C-termini of proteins often play an important role in various biological processes, such as the transcription and translation from DNA to protein and also participating in various biological regulations. The determination of protein C-terminus is so crucial because it provides not only distinct functional annotation, but also a way to monitor the proteolysis-modified proteins. Based on the biological mass spectrometry, a series of novel methods and technologies were developed both for qualitative and quantitative analyses of protein C-terminus. These methods or technologies can be applied to accurate and effective protein C-terminus profiling, including the sequences and quantitative information of C-termini, which reveals the biological function of C-termini in life's activities and provides a better understanding of the degradation of mature proteins. Combined with our research, this review highlights the improvements in C-terminal proteomics study in the past decades, including the methodologies for recognition and identification of C-terminus, as well as the enrichment strategies for protein C-terminus.
Mass Spectrometry ; Protein Processing, Post-Translational ; Proteins ; chemistry ; Proteolysis ; Proteomics

Protein N-termini, as the beginning of translation, has a major impact on protein's biological functions. Its sequence and various post-translational modifications often affect protein activation, stability and cellular-localization, regulate the signal transduction, and even determine protein's final destiny. The systematic study of protein N-termini can clarify the vital function of the N-terminus, and provide in-depth knowledge of the multifunctional roles that protein has played in diverse biological processes. In addition, N-terminal research may help us to achieve high-coverage proteomics and re-annotate genomics. Combined with our own research, this review highlights recent progress of N-terminomics, especially some important enrichment strategies and technologies based on mass spectrometry.
Mass Spectrometry ; Protein Processing, Post-Translational ; Proteins ; chemistry ; Proteomics

Proteins, which exist mainly in linear form in vivo, are easily affected by the change of ambient temperature and pH. The application of proteins (enzymes) in the fields of industrial catalyzing, food manufacturing and medicine are restricted due to their properties. The cyclic structure of natural cyclic peptides confers high thermal stability on itself; such mechanism can be referred to in further enhancement of the thermal stability and transformation of the structure of enzymes. This article reviewed the latest progress in the domestic and international studies on protein cyclization and summarized the traditional methods (such as protein trans-splicing, expressed protein ligation and sortase-catalyzed transpeptidation) in protein cyclization. A novel method based on SpyTag/SpyCather-mediated enzyme cyclization was discussed in more detail.
Cyclization ; Peptides, Cyclic ; chemistry ; Protein Processing, Post-Translational ; Proteins ; chemistry

Cardiovascular Diseases ; Extracellular Vesicles/metabolism* ; Humans ; Protein Processing, Post-Translational

Ubiquitination is a post-translational modification of proteins in eukaryotes, which mediates the specific degradation and signal transduction of proteins to regulate a variety of life processes and thus affects functions of the body. The disorder and imbalance of ubiquitination network is a major cause of serious human diseases. Ubiquitin molecules can form eight homogeneous ubiquitin chains with different topological structures, which vary greatly in abundance and function. At present, the classical ubiquitin chains K48 and K63 with high abundance and rich substrates have been intensively studied, while other atypical ubiquitin chains with low content remain to be studied. However, it has been proved that atypical ubiquitin chains play a key role in intracellular regulation. K6 is an important atypical ubiquitin chain, which is similar to K48 chain and has a tight spatial structure. It plays a role in DNA damage repair, mitochondrial quality control, the occurrence and development of tumor, and the pathogenesis of Parkinson's disease. Due to the lack of specific antibodies and effective enrichment methods for K6, little is known about its substrate and regulatory mechanism. This paper systematically reviews the structural characteristics, regulatory mechanism, biological functions, and relevant diseases of atypical K6 linkages, aiming to provide reference for the functional study of K6.
Humans ; Protein Processing, Post-Translational ; Signal Transduction ; Ubiquitin/chemistry* ; Ubiquitination

Proteolytic cleavage is one of the post-translational modifications and plays important roles in many biological processes, such as apoptosis and tumor cell metastasis. The identification of the cleavage events can improve our understanding of their biological functions in these processes. Although proteomic approaches using N-terminal labeling have resulted in the discovery of many proteolytic cleavages, this strategy has its own inherent drawbacks. Labeling of protein C-termini is an alternative approach. Here, we optimized the labeling procedure in the profiling protein C-termini by enzymatic labeling (ProC-TEL) and improved the labeling efficiency for the positive isolation of protein C-terminal peptides and mass spectrometric identification. We applied this approach to a complex protein mixture from Escherichia coli and identified many C-terminal peptides and internal cleaved peptides from more than 120 proteins. From the identified cleavages, we found several previously known internal proteolytic cleavage sites and many novel ones which may play roles in regulating normal biological processes. This work provides a potential new way, complementary to the N-terminomics, for the identification of proteolytic cleavages in complex biological systems.
Cathepsin A ; chemistry ; Protein C ; chemistry ; Protein Processing, Post-Translational ; Proteolysis ; Proteomics

Abstract　　The promyelocytic leukemia (PML) gene encoded PML protein as a tumor suppressor protein, plays important roles in the occurrence and development of various cancers including acute promyelocytic leukemia. Recent studies have indicated that there are a variety of post-translational modifications of the PML protein, such as SUMOylation, ubiquitination, phosphorylation, and acetylation in cells. These modifications of the PML protein can directly affect the formation of PML nuclear bodies (PML-NBs), repair DNA damage, and modulate cell apoptosis. Furthermore, the abnormal modifications of PML not only result in the occurrence of hematopoietic tumors, but also are closely related to the drug-resistance of cancer. Therefore, investigating the post-translational modifications of PML is significant to uncover the mechanism of formation and functions of PML-NBs, thus contributing to the prevention and treatment of related hematopoietic tumors. In this review, the characteristics of the post-translational modifications of PML protein and the relationship between these modifications and functions of PML-NBs are summarized so as to provide the potential targets for the treatment of related cancers.
Humans ; Intranuclear Inclusion Bodies ; Leukemia, Promyelocytic, Acute ; Nuclear Proteins ; Promyelocytic Leukemia Protein ; Protein Processing, Post-Translational

Activator protein-1 (AP-1) is an inducible transcription factor that contributes to the generation of chronic inflammation in response to oxidative and electrophilic stress. Previous studies have demonstrated that the PI3K/Akt1 pathway plays an important role in the transcriptional regulation of AP-1 expression. Although the histone post-translational modifications (PTMs) are assumed to affect the AP-1 transcriptional regulation by the PI3K/Akt pathway, the detailed mechanisms are completely unknown. In the present study, we show that heterochromatin 1 gamma (HP1gamma) plays a negative role in TPA-induced c-Jun and c-Fos expression. We show that TPA-induced Akt1 directly phosphorylates HP1gamma, abrogates its suppressive function and increases the interaction between histone H3 and 14-3-3epsilon. Collectively, these our data illustrate that the activation of PI3K/Akt pathway may play a permissive role in the recruitment of histone readers or other coactivators on the chromatin, thereby affecting the degree of AP-1 transcription.
Chromatin ; Heterochromatin ; Histones ; Inflammation ; Phosphorylation* ; Protein Processing, Post-Translational ; Transcription Factor AP-1* ; Transcription Factors

Endocytosis is a process by which cells absorb extracellular materials via the inward budding of vesicles formed from the plasma membrane. Receptor-mediated endocytosis is a highly selective process where receptors with specific binding sites for extracellular molecules internalize via vesicles. G protein-coupled receptors (GPCRs) are the largest single family of plasma-membrane receptors with more than 1000 family members. But the molecular mechanisms involved in the regulation of GPCRs are believed to be highly conserved. For example, receptor phosphorylation in collaboration with β-arrestins plays major roles in desensitization and endocytosis of most GPCRs. Nevertheless, a number of subsequent studies showed that GPCR regulation, such as that by endocytosis, occurs through various pathways with a multitude of cellular components and processes. This review focused on i) functional interactions between homologous and heterologous pathways, ii) methodologies applied for determining receptor endocytosis, iii) experimental tools to determine specific endocytic routes, iv) roles of small guanosine triphosphate-binding proteins in GPCR endocytosis, and v) role of post-translational modification of the receptors in endocytosis.
Binding Sites ; Cell Membrane ; Cooperative Behavior ; Endocytosis* ; Glycosylation ; Guanosine ; Humans ; Lipoylation ; Phosphorylation ; Protein Processing, Post-Translational

As a consequence of the Earth's rotation, almost all organisms experience day and night cycles within a 24-hr period. To adapt and synchronize biological rhythms to external daily cycles, organisms have evolved an internal time-keeping system. In mammals, the master circadian pacemaker residing in the suprachiasmatic nucleus (SCN) of the anterior hypothalamus generates circadian rhythmicity and orchestrates numerous subsidiary local clocks in other regions of the brain and peripheral tissues. Regardless of their locations, these circadian clocks are cell-autonomous and self-sustainable, implicating rhythmic oscillations in a variety of biochemical and metabolic processes. A group of core clock genes provides interlocking molecular feedback loops that drive the circadian rhythm even at the single-cell level. In addition to the core transcription/translation feedback loops, post-translational modifications also contribute to the fine regulation of molecular circadian clocks. In this article, we briefly review the molecular mechanisms and post-translational modifications of mammalian circadian clock regulation. We also discuss the organization of and communication between central and peripheral circadian oscillators of the mammalian circadian clock.
Brain ; Circadian Clocks ; Circadian Rhythm ; Hypothalamus, Anterior ; Mammals ; Protein Processing, Post-Translational ; Suprachiasmatic Nucleus