A high-throughput fully automatic biosensing platform for efficient COVID-19 detection.

We propose a label-free biosensor based on a porous silicon resonant microcavity and localized surface plasmon resonance. The biosensor detects SARS-CoV-2 antigen based on engineered trimeric angiotensin converting enzyme-2 binding protein, which is conserved across different variants. Robotic arms run the detection process including sample loading, incubation, sensor surface rinsing, and optical measurements using a portable spectrometer. Both the biosensor and the optical measurement system are readily scalable to accommodate testing a wide range of sample numbers. The limit of detection is 100 TCID50/ml. The detection time is 5 min, and the throughput of one single robotic site is up to 384 specimens in 30 min. The measurement interface requires little training, has standard operation, and therefore is suitable for widespread use in rapid and onsite COVID-19 screening or surveillance.

Copper assisted sequence-specific chemical protein conjugation at a single backbone amide.

Direct, site-specific methods of protein functionalization are highly desirable for biotechnology. However, such methods are challenging due to the difficulty of chemically differentiating a single site within a large protein. Herein, we propose "metal binding targeting" strategy and develop a Copper Assisted Sequence-specific conjugation Tag (CAST) method to achieve rapid (second order rate 8.1 M-1 s-1), site-specific protein backbone chemical modification with pinpoint accuracy. We demonstrate the versatility of CAST conjugation by preparing various on-demand modified recombinant proteins, including a homogeneous antibody-drug conjugate with high plasma stability and potent efficacy in vitro and in vivo. Thus, CAST provides an efficient and quantitative method to site-specifically attach payloads on large, native proteins.

Precise control of embolic stroke with magnetized red blood cells in mice.

Precise embolism control in immature brains can facilitate mechanistic studies of brain damage and repair after perinatal arterial ischemic stroke (PAIS), but it remains a technical challenge. Microhemorrhagic transformation is observed in one-third of infant patients who have suffered PAIS, but the underlying mechanism remains elusive. Building on an established approach that uses magnetic nanoparticles to induce PAIS, we develop a more advanced approach that utilizes magnetized erythrocytes to precisely manipulate de novo and in situ embolus formation and reperfusion in perinatal rodent brains. This approach grants spatiotemporal control of embolic stroke without any transarterial delivery of pre-formed emboli. Transmission electron microscopy revealed that erythrocytes rather than nanoparticles are the main material obstructing the vessels. Both approaches can induce microbleeds as an age-dependent complication; this complication can be prevented by microglia and macrophage depletion. Thus, this study provides an animal model mimicking perinatal embolic stroke and implies a potential therapeutic strategy for the treatment of perinatal stroke.

Cu-Catalyzed cyanoalkylation of electron-deficient alkenes with unactivated alkyl bromides.

We here report the photoinduced Cu-catalyzed cyanoalkylation of electron-deficient alkenes by using alkyl bromides as alkylation reagents. In the reactions, 1°, 2°, and 3° unactivated alkyl bromides with various sensitive functional groups were well tolerated with good yields. Notably, terminal and internal alkenes, as well as alkene-containing peptides, were all tolerated well.

Photoinduced, Copper-Promoted Regio- and Stereoselective Decarboxylative Alkylation of α,ß-Unsaturated Acids with Alkyl Iodides.

The first example of UV light-induced, copper-catalyzed regio- and stereoselective decarboxylative coupling of α,ß-unsaturated acids with alkyl iodides was reported. Under standard conditions, the 1°, 2°, and 3° alkyl iodides proceeded smoothly with the E-selective alkenes obtained in uniformly good yields and high stereoselectivities.