MS‐based strategies for identification of protein SUMOylation modification

Protein SUMOylation modification conjugated with small ubiquitin‐like modifiers (SUMOs) is one kind of PTMs, which exerts comprehensive roles in cellular functions, including gene expression regulation, DNA repair, intracellular transport, stress responses, and tumorigenesis. With the development of the peptide enrichment approaches and MS technology, more than 6000 SUMOylated proteins and about 40 000 SUMO acceptor sites have been identified. In this review, we summarize several popular approaches that have been developed for the identification of SUMOylated proteins in human cells, and further compare their technical advantages and disadvantages. And we also introduce identification approaches of target proteins which are co‐modified by both SUMOylation and ubiquitylation. We highlight the emerging trends in the SUMOylation field as well. Especially, the advent of the clustered regularly interspaced short palindromic repeats/ Cas9 technique will facilitate the development of MS for SUMOylation identification.     

Dissecting cellular processes using small molecules: identification of colchicine-like, taxol-like and other small molecules that perturb mitosis

BACKGROUND: Understanding the molecular mechanisms of complex cellular processes requires unbiased means to identify and to alter conditionally gene products that function in a pathway of interest. Although random mutagenesis and screening (forward genetics) provide a useful means to this end, the complexity of the genome, long generation time and redundancy of gene function have limited their use with mammalian systems. We sought to develop an analogous process using small molecules to modulate conditionally the function of proteins. We hoped to identify simultaneously small molecules that may serve as leads for the development of therapeutically useful agents. RESULTS: We report the results of a high-throughput, phenotype-based screen for identifying cell-permeable small molecules that affect mitosis of mammalian cells. The predominant class of compounds that emerged directly alters the stability of microtubules in the mitotic spindle. Although many of these compounds show the colchicine-like property of destabilizing microtubules, one member shows the taxol-like property of stabilizing microtubules. Another class of compounds alters chromosome segregation by novel mechanisms that do not involve direct interactions with microtubules. CONCLUSIONS: The identification of structurally diverse small molecules that affect the mammalian mitotic machinery from a large library of synthetic compounds illustrates the use of chemical genetics in dissecting an essential cellular pathway. This screen identified five compounds that affect mitosis without directly targeting microtubules. Understanding the mechanism of action of these compounds, along with future screening efforts, promises to help elucidate the molecular mechanisms involved in chromosome segregation during mitosis.     

Characterization of mitosis-specific phosphorylation of tumor-associated microtubule-associated protein

Tumor-associated microtubule-associated protein (TMAP), also known as cytoskeleton associated protein 2 (CKAP2), has been recently shown to be involved in the assembly and maintenance of mitotic spindle and also plays an essential role in maintaining the fidelity of chromosome segregation during mitosis. We have previously reported that TMAP is phosphorylated at multiple residues specifically during mitosis, and characterized the mechanism and functional importance of phosphorylation at one of the mitosis-specific phosphorylation residues (i.e., Thr-622). However, the phosphorylation events at the remaining mitotic phosphorylation sites of TMAP have not been fully characterized in detail. Here, we report on generation and characterization of phosphorylated Thr-578- and phosphorylated Thr-596-specific antibodies. Using the antibodies, we show that phosphorylation of TMAP at Thr-578 and Thr-596 indeed occurs specifically during mitosis. Immunofluorescent staining using the antibodies shows that these residues become phosphorylated starting at prophase and then become rapidly dephosphorylated soon after initiation of anaphase. Subtle differences in the kinetics of phosphorylation between Thr-578 and Thr-596 imply that they may be under different mechanisms of phosphorylation during mitosis. Unlike the phosphorylation-deficient mutant form for Thr-622, the mutant in which both Thr-578 and Thr-596 had been mutated to alanines did not induce significant delay in progression of mitosis. These results show that the majority of mitosis-specific phosphorylation of TMAP is limited to pre-anaphase stages and suggest that the multiple phosphorylation may not act in concert but serve diverse functions.     

A Pyranoxanthone as a Potent Antimitotic and Sensitizer of Cancer Cells to Low Doses of Paclitaxel

Microtubule-targeting agents (MTAs) remain a gold standard for the treatment of several cancer types. By interfering with microtubules dynamic, MTAs induce a mitotic arrest followed by cell death. This antimitotic activity of MTAs is dependent on the spindle assembly checkpoint (SAC), which monitors the integrity of the mitotic spindle and proper chromosome attachments to microtubules in order to ensure accurate chromosome segregation and timely anaphase onset. However, the cytotoxic activity of MTAs is restrained by drug resistance and/or toxicities, and had motivated the search for new compounds and/or alternative therapeutic strategies. Here, we describe the synthesis and mechanism of action of the xanthone derivative pyranoxanthone 2 that exhibits a potent anti-growth activity against cancer cells. We found that cancer cells treated with the pyranoxanthone 2 exhibited persistent defects in chromosome congression during mitosis that were not corrected over time, which induced a prolonged SAC-dependent mitotic arrest followed by massive apoptosis. Importantly, pyranoxanthone 2 was able to potentiate apoptosis of cancer cells treated with nanomolar concentrations of paclitaxel. Our data identified the potential of the pyranoxanthone 2 as a new potent antimitotic with promising antitumor potential, either alone or in combination regimens.     

Traceless Preparation of C‐Terminal α‐Ketoacids for Chemical Protein Synthesis by α‐Ketoacid–Hydroxylamine Ligation: Synthesis of SUMO2/3

A novel protecting group for enantiopure α‐ketoacids delivers C‐terminal peptide α‐ketoacids directly upon resin cleavage and allows the inclusion of all canonical amino acids, including cysteine and methionine. By using this approach, SUMO2 and SUMO3 proteins were prepared by KAHA ligation with 5‐oxaproline. The synthetic proteins containing homoserine residues were recognized by and conjugated to RanGAP1 by SUMOylation enzymes.     

A Small-Molecule AIE Chromosome Periphery Probe for Cytogenetic Studies

The chromosome periphery (CP) is a complex network that covers the outer surface of chromosomes. It acts as a carrier of nucleolar components, helps maintain chromosome structure, and plays an important role in mitosis. Current methods for fluorescence imaging of CP largely rely on immunostaining. We herein report a small-molecule fluorescent probe, ID-IQ , which possesses aggregation-induced emission (AIE) property, for CP imaging. By labelling the CP, ID-IQ sharply highlighted the chromosome boundaries, which enabled rapid segmentation of touching and overlapping chromosomes, direct identification of the centromere, and clear visualization of chromosome morphology. ID-IQ staining was also compatible with fluorescence in situ hybridization and could assist the precise location of the gene in designated chromosome. Altogether, this study provides a versatile cytogenetic tool for improved chromosome analysis, which greatly benefits the clinical diagnostic testing and genomic research.     

Acrylamide-Derived Ionome,Metabolic, and Cell Cycle Alterations Are Alleviated by Ascorbic Acid in the Fission Yeast

Acrylamide (AA), is a chemical with multiple industrial applications, however, it can be found in foods that are rich in carbohydrates. Due to its genotoxic and cytotoxic effects, AA has been classified as a potential carcinogen. With the use of spectrophotometry, ICP-OES, fluorescence spectroscopy, and microscopy cell growth, metabolic activity, apoptosis, ROS production, MDA formation, CAT and SOD activity, ionome balance, and chromosome segregation were determined in Schizosaccharomyces pombe. AA caused growth and metabolic activity retardation, enhanced ROS and MDA production, and modulated antioxidant enzyme activity. This led to damage to the cell homeostasis due to ionome balance disruption. Moreover, AA-induced oxidative stress caused alterations in the cell cycle regulation resulting in chromosome segregation errors, as 4.07% of cells displayed sister chromatid non-disjunction during mitosis. Ascorbic acid (AsA, Vitamin C), a strong natural antioxidant, was used to alleviate the negative impact of AA. Cell pre-treatment with AsA significantly improved AA impaired growth, and antioxidant capacity, and supported ionome balance maintenance mainly due to the promotion of calcium uptake. Chromosome missegregation was reduced to 1.79% (44% improvement) by AsA pre-incubation. Results of our multiapproach analyses suggest that AA-induced oxidative stress is the major cause of alteration to cell homeostasis and cell cycle regulation.     

A Small‐Molecule AIE Chromosome Periphery Probe for Cytogenetic Studies

The chromosome periphery (CP) is a complex network that covers the outer surface of chromosomes. It acts as a carrier of nucleolar components, helps maintain chromosome structure, and plays an important role in mitosis. Current methods for fluorescence imaging of CP largely rely on immunostaining. We herein report a small‐molecule fluorescent probe, ID‐IQ , which possesses aggregation‐induced emission (AIE) property, for CP imaging. By labelling the CP, ID‐IQ sharply highlighted the chromosome boundaries, which enabled rapid segmentation of touching and overlapping chromosomes, direct identification of the centromere, and clear visualization of chromosome morphology. ID‐IQ staining was also compatible with fluorescence in situ hybridization and could assist the precise location of the gene in designated chromosome. Altogether, this study provides a versatile cytogenetic tool for improved chromosome analysis, which greatly benefits the clinical diagnostic testing and genomic research.     

Chromosome segregation in Escherichia coli division: a free energy-driven string model

Although the mechanisms of eukaryotic chromosome segregation and cell division have been elucidated to a certain extent, those for bacteria remain largely unknown. Here we present a computational string model for simulating the dynamics of Escherichia coli chromosome segregation. A novel thermal-average force field accounting for stretching, bending, volume exclusion, friction and random fluctuation is introduced. A Langevin equation is used to simulate the chromosome structural changes. The mechanism of chromosome segregation is thereby postulated as a result of free energy-driven structural optimization with replication introduced chromosomal mass increase. Predictions of the model agree well with observations of fluorescence labeled chromosome loci movement in living cells. The results demonstrate the possibility of a mechanism of chromosome segregation that does not involve cytoskeletal guidance or advanced apparatus in an E. coli cell. The model also shows that DNA condensation of locally compacted domains is a requirement for successful chromosome segregation. Simulations also imply that the shape-determining protein MreB may play a role in the segregation via modification of the membrane pressure.     

The 48 bp centromeric repeat is a functionally conserved motif in great apes and man showing protein-binding properties

The centromere-kinetochore complex is a chromosomal assembly site including repeat motifs and protein binding properties thus mediating chromosome motility and mitotic regulation. Next to the alpha-satellite DNA family as well as human satellite III DNA, contribution of other repetitive sequences has to be strongly considered in centromere function. Here, we report the identification of centromeric 48 bp motifs, isolated from chimpanzee and orang-utan using an orthologous human DNA probe. Applying Southern hybridization we show that these sequences are restricted to hominoid species. Diminishing hybrid formation in gibbons suggested that the 48 bp repeat originated approximately 25-20 million years ago. Consistently, both chimpanzee as well as human repeat probes failed to generate any hybridization signal with the monkey species Cercopithecus aethiops and Aotes trivirgatus. Sequence deviations from the consensus of human repeat monomers of 6% and 10.4% in chimpanzee and orang-utan, respectively, were found within a 16 bp region of the 48 bp repeat units. Gel mobility shift assays using chimpanzee repeat dimers as probes revealed peptide-binding properties with human and chimpanzee nuclear extracts. Species-specific DNA-protein complexes remained unaffected by competition studies and indicated the presence of at least one novel interacting protein consisting of two subunits with 90 and 95 kDa. Our data suggest that the 48 bp repeat, next to alpha-satellite DNA, provides essential sequence information for specific DNA-protein interaction and they imply phylogenetic conservation of these binding properties in primates. The complex is likely involved in the proper formation and/or function of mammalian centromeres.     

Gas-Phase Enrichment of Multiply Charged Peptide Ions by Differential Ion Mobility Extend the Comprehensiveness of SUMO Proteome Analyses

The small ubiquitin-like modifier (SUMO) is a member of the family of ubiquitin-like modifiers (UBLs) and is involved in important cellular processes, including DNA damage response, meiosis and cellular trafficking. The large-scale identification of SUMO peptides in a site-specific manner is challenging not only because of the low abundance and dynamic nature of this modification, but also due to the branched structure of the corresponding peptides that further complicate their identification using conventional search engines. Here, we exploited the unusual structure of SUMO peptides to facilitate their separation by high-field asymmetric waveform ion mobility spectrometry (FAIMS) and increase the coverage of SUMO proteome analysis. Upon trypsin digestion, branched peptides contain a SUMO remnant side chain and predominantly form triply protonated ions that facilitate their gas-phase separation using FAIMS. We evaluated the mobility characteristics of synthetic SUMO peptides and further demonstrated the application of FAIMS to profile the changes in protein SUMOylation of HEK293 cells following heat shock, a condition known to affect this modification. FAIMS typically provided a 10-fold improvement of detection limit of SUMO peptides, and enabled a 36% increase in SUMO proteome coverage compared to the same LC-MS/MS analyses performed without FAIMS.

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Sec13 induces genomic instability in U2OS cells

Sec13p has been known as an endoplasmic reticulum-Golgi transport protein. Recently, it has also been shown to be required for the formation of septation in the fission yeast Schizosaccharomyces pombe. In the present study, we focused on the role of a human homolog of Saccharomyces cerevisiae SEC13, Sec13 protein during mitosis in U2OS cells. We found that the expression of Sec13 was constant throughout the cell cycle, and localized to the kinetochores at metaphase during mitosis. By using green fluorescent protein technology, we observed that Sec13 is required for evasion of mitotic arrest in response to spindle damage, leading to G1-like phase and apoptotic cell death. In addition, cells expressing exogenous Sec13 showed giant nuclei compared to endogenous ones in the absence of nocodazole. These results demonstrate that Sec13 is involved in the regulation of the metaphase/anaphase transition and may be functionally associated with mitotic machinery to maintain genomic stability during mitosis.     

A novel technique for observing the internal ultrastructure of human chromosomes with known karyotype

Observation of the internal ultrastructure of human chromosomes by transmission electron microscopy (TEM) has frequently been attempted in spite of the difficulties in detaching metaphase chromosome spreads from the glass slide for further processing. In this study we have used a method in which metaphase chromosome spreads were prepared on a flexible thermoplastic membrane (ACLAR) film. To assess chromosome identity, a diamidino-phenylindole staining and karyotying was first done using a conventional cytogenetic system. The chromosome spreads were then fixed with 1% osmium tetroxide, stained with freshly prepared 2% tannic acid, dehydrated, and flat-embedded in epoxy resin. The resin sheet was easily detachable and carried whole chromosome spreads. By this method, TEM observation of chromosomes from normal human lymphocytes allowed a thorough examination of the ultrastructure of centromeres, telomeres, fragile sites, and other chromosomal regions. Various ultrastructural patterns including thick electron dense boundaries, less dense internal regions, and extended chromatin loops at the periphery of the chromosomes were discernible. Application of the present method to chromosome research is expected to provide comprehensive information on the internal ultrastructure of different chromosomal regions in relation to function.     

BP-M345, a New Diarylpentanoid with Promising Antimitotic Activity

Previously, we reported the in vitro growth inhibitory effect of diarylpentanoid BP-M345 on human cancer cells. Nevertheless, at that time, the cellular mechanism through which BP-M345 exerts its growth inhibitory effect remained to be explored. In the present work, we report its mechanism of action on cancer cells. The compound exhibits a potent tumor growth inhibitory activity with high selectivity index. Mechanistically, it induces perturbation of the spindles through microtubule instability. As a consequence, treated cells exhibit irreversible defects in chromosome congression during mitosis, which induce a prolonged spindle assembly checkpoint-dependent mitotic arrest, followed by massive apoptosis, as revealed by live cell imaging. Collectively, the results indicate that the diarylpentanoid BP-M345 exerts its antiproliferative activity by inhibiting mitosis through microtubule perturbation and causing cancer cell death, thereby highlighting its potential as antitumor agent.