Discovery of a Monoclonal Antibody That Targets Cell-Surface Pseudaminic Acid of Acinetobacter baumannii with Direct Bactericidal Effect.

The therapeutic effects of antibodies include neutralization of pathogens, activation of the host complement system, and facilitation of phagocytosis of pathogens. However, antibody alone has never been shown to exhibit bactericidal activity. In this study, we developed a monoclonal antibody that targets the bacterial cell surface component Pseudaminic acid (Pse). This monoclonal antibody, Pse-MAB1, exhibited direct bactericidal activity on Acinetobacter baumannii strains, even in the absence of the host complements or other immune factors, and was able to confer a protective effect against A. baumannii infections in mice. This study provides new insight into the potential of developing monoclonal antibody-based antimicrobial therapy of multidrug resistant bacterial infections, especially those which occurred among immunocompromised patients.

Revealing the extracellular function of HMGB1 N-terminal region acetylation assisted by a protein semi-synthesis approach.

HMGB1 (high-mobility group box 1) is a non-histone chromatin-associated protein that has been widely reported as a representative damage-associated molecular pattern (DAMP) and to play a pivotal role in the proinflammatory process once it is in an extracellular location. Accumulating evidence has shown that HMGB1 undergoes extensive post-translational modifications (PTMs) that actively regulate its conformation, localization, and intermolecular interactions. However, fully characterizing the functional implications of these PTMs has been challenging due to the difficulty in accessing homogeneous HMGB1 with site-specific PTMs of interest. In this study, we developed a streamlined protein semi-synthesis strategy via salicylaldehyde ester-mediated chemical ligations (Ser/Thr ligation and Cys/Pen ligation, STL/CPL). This methodology enabled us to generate a series of N-terminal region acetylated HMGB1 proteins. Further studies revealed that acetylation regulates HMGB1-heparin interaction and modulates HMGB1's stability against thrombin, representing a regulatory switch to control HMGB1's extracellular activity.

Protocol for semisynthesis of serotonylated histone H3 by rapid protein desulfurization in tandem with native chemical ligation.

Here, we present a protocol of rapid protein desulfurization in tandem with native chemical ligation for facile syntheses of proteins with site-specific modifications. We describe using sodium tetraethylborate (NaBEt4) to carry out this desulfurization in an add-and-done manner under ambient conditions without requirement of inert atmosphere protection, UV irradiation, heating, or exogenous thiol additives. Specifically, we detail the semisynthesis of serotonylated histone H3(H3Q5ser) via one-pot ligation desulfurization. This protocol can be applied to synthesize proteins of interest with homogenous post-translational modifications. For complete information on the generation and use of this protocol, please refer to Sun et al. (2022).1.

Guanidine Additive Enabled Intermolecular ortho-Phthalaldehyde-Amine-Thiol Three-Component Reactions for Modular Constructions.

Recently, ortho-phthalaldehyde (OPA) is experiencing a renascence for the modification of proteins and peptides through OPA-amine two-component reactions for bioconjugation and intramolecular OPA-amine-thiol three-component reactions for cyclization. Historically, small thiol molecules were used in large excess to allow for the intermolecular OPA-amine-thiol reaction forming 1-thio-isoindole derivatives. In this study, we discovered that guanidine could serve as an effective additive to switch the intermolecular OPA-amine-thiol three-component reaction to a stoichiometric process and enable the modular construction of peptide-peptide, and peptide-drug conjugate structures. Thus, 12 model peptide-peptide conjugates have been synthesized from unprotected peptides featuring all proteinogenic residues. Besides, 6 peptide-drug conjugates have been prepared in one step, with excellent conversions and isolated yields. In addition, a conjugate product has been further functionalized by utilizing a premodified OPA derivative, demonstrating the versatility and flexibility of this reaction.

Chemical Synthesis of Proteins with Base-Labile Posttranslational Modifications Enabled by a Boc-SPPS Based General Strategy Towards Peptide C-Terminal Salicylaldehyde Esters.

Chemical synthesis of proteins bearing base-labile post-translational modifications (PTMs) is a challenging task. For instance, O-acetylation and S-palmitoylation PTMs cannot survive Fmoc removal conditions during Fmoc-solid phase peptide synthesis (SPPS). In this work, we developed a new Boc-SPPS-based strategy for the synthesis of peptide C-terminal salicylaldehyde (SAL) esters, which are the key reaction partner in Ser/Thr ligation and Cys/Pen ligation. The strategy utilized the semicarbazone-modified aminomethyl (AM) resin, which could support the Boc-SPPS and release the peptide SAL ester upon treatment with TFA/H2 O and pyruvic acid. The non-oxidative aldehyde regeneration was fully compatible with all the canonical amino acids. Armed with this strategy, we finished the syntheses of the O-acetylated protein histone H3(S10ac, T22ac) and the hydrophobic S-palmitoylated peptide derived from caveolin-1.

Thiophene-2,3-Dialdehyde Enables Chemoselective Cyclization on Unprotected Peptides, Proteins, and Phage Displayed Peptides.

Ortho-phthalaldehyde has recently found wide potentials for protein bioconjugation and peptide cyclization. Herein, the second-generation dialdehyde-based peptide cyclization method is reported. The thiophene-2,3-dialdehyde (TDA) reacts specifically with the primary amine (from Lys side chain or peptide N-terminus) and thiol (from Cys side chain) within unprotected peptides to generate a highly stable thieno[2,3-c]pyrrole-bridged cyclic structure, while it does not react with primary amine alone. This reaction is carried out in the aqueous buffer and features tolerance of diverse functionalities, rapid and clean transformation, and operational simplicity. The features allow TDA to be used for protein stapling and phage displayed peptide cyclization.

Ligation Embedding Aggregation Disruptor Strategy Enables the Chemical Synthesis of PD-1 Immunoglobulin and Extracellular Domains.

Chemical synthesis of proteins with aggregable or colloidal peptide segments presents a formidable task, as such peptides prove to be difficult for both solid-phase peptide synthesis and peptide ligation. To address this issue, we have developed ligation embedding aggregation disruptor (LEAD) as an effective strategy for the chemical synthesis of difficult-to-obtain proteins. The N,O/S-benzylidene acetals generated from Ser/Thr ligation and Cys/Pen ligation are found to effectively disrupt peptide aggregation, and they can be carried for sequential ligations toward protein synthesis. The effectiveness and generality of this strategy have been demonstrated with total syntheses of programmed cell death protein 1 immunoglobulin like V-type domain and extracellular domain.

Enabling chemical protein (semi)synthesis via reducible solubilizing tags (RSTs).

Chemical synthesis of proteins with poor solubility presents a challenging task. The existing solubilizing tag strategies are not suitable for the expressed protein segment. To address this issue, we report herein that solubilizing tags could be introduced at the side chain of the peptide and C-terminal peptide salicylaldehyde esters via a disulfide linker. Such reducible solubilizing tags (RSTs) are compatible with peptide salicylaldehyde ester-mediated Ser/Thr ligation and Cys/Pen ligation for purifying and ligating peptides with poor solubility. This strategy features operational simplicity and readily accessible materials. Both the protein 2B4 cytoplasmic tail and FCER1G protein have been successfully synthesized via this strategy. Of particular note, the RST strategy could be used for solubilizing the expressed protein segment for protein semi-synthesis of the HMGB1 protein.

Synthetic Pseudaminic-Acid-Based Antibacterial Vaccine Confers Effective Protection against Acinetobacter baumannii Infection.

Acinetobacter baumannii exhibits resistance to most first-line antibiotics; thus, development of new antibacterial agents is urgently required. Pseudaminic acid exists as the surface glycan of A. baumannii. In this study, we chemically synthesized pseudaminic acid, conjugated it to carrier protein CRM197 using the OPA (ortho-phthalaldehyde) chemistry, and obtained three Pse-CRM197 conjugates with different Pse loadings. These Pse-CRM197 conjugates were found to stimulate high immune responses in mice, which protected the vaccinated mice from infections caused by Pse-producing A. baumannii. Our data indicate that chemically synthesized Pse-CRM197 conjugates can be developed into vaccines against Pse-bearing pathogens, thus offering a feasible alternative for the control of clinical infections caused by multidrug-resistant (MDR) A. baumannii, for which current treatment options are extremely limited.

Serine/threonine ligation-assisted chemical synthesis of HMGA1a protein with site-specific post-translational modifications.

Dissecting the function of proteins' post-translational modifications (PTMs) is seriously hindered by the difficulty in obtaining the homogeneous protein with the PTMs of interest. Chemical protein synthesis offers a great potential to overcome this limitation. Here, a detailed protocol is introduced for chemical synthesis of HMGA1a protein with site-specific modifications via Ser/Thr ligation strategy, by which we can systematically study the function of the triple phosphorylation (3pSer) in the HMGA1a acidic tail. For complete details on the use and execution of this protocol, please refer to Wei et al. (2021).

Phosphorylation-regulated HMGA1a-P53 interaction unveils the function of HMGA1a acidic tail phosphorylations via synthetic proteins.

As a typical member of intrinsically disordered proteins (IDPs), HMGA1a carries many post-translational modifications (PTMs). To study the undefined function of acidic tail phosphorylations, seven HMGA1a proteins with site-specific modification(s) were chemically synthesized via Ser/Thr ligation. We found that the phosphorylations significantly inhibit HMGA1a-P53 interaction and the phosphorylations can induce conformational change of HMGA1a from an "open state" to a "close state." Notably, the positively charged lysine-arginine (KR) clusters are responsible for modulating HMGA1a conformation via electrostatic interaction with the phosphorylated acidic tail. Finally, we used a synthetic protein-affinity purification mass spectrometry (SP-AP-MS) methodology to profile the specific interactors, which further supported the function of HMGA1a phosphorylation. Collectively, this study highlights a mechanism for regulating IDPs' conformation and function by phosphorylation of non-protein-binding domain and showcases that the protein chemical synthesis in combination with mass spectrometry can serve as an efficient tool to study the IDPs' PTMs.

The Antidepressant Effects of Resveratrol are Accompanied by the Attenuation of Dendrite/Dendritic Spine Loss and the Upregulation of BDNF/p-cofilin1 Levels in Chronic Restraint Mice.

Depression afflicts more than 300 million people worldwide, but there is currently no universally effective drug in clinical practice. In this study, chronic restraint stress (CRS)-induced mice depression model was used to study the antidepressant effects of resveratrol and its mechanism. Our results showed that resveratrol significantly attenuated depression-like behavior in mice. Consistent with behavioral changes, resveratrol significantly attenuated CRS-induced reduction in the density of dendrites and dendritic spines in both hippocampus and medial prefrontal cortex (mPFC). Meanwhile, in hippocampus and mPFC, resveratrol consistently alleviated CRS-induced cofilin1 activation by increasing its ser3 phosphorylation. In addition, cofilin1 immunofluorescence distribution in neuronal inner peri-membrane in controls, and cofilin1 diffusely distribution in the cytoplasm in CRS group were common in hippocampus. However, the distribution of cofilin1 in mPFC was reversed. Pearson's correlation analysis revealed that there was a significant positive correlation found between the sucrose consumption in sucrose preference test and the dendrite density in multiple sub-regions of hippocampus and mPFC, and a significant negative correlation between the immobility time in tail suspension test and the dendrite/dendritic spine density in several different areas of hippocampus and mPFC. P-cofilin1 was significantly positively correlated with the overall dendritic spine density in mPFC as well as with the overall dendrite density or BDNF in the hippocampus. Our results suggest that the BDNF/cofilin1 pathway, in which cofilin1 may be activated in a brain-specific manner, was involved in resveratrol's attenuating the dendrite and dendritic spine loss and behavioral abnormality.

Chemical Protein Synthesis: Advances, Challenges, and Outlooks.

Contemporary chemical protein synthesis has been dramatically advanced over the past few decades, which has enabled chemists to reach the landscape of synthetic biomacromolecules. Chemical synthesis can produce synthetic proteins with precisely controlled structures which are difficult or impossible to obtain via gene expression systems. Herein, we summarize the key enabling ligation technologies, major strategic developments, and some selected representative applications of synthetic proteins and provide an outlook for future development.