Actin R256 Mono-methylation Is a Conserved Post-translational Modification Involved in Transcription.

Nuclear actin has been elusive due to the lack of knowledge about molecular mechanisms. From actin-containing chromatin remodeling complexes, we discovered an arginine mono-methylation mark on an evolutionarily conserved R256 residue of actin (R256me1). Actin R256 mutations in yeast affect nuclear functions and cause diseases in human. Interestingly, we show that an antibody specific for actin R256me1 preferentially stains nuclear actin over cytoplasmic actin in yeast, mouse, and human cells. We also show that actin R256me1 is regulated by protein arginine methyl transferase-5 (PRMT5) in HEK293 cells. A genome-wide survey of actin R256me1 mark provides a landscape for nuclear actin correlated with transcription. Further, gene expression and protein interaction studies uncover extensive correlations between actin R256me1 and active transcription. The discovery of actin R256me1 mark suggests a fundamental mechanism to distinguish nuclear actin from cytoplasmic actin through post-translational modification (PTM) and potentially implicates an actin PTM mark in transcription and human diseases.

Safety and efficacy of galcanezumab in Taiwanese patients: a post-hoc analysis of phase 3 studies in episodic and chronic migraine.

OBJECTIVE: Migraine is a chronic, disabling neurological disease affecting >1 billion people worldwide. Migraine remains undertreated in Asia, including Taiwan. Galcanezumab is a humanized monoclonal antibody that selectively binds calcitonin gene-related peptide, a peptide firmly established in the pathophysiology of migraine, with demonstrated efficacy and safety in patients with episodic or chronic migraine. Our objective was to evaluate the efficacy and safety of galcanezumab in Taiwanese patients with episodic or chronic migraine. METHODS: We conducted a sub-group analysis of the Taiwanese cohort from two double-blind, placebo-controlled, Phase 3 clinical trials of galcanezumab in the prevention of episodic and chronic migraine, EVOLVE-2 (NCT02614196) and REGAIN (NCT02614261), respectively. During the EVOLVE-2 and REGAIN double-blind periods, 2092 patients were randomly assigned to receive monthly injections of either placebo, 120 mg galcanezumab (240 mg loading dose), or 240 mg galcanezumab. In REGAIN, a 9-month open-label period followed. Post-hoc analysis on the Taiwanese population across both trials included 106 patients, 45 of whom continued into the open-label period in REGAIN. RESULTS: Our findings show that galcanezumab has similar efficacy and safety in the Taiwanese population, as compared to the "All Patients" population included in the study. Galcanezumab treatment reduced the number of monthly migraine headache days, determined a higher percentage of patients with a ≥ 50% response, and positively impacted quality of life. CONCLUSION: Galcanezumab is a promising therapeutic for the preventive treatment of migraine in the Taiwanese population.

Acid Sphingomyelinase regulates the localization and trafficking of palmitoylated proteins.

In human, loss of Acid Sphingomeylinase (ASM/SMPD1) causes Niemann-Pick Disease, type A. ASM hydrolyzes sphingomyelins to produce ceramides but protein targets of ASM remain largely unclear. Our mass-spectrometry-based proteomic analyses have identified >100 proteins associated with the ASM-dependent, detergent-resistant membrane microdomains (lipid rafts), with >60% of these proteins being palmitoylated, including SNAP23, Src-family kinases Yes and Lyn, and Ras and Rab family small GTPases. Inactivation of ASM abolished the presence of these proteins in the plasma membrane, with many of them trapped in the Golgi. While palmitoylation inhibitors and palmitoylation mutants phenocopied the effects of ASM inactivation, we demonstrated that ASM is required for the transport of palmitoylated proteins, such as SNAP23 and Lyn, from the Golgi to the plasma membrane without affecting palmitoylation directly. Importantly, ASM delivered extracellularly can regulate the trafficking of SNAP23 from the Golgi to the plasma membrane. Our studies suggest that ASM, acting at the plasma membrane to produce ceramides, regulates the localization and trafficking of the palmitoylated proteins.

Quantitative phosphoproteome analysis of embryonic stem cell differentiation toward blood.

Murine embryonic stem (ES) cells can differentiate in vitro into three germ layers (endodermic, mesodermic, ectodermic). Studies on the differentiation of these cells to specific early differentiation stages has been aided by an ES cell line carrying the Green Fluorescent Protein (GFP) targeted to the Brachyury (Bry) locus which marks mesoderm commitment. Furthermore, expression of the Vascular Endothelial Growth Factor receptor 2 (Flk1) along with Bry defines hemangioblast commitment. Isobaric-tag for relative and absolute quantification (iTRAQ(TM)) and phosphopeptide enrichment coupled to liquid chromatography separation and mass spectrometry allow the study of phosphorylation changes occurring at different stages of ES cell development using Bry and Flk1 expression respectively. We identified and relatively quantified 37 phosphoentities which are modulated during mesoderm-induced ES cells differentiation, comparing epiblast-like, early mesoderm and hemangioblast-enriched cells. Among the proteins differentially phosphorylated toward mesoderm differentiation were: the epigenetic regulator Dnmt3b, the protein kinase GSK3b, the chromatin remodeling factor Smarcc1, the transcription factor Utf1; as well as protein specifically related to stem cell differentiation, as Eomes, Hmga2, Ints1 and Rif1. As most key factors regulating early hematopoietic development have also been implicated in various types of leukemia, understanding the post-translational modifications driving their regulation during normal development could result in a better comprehension of their roles during abnormal hematopoiesis in leukemia.

Nanog1 in NTERA-2 and recombinant NanogP8 from somatic cancer cells adopt multiple protein conformations and migrate at multiple M.W species.

Human Nanog1 is a 305-amino acid (aa) homeodomain-containing transcription factor critical for the pluripotency of embryonic stem (ES) and embryonal carcinoma (EC) cells. Somatic cancer cells predominantly express a retrogene homolog of Nanog1 called NanogP8, which is ~99% similar to Nanog at the aa level. Although the predicted M.W of Nanog1/NanogP8 is ∼35 kD, both have been reported to migrate, on Western blotting (WB), at apparent molecular masses of 29-80 kD. Whether all these reported protein bands represent authentic Nanog proteins is unclear. Furthermore, detailed biochemical studies on Nanog1/NanogpP8 have been lacking. By combining WB using 8 anti-Nanog1 antibodies, immunoprecipitation, mass spectrometry, and studies using recombinant proteins, here we provide direct evidence that the Nanog1 protein in NTERA-2 EC cells exists as multiple M.W species from ~22 kD to 100 kD with a major 42 kD band detectable on WB. We then demonstrate that recombinant NanogP8 (rNanogP8) proteins made in bacteria using cDNAs from multiple cancer cells also migrate, on denaturing SDS-PAGE, at ~28 kD to 180 kD. Interestingly, different anti-Nanog1 antibodies exhibit differential reactivity towards rNanogP8 proteins, which can spontaneously form high M.W protein species. Finally, we show that most long-term cultured cancer cell lines seem to express very low levels of or different endogenous NanogP8 protein that cannot be readily detected by immunoprecipitation. Altogether, the current study reveals unique biochemical properties of Nanog1 in EC cells and NanogP8 in somatic cancer cells.

Phosphorylation-regulated transitions in an oligomeric state control the activity of the Sae2 DNA repair enzyme.

In the DNA damage response, many repair and signaling molecules mobilize rapidly at the sites of DNA double-strand breaks. This network of immediate responses is regulated at the level of posttranslational modifications that control the activation of DNA processing enzymes, protein kinases, and scaffold proteins to coordinate DNA repair and checkpoint signaling. Here we investigated the DNA damage-induced oligomeric transitions of the Sae2 protein, an important enzyme in the initiation of DNA double-strand break repair. Sae2 is a target of multiple phosphorylation events, which we identified and characterized in vivo in the budding yeast Saccharomyces cerevisiae. Both cell cycle-dependent and DNA damage-dependent phosphorylation sites in Sae2 are important for the survival of DNA damage, and the cell cycle-regulated modifications are required to prime the damage-dependent events. We found that Sae2 exists in the form of inactive oligomers that are transiently released into smaller active units by this series of phosphorylations. DNA damage also triggers removal of Sae2 through autophagy and proteasomal degradation, ensuring that active Sae2 is present only transiently in cells. Overall, this analysis provides evidence for a novel type of protein regulation where the activity of an enzyme is controlled dynamically by posttranslational modifications that regulate its solubility and oligomeric state.

Proteome-wide detection and quantitative analysis of irreversible cysteine oxidation using long column UPLC-pSRM.

Reactive oxygen species (ROS) play an important role in normal biological functions and pathological processes. ROS is one of the driving forces for oxidizing proteins, especially on cysteine thiols. The labile, transient, and dynamic nature of oxidative modifications poses enormous technical challenges for both accurate modification site determination and quantitation of cysteine thiols. The present study describes a mass spectrometry-based approach that allows effective discovery and quantification of irreversible cysteine modifications. The utilization of a long reverse phase column provides high-resolution chromatography to separate different forms of modified cysteine thiols from protein complexes or cell lysates. This Fourier transform mass spectrometry (FT-MS) approach enabled detection and quantitation of ataxia telangiectasia mutated (ATM) complex cysteine sulfoxidation states using Skyline MS1 filtering. When we applied the long column ultra high pressure liquid chromatography (UPLC)-MS/MS analysis, 61 and 44 peptides from cell lysates and cells were identified with cysteine modifications in response to in vitro and in vivo H2O2 oxidation, respectively. Long column ultra high pressure liquid chromatography pseudo selected reaction monitoring (UPLC-pSRM) was then developed to monitor the oxidative level of cysteine thiols in cell lysate under varying concentrations of H2O2 treatment. From UPLC-pSRM analysis, the dynamic conversion of sulfinic (S-O2H) and sulfonic acid (S-O3H) was observed within nucleoside diphosphate kinase (Nm23-H1) and heat shock 70 kDa protein 8 (Hsc70). These methods are suitable for proteome-wide studies, providing a highly sensitive, straightforward approach to identify proteins containing redox-sensitive cysteine thiols in biological systems.

Assessment of downstream effectors of BCR/ABL protein tyrosine kinase using combined proteomic approaches.

Leukaemic transformation is frequently associated with the aberrant activity of a protein tyrosine kinase (PTK). As such it is of clinical relevance to be able to map the effects of these leukaemogenic PTKs on haemopoietic cells at the level of phosphorylation modulation. In this paradigm study we have employed a range of proteomic approaches to analyse the effects of one such PTK, BCR/ABL. We have employed phosphoproteome enrichment techniques allied to peptide and protein quantification to identify proteins and pathways involved in cellular transformation. Amongst the proteins shown to be regulated at the post-translational level were cofilin, an actin-severing protein thus linked to altered motility and Cbl an E3 ubiquitin ligase integrally linked to the control of tyrosine kinase signalling (regulated by 5 and 6 PTKs respectively). The major class of proteins identified however were molecular chaperones. We also showed that HSP90 phosphorylation is altered by BCR/ABL action and that HSP90 plays a crucial role in oncogene stability. Further investigation with another six leukaemogenic PTKs demonstrates that this HSP90 role in oncogene stability appears to be a common phenomenon in a range of leukaemias. This opens up the potential opportunity to treat different leukaemias with HSP90 inhibitors.

Eight-channel iTRAQ enables comparison of the activity of six leukemogenic tyrosine kinases.

There are a number of leukemogenic protein-tyrosine kinases (PTKs) associated with leukemic transformation. Although each is linked with a specific disease their functional activity poses the question whether they have a degree of commonality in their effects upon target cells. Exon array analysis of the effects of six leukemogenic PTKs (BCR/ABL, TEL/PDGFRbeta, FIP1/PDGFRalpha, D816V KIT, NPM/ALK, and FLT3ITD) revealed few common effects on the transcriptome. It is apparent, however, that proteome changes are not directly governed by transcriptome changes. Therefore, we assessed and used a new generation of iTRAQ tagging, enabling eight-channel relative quantification discovery proteomics, to analyze the effects of these six leukemogenic PTKs. Again these were found to have disparate effects on the proteome with few common targets. BCR/ABL had the greatest effect on the proteome and had more effects in common with FIP1/PDGFRalpha. The proteomic effects of the four type III receptor kinases were relatively remotely related. The only protein commonly affected was eosinophil-associated ribonuclease 7. Five of six PTKs affected the motility-related proteins CAPG and vimentin, although this did not correspond to changes in motility. However, correlation of the proteomics data with that from the exon microarray not only showed poor levels of correlation between transcript and protein levels but also revealed alternative patterns of regulation of the CAPG protein by different oncogenes, illustrating the utility of such a combined approach.

Effects of multiple virus coinfections on disease progression in HIV-positive patients.

OBJECTIVE: Since virus infections in AIDS patients are mostly inevitable and as they frequently cause disease deterioration and therapeutic failures, a comprehensive investigation was made of the influence of the coinfections of 9 well-known viruses on disease progression in patients infected with human immunodeficiency virus type 1 (HIV). METHODS: A cross-sectional study of 62 HIV-positive patients was conducted to correlate the prevalence rates for the 9 viruses with the alanine aminotransferase (ALT) levels and CD4 cell counts of the patients. RESULTS: The rates of HIV-positive patients infected with the 9 viruses are significantly higher than those of the control groups. Furthermore, almost one third of the patients in the studied group was coinfected with transfusion-transmitted virus (TTV) and manifested significantly higher ALT levels (p = 0.020), and these were raised further if coinfection with TTV and human hepatitis C virus had occurred (p = 0.010). By analyzing CD4 cell counts, the only significant effect on AIDS progression which could be detected was coinfection with human herpesvirus 8. CONCLUSION: This result confirmed that immune-suppressed persons are more vulnerable to common virus infections. Unlike hepatitis B or C virus, TTV seems to accelerate the progression of chronic hepatitis in HIV-infected patients.