Development of rabbit monoclonal and polyclonal antibodies for detection of site-specific histone modifications and their application in analyzing overall modification levels

In addition to DNA sequence information, site-specific histone modifications are another important determinant of gene expression in a eukaryotic organism. We selected four modification sites in common histones that are known to significantly impact chromatin function and generated monoclonal or polyclonal antibodies that recognize each of those site-specific modifications. We used these antibodies to demonstrate that the site-specific histone modification levels remain relatively constant in different organs of the same organism. We also compared the levels of selected histone modifications among several representative organisms and found that site-specific modifications are highly variable among different organisms, providing new insight into the evolutionary divergence of specific histone modifications.

Advances of N-terminal modifications of GLP-1 and their applications for the treatment of type 2 diabetes

Glucagon-like peptide-1（GLP-1） is an endogenous insulinotropic hormone with excellent blood glucose-lowering activity, however, it is rapidly inactivated in the plasma mainly by dipeptidyl peptidase IV（DPP-IV）. To overcome this problem, various N-terminal modifications of GLP-1 have been performed to prolong the in vivo biological activity, by improving the DPP-IV resistance while retaining receptor affinity and receptor activation. These studies have included modifications of His7, Ala8 or Glu9 at the N-terminus of GLP-1 and some other modifications. Among them, Ala8 substitutions with glycine（Gly8） and α-aminoisobutyric acid（Aib8） have been clinically applied in the development of diabetic therapy, such as Exenatide, Semaglutide, Albiglutide and Taspoglutide. In this review, we introduce N-terminal modifications of GLP-1 that have been reported, and discuss their potential and challenges for the treatment of type 2 diabetes.

Selective N-terminal modification of peptides and proteins:Recent progresses and applications

Numerous strategies for linking desired chemical probes with target peptides and proteins have been developed and applied in the field of biological chemistry.Approaches for site-specific modification of native amino acid residues in test tubes and biological contexts represent novel biological tools for understanding the role of peptides and proteins.Selective N-terminal modification strategies have been broadly studied especially in the last 10 years,as N-terminal positions are typically solvent exposed and provide chemically distinct sites for many peptide and protein targets,making N terminus distinct from other functional groups.A growing number of chemical and enzymatic techniques have been developed to modify N-terminal amino acids,and those techniques have the potential in the fields of medicine,basic research and applied materials science.This review focuses on appraising modification methodologies with the potential for biological applications from the past 10 years.

Recent advances in enzyme-mediated peptide ligation

Artificial synthesis and site-specific modification of peptides and proteins has evolved into an indispensable tool for protein engineers and chemical biologists. Chemical and enzymatic approaches to peptide ligation are important alternatives of recombinant DNA technology for protein synthesis and modification. Although as old as that of chemical procedures, enzyme-mediated peptide ligation is far less developed than that of chemical counterpart due to the difficult availability of peptide ligase.Fortunately, this situation has been changed slowly with the fast development of biological techniques. In the past decades, several natural peptide ligases have been discovered. Protein engineering to improve the ligation efficiencies of the natural peptide ligase and to reverse the functionality of protease provide more powerful peptide ligases. In this review, the advances of enzyme-mediated peptide ligation and their application in protein synthesis and modification will be discussed.

Peptide asparaginyl ligases——renegade peptide bond makers

Making peptide bonds is tightly controlled by genetic code and machinery which includes cofactors,ATP,and RNAs.In this regard,the stand-alone and genetic-code-independent peptide ligases constitute a new family of renegade peptide-bond makers.A prime example is butelase-1,an Asn/Asp(Asx)-specific ligase that structurally belongs to the asparaginyl endopeptidase family.Butelase-1 specifically recognizes a C-terminal Asx-containing tripeptide motif,Asn/Asp-Xaa-Yaa(Xaa and Yaa are any amino acids),to form a site-specific Asn-Xaa peptide bond either intramolecularly as cyclic proteins or intermolecularly as modified proteins.Our work in the past five years has validated that butelase-1 is a potent and versatile ligase.Here we review the advances in ligases,with a focus on butelase-1,and their applications in engineering bioactive peptides and precision protein modifications,antibody-drug conjugates,and live-cell labeling.

Supramolecular approaches for insulin stabilization without prolonged duration of action

Aggregation represents a significant challenge for the long-term formulation stability of insulin therapeutics.The supramolecular PEGylation of insulin with conjugates of cucurbit[7]uril and polyethylene glycol(CB[7]-PEG)has been shown to stabilize insulin formulations by reducing aggregation propensity.Yet prolonged in vivo duration of action,arising from sustained complex formation in the subcutaneous depot,limits the application scope for meal-time insulin uses and could increase hypoglycemic risk several hours after a meal.Supramolecular affinity of CB[7]in binding the B1-Phe residue on insulin is central to supramolecular PEGylation using this approach.Accordingly,here we synthesized N-terminal acid-modified insulin analogs to reduce CB[7]interaction affinity at physiological pH and reduce the duration of action by decreasing the subcutaneous depot effect of the formulation.These insulin analogs show weak to no interaction with CB[7]-PEG at physiological pH but demonstrate high formulation stability at reduced pH.Accordingly,N-terminal modified analogs have in vitro and in vivo bioactivity comparable to native insulin.Furthermore,in a rat model of diabetes,the acid-modified insulin formulated with CB[7]-PEG offers a reduced duration of action compared to native insulin formulated with CB[7]-PEG.This work extends the application of supramolecular PEGylation of insulin to achieve enhanced stability while reducing the risks arising from a subcutaneous depot effect prolonging in vivo duration of action.

A Proximity-Triggered Strategy toward Transferable Proteolysis Targeting Chimeras

Over the past 20 years,great efforts have been invested in developing site-specific approaches to protein modification to dissect protein functions directly and accurately.Here,we report a proximitytriggered group transfer strategy from a sulfonium warhead to a Cysteine(Cys)residue of the target protein.With a guiding ligand,cargoes could be transferred selectively from a sulfonium center onto the Cys residue in the vicinity of their binding interface.The successful thalidomide transfer of sulfonium 1-X could be applied intracellularly for epidermal growth factor receptor degradation,highlighting the potential of group transfer strategy as a suite of chemical biology studies,including cell imaging,protein profiling,and protein degradation by simply employing different transferrable groups.