A peroxisomal long-chain acyl-CoA synthetase from Glycine max involved in lipid degradation.

Seed storage oil, in the form of triacylglycerol (TAG), is degraded to provide carbon and energy during germination and early seedling growth by the fatty acid β-oxidation in the peroxisome. Although the pathways for lipid degradation have been uncovered, understanding of the exact involved enzymes in soybean is still limited. Long-chain acyl-CoA synthetase (ACSL) is a critical enzyme that activates free fatty acid released from TAG to form the fatty acyl-CoA. Recent studies have shown the importance of ACSL in lipid degradation and synthesis, but few studies were focused on soybean. In this work, we cloned a ACSL gene from soybean and designated it as GmACSL2. Sequence analysis revealed that GmACSL2 encodes a protein of 733 amino acid residues, which is highly homologous to the ones in other higher plants. Complementation test showed that GmACSL2 could restore the growth of an ACS-deficient yeast strain (YB525). Co-expression assay in Nicotiana benthamiana indicated that GmACSL2 is located at peroxisome. Expression pattern analysis showed that GmACSL2 is highly expressed in germinating seedling and strongly induced 1 day after imbibition, which indicate that GmACSL2 may take part in the seed germination. GmACSL2 overexpression in yeast and soybean hairy root severely reduces the contents of the lipids and fatty acids, compared with controls in both cells, and enhances the β-oxidation efficiency in yeast. All these results suggest that GmACSL2 may take part in fatty acid and lipid degradation. In conclusion, peroxisomal GmACSL2 from Glycine max probably be involved in the lipid degradation during seed germination

Real-time PCR analysis of expression of <i>GmACSL2</i> in soybean.

<p>(A) Expression pattern of <i>GmACSL2</i> in different tissues. Total RNA was extracted from young roots, senescent roots, stems, young leaves, senescent leaves, flowers, developing seeds, and germinating seedlings. The actin gene served as the positive control. (B) Expression pattern of <i>GmACSL2</i> in germination seedling under dark. (a) Germination seedlings from 0 day to 6 day after imbibition (DAI). (b) Lipids content of seedlings from 0 day to 6 day after imbibition. (c) Real-time analysis of the expression of <i>GmACSL2</i> from 0 day to 6 day after imbibition. Data are presented as the mean ± SEM of three experiments.</p

Sequence analysis of GmACSL2.

<p>(A) Multiple amino acid sequences alignment of <i>Glycine max</i> GmACSL2 and GmLACS sequence with <i>Arabidopsis thaliana</i> AtLACS1 to AtLACS9. The <b>I</b> and <b>II</b> blocks of shadows indicate the highly conserved acid residues in AMP-binding protein. Black frames indicate the higher conservation amino acids, dark grey frames indicate the conservation amino acids, and light frames indicate the lower conservation amino acids. The protein follows: AtLACS1 (AAM28868), AtLACS2 (AAM288689), AtLACS3 (AAM28870), AtLACS4 (AAM28871), AtLACS5 (AAM28872), AtLACS6 (AAM28873), AtLACS7 (AAM28874), AtLACS8 (AAM28875), and AtLACS9 (AAM28876). (B) Phylogenic analysis between GmACSL2 with other ACSL enzymes and FATP proteins from plant, mammalian and yeast. The ACSL enzymes include <i>G.max</i> GmLACS, <i>Arabidopsis thaliana</i> AtLACS1-9, <i>Gossypium hirsutum</i> GhACS1 (ABA00144), <i>Brassica napus</i> BnACS6 (CAC19877), <i>Ricinus communis</i> RcACSL1 (XP_002520618) and RcACSL2 (XP_002520615), <i>Aegilops tauschii</i> AeACSL1 (EMT11835), <i>Triticum urartu</i> TuACSL (EMS60031), <i>Homo sapiens</i> HsACSL1 (NP_001986), HsACSL3 (NP_004448), HsACSL4 (NP_004449), HsACSL5 (NP_057318), and HsACSL6 (NP_056071), <i>Mus musculus</i> MmACSL1 (NP_032007), MmACSL3 (XP_129894), MmACSL4 (NP_062350), MmACSL5 (AAH31544), and MmACSL6 (NP_659072), <i>Rattus norvegicus</i> RnACSL1 (NP_036952), RnACSL3 (NP_476448), RnACSL4 (NP_446075), RnACSL5 (NP_446059), and RnACSL6 (NP_570095), and <i>Saccharomyces cerevisiae</i> ScFAA1 (P30624), ScFAA2 (P39518), ScFAA3 (P39002), and ScFAA4 (P47912). FATP proteins include <i>Homo sapiens</i> HsFATP1 (NP_940982), HsFATP3 (NP_077306), HsFATP4 (Q6P1M0), HsFATP5 (Q9Y2P5), and HsFATP6 (NP_001017372), <i>Mus musculus</i> MmFATP1 (NP_036107), MmFATP2 (AAC40186), and MmFATP4 (XP_130079), <i>Rattus norvegicus</i> RnFATP1 (NP_036119), and <i>Saccharomyces cerevisiae</i> (EWH19453). The bars stand for evolutionary distance. Bar = 0.2.</p

Overexpression of <i>GmACSL2</i> in yeast <i>pep4</i>.

<p>(A) Expression analysis of <i>GmACSL2</i> in different transformed lines by RT-PCR. Numbers 1 to 5 represents the five lines, GmACSL2-1, GmACSL2-2, GmACSL2-3, GmACSL2-4, and GmACSL2-5, transformed with pYES2-GmACSL2. The actin gene was used as the control for equal gel loading. Control represents the line transformed with pYES2 empty vector. (B) Sudan black B staining. The cells of three transformed lines GmACSL2-1, GmACSL2-3, and GmACSL2-4 were stained with Sudan Black B. The absorbance was measured at 580 nm and the line transformed with pYES2 vectors as control. (C) Fatty acids analysis comparisons between three transformed lines GmACSL2-1, GmACSL2-3 and GmACSL2-4 and the control line. The four major fatty acid species C16∶0, C16∶1, C18∶0, C18∶1 and the total fatty acids content in the yeast were detected by gas chromatography-mass spectrometry. (D) β-oxidation assay comparisons between three transformed lines GmACSL2-1, GmACSL2-3 and GmACSL2-4 and the control line. Oleate β-oxidation measurements in cells were followed by quantification of [¹⁴C] CO₂ and ¹⁴C-labelled β-oxidation products in a liquid scintillation counter. The β-oxidation activity in control cells in each experiment was taken as reference (100%). Data are presented as the mean ± SEM of the three experiments. *<i>p</i><0.05, **<i>p</i><0.01.</p

Overexpression of <i>GmACSL2</i> in transgenic hairy roots.

<p>(A) Hairy roots were induced by <i>A. rhizogenes.</i> (a) Soybean seeds were germinated in germination medium; (b) Explants were co-cultivated with <i>A. rhizogenes in</i> MCC medium; (c) Hairy roots were induced in 1/2 MS medium with hygromycin B as selection; (d) Hairy roots appeared at the wounding sites of the explants in 1/2 MS medium; (e) Hairy roots were grown in 1/2 MS solid medium; (f) Hairy roots were propagated in 1/2 MS liquid medium. (B) GUS histochemical staining and GFP fluorescence signal detection. After extended culture, the hairy roots were stained by GUS (upper panel) and signals of fluorescence GFP observation (lower panel). WT represents the wild-type soybean hairy roots hairy roots transformed with the K599 strain only; Trans represents the soybean hairy roots transformed with the control vector pGFPGUS and pGFPGUS-GmACSL2. (C) PCR analysis of hairy roots DNA using the primers to amplify the fragment of the GmACSL2 gene to further identify the transgenic line of GmACSL2. M, Marker; “+”, positive control, GmACSL2 plasmid was used as template; “−”, negative control, soybean hairy roots were transformed with the control vector pGFPGUS as template; 1–8, 1–11, 1–13, 1–17, 2–2, 2–4, 2–9, 2–22, 3–5, 3–14, and 3–33, individual lines transformed with the binary vector pGFPGUS-GmACSL2. (D) Expression analysis of <i>GmACSL2</i> in different transformed lines by RT-PCR. 1–8, 1–11, 1–13, 1–17, 2–2, 2–4, 2–9, 2–22, 3–5, 3–14, and 3–33 represent different transformed hairy roots overexpression with <i>GmACSL2</i>. Soybean actin gene was used as an internal control. (E) Analysis of lipid contents in the transgenic lines by soxhlet extraction. Control, transgenic hairy roots induced by pGFPGUS control vector; GmACSL2-1-8, GmACSL2-2-4, GmACSL2-3-5, GmACSL2-3-14, and GmACSL2-3-33, the five transgenic hairy root lines, induced by the pGFPGUS-GmACSL2 vector. (F) Fatty acid analysis comparisons between five <i>GmACSL2</i> transgenic lines and the control. The five major fatty acid species C16∶0, C18∶0, C18∶1, C18∶2, C18∶3 and the total fatty acids content in hairy roots were detected by gas chromatography-mass spectrometry. Data are the mean ± SEM of the three experiments. *<i>p</i><0.05, **<i>p</i><0.01.</p

Yeast complementation test and subcellular localization.

<p>(A) Yeast complementation test. Right: culture of yeast strain YB525 cells containing the pYES2 empty plasmids; Left: culture of yeast strain YB525 cells containing the pYES2-GmACSL2 plasmids. Bar = 1 cm. (B) ACS enzyme activities. YB525 cells carrying the pYES2 or pYES2-GmACSL2 plasmids were harvested after galactose induction for 18 h. 1-[¹⁴C] oleic acid was used as a substrate. Enzyme activities were measured based on the [¹⁴C] label incorporated into the acyl-CoA fraction per assay. Values are means of triplicate with standard deviation (SD). (C) Growth rate of the transformed line in different fatty acid culture medium. YB525 was transformed with the pYES2-GmACSL2 and empty PYES2 plasmids, which were cultured in liquid medium with various fatty acids (12∶0, 14∶0, 16∶0, 18∶0, 18∶1, and 22∶1) as the sole carbon source. (D) Subcellular localization. Fluorescence signals of eGFP were detected in cells expressing GmACSL2-eGFP fusion protein by Leica TCS scanning confocal microscope (left panels). Fluorescence signals of dsRFP were detected in cells expressing SSE1-dsRFP fusion protein (middle panels). Right panels were merged by left and middle panels. The upper panels are low-resolution pictures and the lower panels were high-resolution pictures. The immunofluorescence was done with tobacco leafs. Bar = 10 µm. Data are presented as the mean ± SEM of three experiments.</p

Allosteric Inhibitors of SHP2 with Therapeutic Potential for Cancer Treatment

SHP2, a cytoplasmic protein-tyrosine phosphatase encoded by the PTPN11 gene, is involved in multiple cell signaling processes including Ras/MAPK and Hippo/YAP pathways. SHP2 has been shown to contribute to the progression of a number of cancer types including leukemia, gastric, and breast cancers. It also regulates T-cell activation by interacting with inhibitory immune checkpoint receptors such as the programmed cell death 1 (PD-1) and B- and T-lymphocyte attenuator (BTLA). Thus, SHP2 inhibitors have drawn great attention by both inhibiting tumor cell proliferation and activating T cell immune responses toward cancer cells. In this study, we report the identification of an allosteric SHP2 inhibitor 1-(4-(6-bromonaphthalen-2-yl)­thiazol-2-yl)-4-methylpiperidin-4-amine (<b>23</b>) that locks SHP2 in a closed conformation by binding to the interface of the N-terminal SH2, C-terminal SH2, and phosphatase domains. Compound <b>23</b> suppresses MAPK signaling pathway and YAP transcriptional activity and shows antitumor activity <i>in vivo</i>. The results indicate that allosteric inhibition of SHP2 could be a feasible approach for cancer therapy