Systematic Investigation of the Trafficking of Glycoproteins on the Cell Surface.

Glycoproteins located on the cell surface play a pivotal role in nearly every extracellular activity. N-glycosylation is one of the most common and important protein modifications in eukaryotic cells, and it often regulates protein folding and trafficking. Glycosylation of cell-surface proteins undergoes meticulous regulation by various enzymes in the endoplasmic reticulum (ER) and the Golgi, ensuring their proper folding and trafficking to the cell surface. However, the impacts of protein N-glycosylation, N-glycan maturity, and protein folding status on the trafficking of cell-surface glycoproteins remain to be explored. In this work, we comprehensively and site-specifically studied the trafficking of cell-surface glycoproteins in human cells. Integrating metabolic labeling, bioorthogonal chemistry, and multiplexed proteomics, we investigated 706 N-glycosylation sites on 396 cell-surface glycoproteins in monocytes, either by inhibiting protein N-glycosylation, disturbing N-glycan maturation, or perturbing protein folding in the ER. The current results reveal their distinct impacts on the trafficking of surface glycoproteins. The inhibition of protein N-glycosylation dramatically suppresses the trafficking of many cell-surface glycoproteins. The N-glycan immaturity has more substantial effects on proteins with high N-glycosylation site densities, while the perturbation of protein folding in the ER exerts a more pronounced impact on surface glycoproteins with larger sizes. Furthermore, for N-glycosylated proteins, their trafficking to the cell surface is related to the secondary structures and adjacent amino acid residues of glycosylation sites. Systematic analysis of surface glycoprotein trafficking advances our understanding of the mechanisms underlying protein secretion and surface presentation.

Mass spectrometry-based methods for investigating the dynamics and organization of the surfaceome: exploring potential clinical implications.

INTRODUCTION: Cell-surface proteins are extremely important for many cellular events, such as regulating cell-cell communication and cell-matrix interactions. Aberrant alterations in surface protein expression, modification (especially glycosylation), and interactions are directly related to human diseases. Systematic investigation of surface proteins advances our understanding of protein functions, cellular activities, and disease mechanisms, which will lead to identifying surface proteins as disease biomarkers and drug targets. AREAS COVERED: In this review, we summarize mass spectrometry (MS)-based proteomics methods for global analysis of cell-surface proteins. Then, investigations of the dynamics of surface proteins are discussed. Furthermore, we summarize the studies for the surfaceome interaction networks. Additionally, biological applications of MS-based surfaceome analysis are included, particularly highlighting the significance in biomarker identification, drug development, and immunotherapies. EXPERT OPINION: Modern MS-based proteomics provides an opportunity to systematically characterize proteins. However, due to the complexity of cell-surface proteins, the labor-intensive workflow, and the limit of clinical samples, comprehensive characterization of the surfaceome remains extraordinarily challenging, especially in clinical studies. Developing and optimizing surfaceome enrichment methods and utilizing automated sample preparation workflow can expand the applications of surfaceome analysis and deepen our understanding of the functions of cell-surface proteins.

The cell surface contains many important proteins such as receptors and transporters. These proteins are responsible for cells to communicate with each other, take nutrients from outside, and interact with their surroundings. Aberrant changes in surface protein expression, modifications, and interactions with other molecules directly result in various diseases, including infections, immune disorders, and cancer. Currently, mass spectrometry (MS)-based proteomics is very powerful to study proteins on a large scale, and there has been a strong interest in employing MS to investigate cell-surface proteins. In this review, we discuss different methods combining mass spectrometry with other approaches to systematically characterize protein abundance, dynamics, modification, and interaction on the cell surface. These methods help uncover protein functions and specific cell-surface proteins related to human diseases. A better understanding of the functions and properties of cell-surface proteins can facilitate the discovery of surface proteins as effective biomarkers for disease early detection and the identification of drug targets for disease treatment.

Systematic Investigation of Dose-Dependent Protein Thermal Stability Changes to Uncover the Mechanisms of the Pleiotropic Effects of Metformin.

Metformin is a widely used drug to treat type II diabetes. Beyond lowering blood sugar, it has been reported to have pleiotropic effects such as suppressing cancer growth and attenuating cell oxidative stress and inflammation. However, the underlying mechanisms of these effects remain to be explored. Here, we systematically study the thermal stability changes of proteins in liver cells (HepG2) induced by a wide dosage range of metformin by using the proteome integral solubility alteration (PISA) assay. The current results demonstrate that, besides the most accepted target of metformin (complex I), low concentrations of metformin (such as 0.2 µM) stabilize the complex IV subunits, suggesting its important role in the sugar-lowering effect. Low-dose metformin also results in stability alterations of ribosomal proteins, correlating with its inhibitive effect on cell proliferation. We further find that low-concentration metformin impacts mitochondrial cargo and vesicle transport, while high-concentration metformin affects cell redox responses and cell membrane protein sorting. This study provides mechanistic insights into the molecular mechanisms of lowering blood sugar and the pleiotropic effects of metformin.

Global quantification of newly synthesized proteins reveals cell type- and inhibitor-specific effects on protein synthesis inhibition.

Manipulation of protein synthesis is commonly applied to uncover protein functions and cellular activities. Multiple inhibitors with distinct mechanisms have been widely investigated and employed in bio-related research, but it is extraordinarily challenging to measure and evaluate the synthesis inhibition efficiencies of individual proteins by different inhibitors at the proteome level. Newly synthesized proteins are the immediate and direct products of protein synthesis, and thus their comprehensive quantification provides a unique opportunity to study protein inhibition. Here, we systematically investigate protein inhibition and evaluate different popular inhibitors, i.e. cycloheximide, puromycin, and anisomycin, through global quantification of newly synthesized proteins in several types of human cells (A549, MCF-7, Jurkat, and THP-1 cells). The inhibition efficiencies of protein synthesis are comprehensively measured by integrating azidohomoalanine-based protein labeling, selective enrichment, a boosting approach, and multiplexed proteomics. The same inhibitor results in dramatic variation of the synthesis inhibition efficiencies for different proteins in the same cells, and each inhibitor exhibits unique preferences. Besides cell type- and inhibitor-specific effects, some universal rules are unraveled. For instance, nucleolar and ribosomal proteins have relatively higher inhibition efficiencies in every type of cells treated with each inhibitor. Moreover, proteins intrinsically resistant or sensitive to the inhibition are identified and found to have distinct functions. Systematic investigation of protein synthesis inhibition in several types of human cells by different inhibitors provides valuable information about the inhibition of protein synthesis, advancing our understanding of inhibiting protein synthesis.

Investigation of Cellular Response to the HSP90 Inhibition in Human Cells Through Thermal Proteome Profiling.

Heat shock proteins are chaperones, and they are responsible for protein folding in cells. Heat shock protein 90 (HSP90) is one of the most important chaperones in human cells, and its inhibition is promising for cancer therapy. However, despite the development of multiple HSP90 inhibitors, none of them has been approved for disease treatment due to unexpected cellular toxicity and side effects. Hence, a more comprehensive investigation of cellular response to HSP90 inhibitors can aid in a better understanding of the molecular mechanisms of the cytotoxicity and side effects of these inhibitors. The thermal stability shifts of proteins, which represent protein structure and interaction alterations, can provide valuable information complementary to the results obtained from commonly used abundance-based proteomics analysis. Here, we systematically investigated cell response to different HSP90 inhibitors through global quantification of protein thermal stability changes using thermal proteome profiling, together with the measurement of protein abundance changes. Besides the targets and potential off-targets of the drugs, proteins with significant thermal stability changes under the HSP90 inhibition are found to be involved in cell stress responses and the translation process. Moreover, proteins with thermal stability shifts under the inhibition are upstream of those with altered expression. These findings indicate that the HSP90 inhibition perturbs cell transcription and translation processes. The current study provides a different perspective for achieving a better understanding of cellular response to chaperone inhibition.

Combining Selective Enrichment and a Boosting Approach to Globally and Site-Specifically Characterize Protein Co-translational O-GlcNAcylation.

Protein O-GlcNAcylation plays extremely important roles in mammalian cells, regulating signal transduction and gene expression. This modification can happen during protein translation, and systematic and site-specific analysis of protein co-translational O-GlcNAcylation can advance our understanding of this important modification. However, it is extraordinarily challenging because normally O-GlcNAcylated proteins are very low abundant and the abundances of co-translational ones are even much lower. Here, we developed a method integrating selective enrichment, a boosting approach, and multiplexed proteomics to globally and site-specifically characterize protein co-translational O-GlcNAcylation. The boosting approach using the TMT labeling dramatically enhances the detection of co-translational glycopeptides with low abundance when enriched O-GlcNAcylated peptides from cells with a much longer labeling time was used as a boosting sample. More than 180 co-translational O-GlcNAcylated proteins were site-specifically identified. Further analyses revealed that among co-translational glycoproteins, those related to DNA binding and transcription are highly overrepresented using the total identified O-GlcNAcylated proteins in the same cells as the background. Compared with the glycosylation sites on all glycoproteins, co-translational sites have different local structures and adjacent amino acid residues. Overall, an integrative method was developed to identify protein co-translational O-GlcNAcylation, which is very useful to advance our understanding of this important modification.

Quantitative Structural Proteomics Unveils the Conformational Changes of Proteins under the Endoplasmic Reticulum Stress.

Protein structures are decisive for their activities and interactions with other molecules. Global analysis of protein structures and conformational changes cannot be achieved by commonly used abundance-based proteomics. Here, we integrated cysteine covalent labeling, selective enrichment, and quantitative proteomics to study protein structures and structural changes on a large scale. This method was applied to globally investigate protein structures in HEK293T cells and protein structural changes in the cells with the tunicamycin (Tm)-induced endoplasmic reticulum (ER) stress. We quantified several thousand cysteine residues, which contain unprecedented and valuable information of protein structures. Combining this method with pulsed stable isotope labeling by amino acids in cell culture, we further analyzed the folding state differences between pre-existing and newly synthesized proteins in cells under the Tm treatment. Besides newly synthesized proteins, unexpectedly, many pre-existing proteins were found to become unfolded upon ER stress, especially those related to gene transcription and protein translation. Furthermore, the current results reveal that N-glycosylation plays a more important role in the folding process of the tertiary and quaternary structures than the secondary structures for newly synthesized proteins. Considering the importance of cysteine in protein structures, this method can be extensively applied in the biological and biomedical research fields.

Cyclic Ureate Tantalum Catalyst for Preferential Hydroaminoalkylation with Aliphatic Amines: Mechanistic Insights into Substrate Controlled Reactivity.

The efficient and catalytic amination of unactivated alkenes with simple secondary alkyl amines is preferentially achieved. A sterically accessible, N,O-chelated cyclic ureate tantalum catalyst was prepared and characterized by X-ray crystallography. This optimized catalyst can be used for the hydroaminoalkylation of 1-octene with a variety of aryl and alkyl amines, but notably enhanced catalytic activity can be realized with challenging N-alkyl secondary amine substrates. This catalyst offers turnover frequencies of up to 60 h-1, affording full conversion at 5 mol% catalyst loading in approximately 20 min with these nucleophilic amines. Mechanistic investigations, including kinetic isotope effect (KIE) studies, reveal that catalytic turnover is limited by protonolysis of the intermediate 5-membered azametallacycle. A Hammett kinetic analysis shows that catalytic turnover is promoted by electron rich amine substrates that enable catalytic turnover. This more active catalyst is shown to be effective for late stage drug modification.

Rationally Designed Small-Molecule Inhibitors Targeting an Unconventional Pocket on the TLR8 Protein-Protein Interface.

Rational designs of small-molecule inhibitors targeting protein-protein interfaces have met little success. Herein, we have designed a series of triazole derivatives with a novel scaffold to specifically intervene with the interaction of TLR8 homomerization. In multiple assays, TH1027 was identified as a highly potent and specific inhibitor of TLR8. A successful solution of the X-ray crystal structure of TLR8 in complex with TH1027 provided an in-depth mechanistic insight into its binding mode, validating that TH1027 was located between two TLR8 monomers and recognized as an unconventional pocket, thereby preventing TLR8 from activation. Further biological evaluations showed that TH1027 dose-dependently suppressed the TLR8-mediated inflammatory responses in both human monocyte cell lines, peripheral blood mononuclear cells, and rheumatoid arthritis patient specimens, suggesting a strong therapeutic potential against autoimmune diseases.