LOX out, histones: a new enzyme is nipping at your tails

In the current issue of Molecular Cell, Herranz et al. (2012) demonstrate that LOXL2 deaminates trimethylated histone 3 lysine 4 (H3K4me3), which uncovers a new chromatin modification and a new enzymatic mechanism with the potential to regulate additional lysine residues.     

赖氨酰氧化酶样蛋白4在人类恶性肿瘤发生与发展中的作用

赖氨酰氧化酶样蛋白4(lysyl oxidase like 4, LOXL4)是一种属于赖氨酰氧化酶(lysyl oxidase, LOX)蛋白质家族的分泌型铜依赖性胺氧化酶,参与细胞外基质(extracellular matrix, ECM)的组装和维持。LOXL4蛋白在人类肝癌、胃癌、乳腺癌、宫颈癌、头颈鳞癌、食管癌和结直肠癌中表达上调,而在人类膀胱癌和肺癌中表达下调并抑制肿瘤的生长,表明LOXL4蛋白在不同类型的人类恶性肿瘤中具有促癌或抑癌的双向作用。肿瘤细胞外泌体中的LOXL4蛋白通过催化作用产生过氧化氢,后者直接激活FAK/Src信号通路,并促进细胞基质粘附和细胞迁移。外泌体介导的LOXL4还可以通过激活PI3K/Akt信号通路来促进肿瘤细胞的增殖和免疫逃逸。肿瘤细胞中的 LOXL4可以经外泌体转运至巨噬细胞,进一步通过STAT1和STAT3介导的信号通路激活细胞免疫抑制功能和激活程序性死亡配体 1(programmed death ligand 1, PD-L1)表达,触发巨噬细胞的免疫抑制功能,促进肿瘤细胞的免疫逃逸。此外,LOXL4蛋白还能通过激活p53蛋白和抑制Ras/ERK信号转导通路发挥抑癌功能。本文主要总结了LOXL4蛋白的结构、功能及其在人类恶性肿瘤发生发展的作用机制,进一步探讨LOXL4蛋白在恶性肿瘤研究中的应用前景,为恶性肿瘤的临床诊断、治疗和筛选预后标志物提供理论基础和参考依据。     

The structure of the core region of the lipopolysaccharide from Klebsiella pneumoniae O3. 3-deoxy-alpha-D-manno-octulosonic acid (alpha-Kdo) residue in the outer part of the core, a common structural element of Klebsiella pneumoniae O1, O2, O3, O4, O5, O8, and O12 lipopolysaccharides.

The structure of lipid A-core region of the lipopolysaccharide (LPS) from Klebsiella pneumoniae serotype O3 was determined using NMR, MS and chemical analysis of the oligosaccharides, obtained by mild acid hydrolysis, alkaline deacylation, and deamination of the LPS: [carbohydrate structure see text] where P is H or alpha-Hep; J is H or beta-GalA; R is H or P (in the deacylated oligosaccharides).Screening of the LPS from K. pneumoniae O1, O2, O4, O5, O8, and O12 using deamination showed that they also contain alpha-Hep-(1-->4)-alpha-Kdo-(2-->6)-GlcN and alpha-Kdo-(2-->6)-GlcN fragments.     

Lysyl oxidase-like 2 is a regulator of angiogenesis through modulation of endothelial-to-mesenchymal transition

Lysyl oxidase-like 2 (LOXL2) belongs to the family of lysyl oxidases, and as such promotes crosslinking of collagens and elastin by oxidative deamination of lysine residues. In endothelial cells (ECs), LOXL2 is involved in crosslinking and scaffolding of collagen IV. Additionally, several reports have shown a role for LOXL2 in other processes, including regulation of gene expression, tumor metastasis, and epithelial-to-mesenchymal transition (EMT). Here, we demonstrate an additional role for LOXL2 in the regulation of angiogenesis by modulation of endothelial-to-mesenchymal transition (EndMT). LOXL2 knockdown in ECs results in decreased migration and sprouting, and concordantly, LOXL2 overexpression leads to an increase in migration and sprouting, independent of its catalytic activity. Furthermore, LOXL2 knockdown resulted in a reduced expression of EndMT markers, and inhibition of transforming growth factor-β (TGF-β)-mediated induction of EndMT. Interestingly, unlike in EMT, overexpression of LOXL2 alone is insufficient to induce EndMT. Further investigation revealed that LOXL2 expression regulates protein kinase B (PKB)/Akt and focal adhesion kinase (FAK) signaling, both pathways that have been implicated in the regulation of EMT. Altogether, our studies reveal a role for LOXL2 in angiogenesis through the modulation of EndMT in ECs, independent of its enzymatic crosslinking activity.     

Glimpses of evolution: heterochromatic histone H3K9 methyltransferases left its marks behind

In eukaryotes, histone methylation is an epigenetic mechanism associated with a variety of functions related to gene regulation or genomic stability. Recently analyzed H3K9 methyltransferases (HMTases) as SUV39H1, Clr4p, DIM-5, Su(var)3-9 or SUVH2 are responsible for the establishment of histone H3 lysine 9 methylation (H3K9me), which is intimately connected with heterochromatinization. In this review, available data will be evaluated concerning (1) the phylogenetic distribution of H3K9me as heterochromatin-specific histone modification and its evolutionary stability in relation to other epigenetic marks, (2) known families of H3K9 methyltransferases, (3) their responsibility for the formation of constitutive heterochromatin and (4) the evolution of Su(var)3-9-like and SUVH-like H3K9 methyltransferases. Compilation and parsimony analysis reveal that histone H3K9 methylation is, next to histone deacetylation, the evolutionary most stable heterochromatic mark, which is established by at least two subfamilies of specialized heterochromatic HMTases in almost all studied eukaryotes.     

Lysyl oxidase-like 2 (LOXL2) controls tumor-associated cell proliferation through the interaction with MARCKSL1

Lysyl oxidase-like 2 (LOXL2) is a member of the lysyl oxidase gene family that contributes to the invasiveness and metastasis in tumor progression. However, the role of LOXL2 in cellular signaling is incompletely understood. In this study, we investigated a possible mechanism of LOXL2 function in tumor metastases in vitro, using a human breast carcinoma cell line. Myristoylated alanine-rich C kinase substrate-like 1 (MARCKSL1), a modulator in the regulation of cellular homeostasis, was identified as a LOXL2 interacting protein. We examined the binding domains that are required for the interaction between LOXL2 and MARCKSL1. The scavenger-receptor domain of LOXL2 was shown to interact with the N-terminal domain of MARCKSL1. Luciferase activity was noticeably reduced by the transfection of MARCKSL1 in a dose-dependent manner. In addition, over-expression of LOXL2 activates cell growth by inhibiting MARCKSL1-induced apoptosis. The effect of LOXL2 on cell cycle and apoptosis-related components was also confirmed through the silencing of LOXL2 expression. LOXL2 activates the FAK/Akt/mTOR signaling pathways, and MARCKSL1 suppresses LOXL2-induced oncogenesis. These insights supply evidence that LOXL2 promotes cell proliferation and inhibits apoptotic cell death. Taken together, our results indicate an underlying mechanism for an increase of LOXL2-related activity in breast tumor cells.     

Salidroside ameliorated hypoxia-induced tumorigenesis of BxPC-3 cells via downregulating hypoxia-inducible factor (HIF)-1α and LOXL2

Herein, we found that salidroside suppressed hypoxia-inducible factor 1 alpha (HIF-1α) and lysyl oxidase-like protein 2 (LOXL2) within human pancreatic cancer BxPC-3 cells cultured both under normoxia and hypoxia condition. To investigate the effect of salidroside on tumorigenesis of BxPC-3 cells and whether HIF-1α and LXCL2 were involved in this process, cells transfected with or without LOXL2 overexpression vector, were treated with 50 μg/mL of salidroside or 50 μM of KC7F2 (a HIF-1α inhibitor) under hypoxia. Cell viability and invasion were assessed using CCK-8 and Transwell chamber assay, respectively. Expression of E-cadherin and matrix metalloproteinase 2/9 (MMP 2/9) was determined, by Western blot analysis, to assess cell mobility at molecular levels. We confirmed that hypoxia increased LOXL2 and induced tumorigenesis of BxPC-3 cells, as evidenced by promoted cell proliferation and invasion, enhanced MMP2/9 while reduced E-cadherin. Interestingly, hypoxia-induced carcinogenesis was significantly retarded by both salidroside and KC7F2, however, enhanced with LOXL2 overexpression. Besides, salidroside and KC7F2 reduced LOXL2, and reversed the tumorigenesis of BxPC-3 cells induced by LOXL2 overexpression. Given the inhibitory effect of salidroside on HIF-1α expression, our data suggested that: (1) LOXL2 was the mechanism, whereby salidroside and KC7F2 showed inhibitory effect on cancer progression of BxPC-3 cells; (2) salidroside exerted its anticancer effect, most likely, by a HIF-1α/LOXL2 pathway. In conclusion, salidroside was a novel therapeutic drug in pancreatic cancer, and downregulation of HIF-1α and LXCL2 was the underlying mechanism.     

Recognition of multivalent histone states associated with heterochromatin by UHRF1 protein

Histone modifications and DNA methylation represent two layers of heritable epigenetic information that regulate eukaryotic chromatin structure and gene activity. UHRF1 is a unique factor that bridges these two layers; it is required for maintenance DNA methylation at hemimethylated CpG sites, which are specifically recognized through its SRA domain and also interacts with histone H3 trimethylated on lysine 9 (H3K9me3) in an unspecified manner. Here we show that UHRF1 contains a tandem Tudor domain (TTD) that recognizes H3 tail peptides with the heterochromatin-associated modification state of trimethylated lysine 9 and unmodified lysine 4 (H3K4me0/K9me3). Solution NMR and crystallographic data reveal the TTD simultaneously recognizes H3K9me3 through a conserved aromatic cage in the first Tudor subdomain and unmodified H3K4 within a groove between the tandem subdomains. The subdomains undergo a conformational adjustment upon peptide binding, distinct from previously reported mechanisms for dual histone mark recognition. Mutant UHRF1 protein deficient for H3K4me0/K9me3 binding shows altered localization to heterochromatic chromocenters and fails to reduce expression of a target gene, p16(INK4A), when overexpressed. Our results demonstrate a novel recognition mechanism for the combinatorial readout of histone modification states associated with gene silencing and add to the growing evidence for coordination of, and cross-talk between, the modification states of H3K4 and H3K9 in regulation of gene expression.     

Primary deuterium isotope effects for the 3-methylaspartase-catalyzed deamination of (2S)-aspartic acid, (2S,3S)-3-methylaspartic acid, and (2S,3S)-3-ethylaspartic acid

3-Methylaspartate ammonia-lyase catalyzes the deamination of (2S)-aspartic acid 137 times more slowly than the deamination of (2S,3S)-3-methylaspartic acid but catalyzes the amination of fumaric acid 1.8 times faster than the amination of mesaconic acid [Botting, N.P., Akhtar, M., Cohen, M. A., & Gani, D. (1988) Biochemistry (preceding paper in this issue)]. In order to understand the mechanistic basis for these observations, the deamination reaction was examined kinetically with (2S)-aspartic acid, (2S,3S)-3-methylaspartic acid, (2S,3S)-3-ethylaspartic acid, and the corresponding C-3-deuteriated isotopomers. Comparison of the double-reciprocal plots of the initial reaction velocities for each of the three pairs of substrates revealed that the magnitude of the primary isotope effect on both Vmax and V/K varied with the substituent at C-3 of the substrate. 3-Methylaspartic acid showed the largest isotope effect (1.7 on Vmax and V/K), 3-ethylaspartic acid showed a smaller isotope effect (1.2 on Vmax and V/K), and aspartic acid showed no primary isotope effect at all. These results, which are inconsistent with earlier reports that there is no primary isotope effect for 3-methylaspartic acid [Bright, H. J. (1964) J. Biol. Chem. 239, 2307], suggest that for both 3-methylaspartic acid and 3-ethylaspartic acid elimination occurs via a predominantly concerted mechanism whereas for aspartic acid an E1cb mechanism prevails.(ABSTRACT TRUNCATED AT 250 WORDS)     

Temporal and spatial changes of histone 3 K4 dimethylation at the IFN-gamma gene during Th1 and Th2 cell differentiation

Covalent modification of nucleosomal histones is an important mechanism for cytokine gene regulation in Th1 and Th2 cells. In this study, we analyzed the kinetics of histone H3 K4 dimethylation (H3K4me2) of the IFN-gamma gene. Minimal levels of H3K4me2 were found in naive CD4 T cells. After 5 days of differentiation, H3K4me2 levels were elevated in both Th1 and Th2 cells at the -5.3 kb, the promoter, the intronic DNase I hypersensitive sites, and 3' distal sites including the +9.5 kb and +16 kb sites. Th1 cells maintained high levels of H3K4me2 after longer time of culture. However, in Th2 cells after 14 days, high levels of H3K4me2 were detected only at the -5.3 kb and the promoter, whereas H3K4me2 was lost at the 3' distal sites and greatly diminished at the DNase I hypersensitive sites. After 28 days, Th2 cells lose H3K4me2 at all sites. Unlike the long-term primary Th2 cells, the Th2 clone D10 showed strong H3K4me2 at the IFN-gamma gene with distinctly high levels at the 3' distal sites. CD4 T cells transgenic for Hlx or infected with T-bet-expressing retrovirus produced IFN-gamma and retained high levels of H3K4me2 even after differentiated under Th2 polarizing conditions, suggesting positive roles of these two factors in maintaining high levels of H3K4me2 at the IFN-gamma gene.     

Discovering common combinatorial histone modification patterns in the human genome

Histone modifications play a crucial role in regulating gene expression and cell lineage determination and maintenance at the epigenetic level. To systematically investigate this phenomenon, this paper presented a statistical hybrid clustering algorithm to identify common combinatorial histone modification patterns. We applied the algorithm to 39 histone modification marks in human CD4 + T cells and detected 854 common combinatorial histone modification patterns. Our results could cover 211 (76.17%) patterns among 277 patterns identified by the tandem mass spectrometry experiments. Based on the frequency statistical analysis, it was found that the co-occurrence frequencies of 20 backbone modifications are greater than or close to 0.2 in the 854 patterns. we also found that 15 modifications (H2BK120ac, H4K91ac, H2BK20ac, etc.), three histone acetylations (H2AK9ac, H4K16ac, and H4K12ac) and five histone methylations (H3K79me1, H3K79me2, 3K79me3, H4K20me1, and H2BK5me1) were most likely prone to coexist respectively in these patterns. In addition, we found that DNA methylation tends to combine with histone acetylation rather than histone methylation.