Reconstitution of the recombinant human γ-tubulin ring complex

Cryo-electron microscopy recently resolved the structure of the vertebrate γ-tubulin ring complex (γ-TuRC) purified from Xenopus laevis egg extract and human cells to near-atomic resolution. These studies clarified the arrangement and stoichiometry of γ-TuRC components and revealed that one molecule of actin and the small protein MZT1 are embedded into the complex. Based on this structural census of γ-TuRC core components, we developed a recombinant expression system for the reconstitution and purification of human γ-TuRC from insect cells. The recombinant γ-TuRC recapitulates the structure of purified native γ-TuRC and has similar functional properties in terms of microtubule nucleation and minus end capping. This recombinant system is a central step towards deciphering the activation mechanisms of the γ-TuRC and the function of individual γ-TuRC core components.     

Recruitment of the γ-Tubulin Ring Complex to Drosophila Salt-stripped Centrosome Scaffolds

Extracting isolated Drosophila centrosomes with 2 M KI generates salt-resistant scaffolds that lack the centrosomal proteins CP190, CP60, centrosomin, and γ-tubulin. To clarify the role of these proteins in microtubule nucleation by centrosomes and to identify additional centrosome components required for nucleation, we have developed an in vitro complementation assay for centrosome function. Centrosome aster formation is reconstituted when these inactive, salt-stripped centrosome scaffolds are supplemented with a soluble fraction of a Drosophila embryo extract. The CP60 and CP190 can be removed from this extract without effect, whereas removing the γ-tubulin destroys the complementing activity. Consistent with these results, we find no evidence that these three proteins form a complex together. Instead, γ-tubulin is found in two distinct protein complexes of 240,000 and ∼3,000,000 D. The larger complex, which is analogous to the Xenopus γ-tubulin ring complex (γTuRC) (Zheng, Y., M.L. Wong, B. Alberts, and T. Mitchison. 1995. Nature. 378:578–583), is necessary but not sufficient for complementation. An additional factor found in the extract is required. These results provide the first evidence that the γTuRC is required for microtubule nucleation at the centrosome.     

Xgrip109: A γ Tubulin–Associated Protein with an Essential Role in γ Tubulin Ring Complex (γTuRC) Assembly and Centrosome Function

Previous studies indicate that γ tubulin ring complex (γTuRC) can nucleate microtubule assembly and may be important in centrosome formation. γTuRC contains approximately eight subunits, which we refer to as Xenopus gamma ring proteins (Xgrips), in addition to γ tubulin. We found that one γTuRC subunit, Xgrip109, is a highly conserved protein, with homologues present in yeast, rice, flies, zebrafish, mice, and humans. The yeast Xgrip109 homologue, Spc98, is a spindle–pole body component that interacts with γ tubulin. In vertebrates, Xgrip109 identifies two families of related proteins. Xgrip109 and Spc98 have more homology to one family than the other. We show that Xgrip109 is a centrosomal protein that directly interacts with γ tubulin. We have developed a complementation assay for centrosome formation using demembranated Xenopus sperm and Xenopus egg extract. Using this assay, we show that Xgrip109 is necessary for the reassembly of salt-disrupted γTuRC and for the recruitment of γ tubulin to the centrosome. Xgrip109, therefore, is essential for the formation of a functional centrosome.     

Casein kinase 2 promotes the TGF-β-induced activation of α-tubulin acetyltransferase 1 in fibroblasts cultured on a soft matrix

Cell signals for growth factors depend on the mechanical properties of the extracellular matrix (ECM) surrounding the cells. Microtubule acetylation is involved in the transforming growth factor (TGF)-β-induced myofibroblast differentiation in the soft ECM. However, the mechanism of activation of α-tubulin acetyltransferase 1 (α-TAT1), a major α-tubulin acetyltransferase, in the soft ECM is not well defined. Here, we found that casein kinase 2 (CK2) is required for the TGF-β-induced activation of α-TAT1 that promotes microtubule acetylation in the soft matrix. Genetic mutation and pharmacological inhibition of CK2 catalytic activity specifically reduced microtubule acetylation in the cells cultured on a soft matrix rather than those cultured on a stiff matrix. Immunoprecipitation analysis showed that CK2α, a catalytic subunit of CK2, directly bound to the C-terminal domain of α-TAT1, and this interaction was more prominent in the cells cultured on the soft matrix. Moreover, the substitution of alanine with serine, the 236th amino acid located at the C-terminus, which contains the CK2-binding site of α-TAT1, sig-nificantly abrogated the TGF-β-induced microtubule acetylation in the soft matrix, indicating that the successful binding of CK2 and the C-terminus of α-TAT1 led to the phosphorylation of serine at the 236th position of amino acids in α-TAT1 and regulation of its catalytic activity. Taken together, our findings provide novel insights into the molecular mechanisms underlying the TGF-β-induced activation of α-TAT1 in a soft matrix.     

Synthesis and bioactivities evaluation of oleanolic acid oxime ester derivatives as α-glucosidase and α-amylase inhibitors

Different oleanolic acid (OA) oxime ester derivatives (3a-3t) were designed and synthesised to develop inhibitors against α-glucosidase and α-amylase. All the synthesised OA derivatives were evaluated against α-glucosidase and α-amylase in vitro. Among them, compound 3a showed the highest α-glucosidase inhibition with an IC₅₀ of 0.35 µM, which was ∼1900 times stronger than that of acarbose, meanwhile compound 3f exhibited the highest α-amylase inhibitory with an IC₅₀ of 3.80 µM that was ∼26 times higher than that of acarbose. The inhibition kinetic studies showed that the inhibitory mechanism of compounds 3a and 3f were reversible and mixed types towards α-glucosidase and α-amylase, respectively. Molecular docking studies analysed the interaction between compound and two enzymes, respectively. Furthermore, cytotoxicity evaluation assay demonstrated a high level of safety profile of compounds 3a and 3f against 3T3-L1 and HepG2 cells.

Highlights

1. Oleanolic acid oxime ester derivatives (3a–3t) were synthesised and screened against α-glucosidase and α-amylase.
2. Compound 3a showed the highest α-glucosidase inhibitory with IC50 of 0.35 µM.
3. Compound 3f presented the highest α-amylase inhibitory with IC50 of 3.80 µM.
4. Kinetic studies and in silico studies analysed the binding between compounds and α-glucosidase or α-amylase.

     

β-cell Smad2 null mice have improved β-cell function and are protected from diet-induced hyperglycemia

Understanding signaling pathways that regulate pancreatic β-cell function to produce, store, and release insulin, as well as pathways that control β-cell proliferation, is vital to find new treatments for diabetes mellitus. Transforming growth factor-beta (TGF-β) signaling is involved in a broad range of β-cell functions. The canonical TGF-β signaling pathway functions through intracellular smads, including smad2 and smad3, to regulate cell development, proliferation, differentiation, and function in many organs. Here, we demonstrate the role of TGF-β/smad2 signaling in regulating mature β-cell proliferation and function using β-cell-specific smad2 null mutant mice. β-cell-specific smad2-deficient mice exhibited improved glucose clearance as demonstrated by glucose tolerance testing, enhanced in vivo and ex vivo glucose-stimulated insulin secretion, and increased β-cell mass and proliferation. Furthermore, when these mice were fed a high-fat diet to induce hyperglycemia, they again showed improved glucose tolerance, insulin secretion, and insulin sensitivity. In addition, ex vivo analysis of smad2-deficient islets showed that they displayed increased glucose-stimulated insulin secretion and upregulation of genes involved in insulin synthesis and insulin secretion. Thus, we conclude that smad2 could represent an attractive therapeutic target for type 2 diabetes mellitus.     

Phosphorylation of human CEACAM1-LF by PKA and GSK3β promotes its interaction with β-catenin

CEACAM1-LF, a homotypic cell adhesion adhesion molecule, transduces intracellular signals via a 72 amino acid cytoplasmic domain that contains two immunoreceptor tyrosine-based inhibitory motifs (ITIMs) and a binding site for β-catenin. Phosphorylation of Ser503 by PKC in rodent CEACAM1 was shown to affect bile acid transport or hepatosteatosis via the level of ITIM phosphorylation, but the phosphorylation of the equivalent residue in human CEACAM1 (Ser508) was unclear. Here we studied this analogous phosphorylation by NMR analysis of the ¹⁵N labeled cytoplasmic domain peptide. Incubation with a variety of Ser/Thr kinases revealed phosphorylation of Ser508 by GSK3bβ but not by PKC. The lack of phosphorylation by PKC is likely due to evolutionary sequence changes between the rodent and human genes. Phosphorylation site assignment by mass spectrometry and NMR revealed phosphorylation of Ser472, Ser461 and Ser512 by PKA, of which Ser512 is part of a conserved consensus site for GSK3β binding. We showed here that only after phosphorylation of Ser512 by PKA was GSK3β able to phosphorylate Ser508. Phosphorylation of Ser512 by PKA promoted a tight association with the armadillo repeat domain of β-catenin at an extended region spanning the ITIMs of CEACAM1. The kinetics of phosphorylation of the ITIMs by Src, as well dephosphorylation by SHP2, were affected by the presence of Ser508/512 phosphorylation, suggesting that PKA and GSK3β may regulate the signal transduction activity of human CEACAM1-LF. The interaction of CEACAM1-LF with β-catenin promoted by PKA is suggestive of a tight association between the two ITIMs of CEACAM1-LF.     

Microtubule-associated proteins promote microtubule generation in the absence of γ-tubulin in human colon cancer cells

The γ-tubulin complex acts as the predominant microtubule (MT) nucleator that initiates MT formation and is therefore an essential factor for cell proliferation. Nonetheless, cellular MTs are formed after experimental depletion of the γ-tubulin complex, suggesting that cells possess other factors that drive MT nucleation. Here, by combining gene knockout, auxin-inducible degron, RNA interference, MT depolymerization/regrowth assay, and live microscopy, we identified four microtubule-associated proteins (MAPs), ch-TOG, CLASP1, CAMSAPs, and TPX2, which are involved in γ-tubulin–independent MT generation in human colon cancer cells. In the mitotic MT regrowth assay, nucleated MTs organized noncentriolar MT organizing centers (ncMTOCs) in the absence of γ-tubulin. Depletion of CLASP1 or TPX2 substantially delayed ncMTOC formation, suggesting that these proteins might promote MT nucleation in the absence of γ-tubulin. In contrast, depletion of ch-TOG or CAMSAPs did not affect the timing of ncMTOC appearance. CLASP1 also accelerates γ-tubulin–independent MT regrowth during interphase. Thus, MT generation can be promoted by MAPs without the γ-tubulin template.     

Induction of γδT cells from HSC‐enriched BMCs co‐cultured with iPSC‐derived thymic epithelial cells

T cells bearing γδ antigen receptors have been investigated as potential treatments for several diseases, including malignant tumours. However, the clinical application of γδT cells has been hampered by their relatively low abundance in vivo and the technical difficulty of inducing their differentiation from hematopoietic stem cells (HSCs) in vitro. Here, we describe a novel method for generating mouse γδT cells by co‐culturing HSC‐enriched bone marrow cells (HSC‐eBMCs) with induced thymic epithelial cells (iTECs) derived from induced pluripotent stem cells (iPSCs). We used BMCs from CD45.1 congenic C57BL/6 mice to distinguish them from iPSCs, which expressed CD45.2. We showed that HSC‐eBMCs and iTECs cultured with IL‐2 + IL‐7 for up to 21 days induced CD45.1⁺ γδT cells that expressed a broad repertoire of Vγ and Vδ T‐cell receptors. Notably, the induced lymphocytes contained few or no αβT cells, NK1.1⁺ natural killer cells, or B220⁺ B cells. Adoptive transfer of the induced γδT cells to leukemia‐bearing mice significantly reduced tumour growth and prolonged mouse survival with no obvious side effects, such as tumorigenesis and autoimmune diseases. This new method suggests that it could also be used to produce human γδT cells for clinical applications.     

In vitro and in silico studies of bis (indol-3-yl) methane derivatives as potential α-glucosidase and α-amylase inhibitors

In this paper, bis (indol-3-yl) methanes (BIMs) were synthesised and evaluated for their inhibitory activity against α-glucosidase and α-amylase. All synthesised compounds showed potential α-glucosidase and α-amylase inhibitory activities. Compounds 5 g (IC₅₀: 7.54 ± 1.10 μM), 5e (IC₅₀: 9.00 ± 0.97 μM), and 5 h (IC₅₀: 9.57 ± 0.62 μM) presented strongest inhibitory activities against α-glucosidase, that were ∼ 30 times stronger than acarbose. Compounds 5 g (IC₅₀: 32.18 ± 1.66 µM), 5 h (IC₅₀: 31.47 ± 1.42 µM), and 5 s (IC₅₀: 30.91 ± 0.86 µM) showed strongest inhibitory activities towards α-amylase, ∼ 2.5 times stronger than acarbose. The mechanisms and docking simulation of the compounds were also studied. Compounds 5 g and 5 h exhibited bifunctional inhibitory activity against these two enzymes. Furthermore, compounds showed no toxicity against 3T3-L1 cells and HepG2 cells.

Highlights

1. A series of bis (indol-3-yl) methanes (BIMs) were synthesised and evaluated inhibitory activities against α-glucosidase and α-amylase.
2. Compound 5g exhibited promising activity (IC₅₀ = 7.54 ± 1.10 μM) against α-glucosidase.
3. Compound 5s exhibited promising activity (IC₅₀ = 30.91 ± 0.86 μM) against α-amylase.
4. In silico studies were performed to confirm the binding interactions of synthetic compounds with the enzyme active site.

     

High temperature promotes amyloid β-protein production and γ-secretase complex formation via Hsp90

Alzheimer''s disease (AD) is characterized by neuronal loss and accumulation of β-amyloid-protein (Aβ) in the brain parenchyma. Sleep impairment is associated with AD and affects about 25–40% of patients in the mild-to-moderate stages of the disease. Sleep deprivation leads to increased Aβ production; however, its mechanism remains largely unknown. We hypothesized that the increase in core body temperature induced by sleep deprivation may promote Aβ production. Here, we report temperature-dependent regulation of Aβ production. We found that an increase in temperature, from 37 °C to 39 °C, significantly increased Aβ production in amyloid precursor protein-overexpressing cells. We also found that high temperature (39 °C) significantly increased the expression levels of heat shock protein 90 (Hsp90) and the C-terminal fragment of presenilin 1 (PS1-CTF) and promoted γ-secretase complex formation. Interestingly, Hsp90 was associated with the components of the premature γ-secretase complex, anterior pharynx-defective-1 (APH-1), and nicastrin (NCT) but was not associated with PS1-CTF or presenilin enhancer-2. Hsp90 knockdown abolished the increased level of Aβ production and the increased formation of the γ-secretase complex at high temperature in culture. Furthermore, with in vivo experiments, we observed increases in the levels of Hsp90, PS1-CTF, NCT, and the γ-secretase complex in the cortex of mice housed at higher room temperature (30 °C) compared with those housed at standard room temperature (23 °C). Our results suggest that high temperature regulates Aβ production by modulating γ-secretase complex formation through the binding of Hsp90 to NCT/APH-1.     

MicroRNA‐181a restricts human γδ T cell differentiation by targeting Map3k2 and Notch2

γδ T cells are a conserved population of lymphocytes that contributes to anti‐tumor responses through its overt type 1 inflammatory and cytotoxic properties. We have previously shown that human γδ T cells acquire this profile upon stimulation with IL‐2 or IL‐15, in a differentiation process dependent on MAPK/ERK signaling. Here, we identify microRNA‐181a as a key modulator of human γδ T cell differentiation. We observe that miR‐181a is highly expressed in patients with prostate cancer and that this pattern associates with lower expression of NKG2D, a critical mediator of cancer surveillance. Interestingly, miR‐181a expression negatively correlates with an activated type 1 effector profile obtained from in vitro differentiated γδ T cells and miR‐181a overexpression restricts their levels of NKG2D and TNF‐α. Upon in silico analysis, we identify two miR‐181a candidate targets, Map3k2 and Notch2, which we validate via overexpression coupled with luciferase assays. These results reveal a novel role for miR‐181a as critical regulator of human γδ T cell differentiation and highlight its potential for manipulation of γδ T cells in next‐generation immunotherapies.     

Moieties of Complement iC3b Recognized by the I-domain of Integrin αXβ2

Complement fragment iC3b serves as a major opsonin for facilitating phagocytosis via its interaction with complement receptors CR3 and CR4, also known by their leukocyte integrin family names, αMβ2 and αXβ2, respectively. Although there is general agreement that iC3b binds to the αM and αX I-domains of the respective β2-integrins, much less is known regarding the regions of iC3b contributing to the αX I-domain binding. In this study, using recombinant αX I-domain, as well as recombinant fragments of iC3b as candidate binding partners, we have identified two distinct binding moieties of iC3b for the αX I-domain. They are the C3 convertase-generated N-terminal segment of the C3b α’-chain (α’NT) and the factor I cleavage-generated N-terminal segment in the CUBf region of α-chain. Additionally, we have found that the CUBf segment is a novel binding moiety of iC3b for the αM I-domain. The CUBf segment shows about a 2-fold higher binding activity than the α’NT for αX I-domain. We also have shown the involvement of crucial acidic residues on the iC3b side of the interface and basic residues on the I-domain side.     

Glycogen synthase kinase 3β promotes osteosarcoma invasion and migration via regulating PTEN and phosphorylation of focal adhesion kinase

Aim: Typical features of human osteosarcoma are highly invasive and migratory capacities. Our study aimed to investigate the roles of glycogen synthase kinase 3β (GSK3β) in human osteosarcoma metastasis.Methods: GSK3β expressions in clinical osteosarcoma tissues with or without metastasis were examined by immunohistochemical staining. The expressions of GSK3β, p-GSK3β^Ser9, and p-GSK3β^Tyr216 in human osteoblast cells (hFOB1.19) and human osteosarcoma cells (MG63, SaOS-2, and U2-OS) were detected by Western blotting. The GSK3β activity was measured by non-radio isotopic in vitro kinase assay. Migration and invasion abilities of MG-63 cells treated with small-molecular GSK3β inhibitors were respectively examined by monolayer-based wound-healing assay and transwell assay. The mRNA expressions of GSK3β, matrix metalloproteinase-2 (MMP-2), MMP-9, phosphatase with tensin homology (PTEN), and focal adhesion kinase (FAK) were detected after siRNA transfection for 72 h. Meanwhile, protein expressions of GSK3β, FAK, p-FAK^Y397, PTEN, MMP-2, and MMP-9 were measured by Western blotting.Results: Clinical osteosarcoma tissues with metastasis showed higher GSK3β expressions. MG63 and U2-OS cells that were easy to occur metastasis showed significantly higher expressions and activities of GSK3β than SaOS-2 cells. Inhibition of GSK3β with small-molecular GSK3β inhibitors in MG63 cells significantly attenuated cell migration and invasion. These effects were associated with reduced expressions of MMP-2 and MMP-9. Moreover, increased PTEN and decreased p-FAK^Y397 expressions were observed following GSK3β knockdown by siRNA transfection. Conclusion: GSK3β might promote osteosarcoma invasion and migration via pathways associated with PTEN and phosphorylation of FAK.     

Second generation β-elemene nitric oxide derivatives with reasonable linkers: potential hybrids against malignant brain glioma

Elemene is a second-line broad-spectrum anti-tumour drug that has been used in China for more than two decades. However, its main anti-tumour ingredient, β-elemene, has disadvantages, including excessive lipophilicity and relatively weak anti-tumour efficacy. To improve the anti-tumour activity of β-elemene, based on its minor molecular weight character, we introduced furoxan nitric oxide (NO) donors into the β-elemene structure and designed six series of new generation β-elemene NO donor hybrids. The synthesised compounds could effectively release NO in vitro, displayed significant anti-proliferative effects on U87MG, NCI-H520, and SW620 cell lines. In the orthotopic glioma model, compound Id significantly and continuously suppressed the growth of gliomas in nude mice, and the brain glioma of the treatment group was markedly inhibited (>90%). In short, the structural fusion design of NO donor and β-elemene is a feasible strategy to improve the in vivo anti-tumour activity of β-elemene.     

An ancient interleukin-16–like molecule regulates hemocyte proliferation via integrin β1 in invertebrates

Interleukins (ILs) are cytokines with crucial functions in innate and adaptive immunity. IL genes are only found in vertebrates, except for IL-16, which has been cloned in some arthropod species. However, the function of this gene in invertebrates is unknown. In the present study, an IL-16–like gene (EsIL-16) was identified from the Chinese mitten crab Eriocheir sinensis. EsIL-16 was predicted to encode a precursor (proEsIL-16) that shares similarities with pro-IL-16 proteins from insects and vertebrates. We show that caspase-3 processes proEsIL-16 into an approximately 144-kDa N-terminal prodomain with nuclear import activity and an approximately 34-kDa mature peptide that might be secreted into the extracellular region. EsIL-16 mRNA could be detected in all analyzed tissues and was significantly upregulated after immune challenge both in vitro and in vivo. T7 phage display library screening suggested potential binding activity between EsIL-16 and integrin, which was confirmed by coimmunoprecipitation assay. Interestingly, EsIL-16 promoted cell proliferation via integrin β1 in primary cultured crab hemocytes and Drosophila S2 cells. Furthermore, the interaction between EsIL-16 and integrin β1 was necessary to efficiently protect the host from bacterial infection. To our knowledge, this study revealed integrin β1 as a receptor for IL-16 and the function of this interaction in hemocyte proliferation in invertebrates for the first time. These results provide new insights into the regulation of innate immune responses in invertebrates and shed the light on the evolution of ILs within the animal kingdom.     

Centrosomes Isolated from Spisula solidissima Oocytes Contain Rings and an Unusual Stoichiometric Ratio of α/β Tubulin

Centrosome-dependent microtubule nucleation involves the interaction of tubulin subunits with pericentriolar material. To study the biochemical and structural basis of centrosome-dependent microtubule nucleation, centrosomes capable of organizing microtubules into astral arrays were isolated from parthenogenetically activated Spisula solidissima oocytes. Intermediate voltage electron microscopy tomography revealed that each centrosome was composed of a single centriole surrounded by pericentriolar material that was studded with ring-shaped structures ~25 nm in diameter and <25 nm in length. A number of proteins copurified with centrosomes including: (a) proteins that contained M-phase–specific phosphoepitopes (MPM-2), (b) α-, β-, and γ-tubulins, (c) actin, and (d) three low molecular weight proteins of <20 kD. γ-Tubulin was not an MPM-2 phosphoprotein and was the most abundant form of tubulin in centrosomes. Relatively little α- or β-tubulin copurified with centrosomes, and the ratio of α- to β-tubulin in centrosomes was not 1:1 as expected, but rather 1:4.6, suggesting that centrosomes contain β-tubulin that is not dimerized with α-tubulin.     

Verteporfin is a substrate-selective γ-secretase inhibitor that binds the amyloid precursor protein transmembrane domain

This work reports substrate-selective inhibition of a protease with broad substrate specificity based on direct binding of a small-molecule inhibitor to the substrate. The target for these studies was γ-secretase protease, which cleaves dozens of different single-span membrane protein substrates, including both the C99 domain of the human amyloid precursor protein and the Notch receptor. Substrate-specific inhibition of C99 cleavage is desirable to reduce production of the amyloid-β polypeptide without inhibiting Notch cleavage, a major source of toxicity associated with broad specificity γ-secretase inhibitors. In order to identify a C99-selective inhibitors of the human γ-secretase, we conducted an NMR-based screen of FDA-approved drugs against C99 in model membranes. From this screen, we identified the small-molecule verteporfin with these properties. We observed that verteporfin formed a direct 1:1 complex with C99, with a K_D of 15–47 μM (depending on the membrane mimetic used), and that it did not bind the transmembrane domain of the Notch-1 receptor. Biochemical assays showed that direct binding of verteporfin to C99 inhibits γ-secretase cleavage of C99 with IC₅₀ values in the range of 15–164 μM, while Notch-1 cleavage was inhibited only at higher concentrations, and likely via a mechanism that does not involve binding to Notch-1. This work documents a robust NMR-based approach to discovery of small-molecule binders to single-span membrane proteins and confirmed that it is possible to inhibit γ-secretase in a substrate-specific manner.