MV-mimicking micelles loaded with PEG-serine-ACP nanoparticles to achieve biomimetic intra/extra fibrillar mineralization of collagen in vitro

Since their discovery, matrix vesicles (MVs) containing minerals have received considerable attention for their role in the mineralization of bone, dentin and calcified cartilage. Additionally, MVs' association with collagen fibrils, which serve as the scaffold for calcification in the organic matrix, has been repeatedly highlighted. The primary purpose of the present study was to establish a MVs–mimicking model (PEG-S-ACP/micelle) in vitro for studying the exact mechanism of MVs-mediated extra/intra fibrillar mineralization of collagen in vivo. In this study, high-concentration serine was used to stabilize the amorphous calcium phosphate (S-ACP), which was subsequently mixed with polyethylene glycol (PEG) to form PEG-S-ACP nanoparticles. The nanoparticles were loaded in the polysorbate 80 micelle through a micelle self-assembly process in an aqueous environment. This MVs–mimicking model is referred to as the PEG-S-ACP/micelle model. By adjusting the pH and surface tension of the PEG-S-ACP/micelle, two forms of minerals (crystalline mineral nodules and ACP nanoparticles) were released to achieve the extrafibrillar and intrafibrillar mineralization, respectively. This in vitro mineralization process reproduced the mineral nodules mediating in vivo extrafibrillar mineralization and provided key insights into a possible mechanism of biomineralization by which in vivo intrafibrillar mineralization could be induced by ACP nanoparticles released from MVs. Also, the PEG-S-ACP/micelle model provides a promising methodology to prepare mineralized collagen scaffolds for repairing bone defects in bone tissue engineering.     

Microcystins: Synthesis and structure–activity relationship studies toward PP1 and PP2A

Microcystins are highly toxic cyanotoxins responsible for plant, animal and human poisoning. Exposure to microcystins, mainly through drinkable water and contaminated food, is a current world health concern. Although it is quite challenging, the synthesis of these potent cyanotoxins, analogs and derivatives helps to evaluate their toxicological properties and to elucidate their binding mechanisms to their main targets Protein Phosphatase-1 (PP1) and -2A (PP2A). This review focuses on synthetic approaches to prepare microcystins and analogs and compiles structure–activity relationship (SAR) studies that describe the unique features of microcystins that make them so potent.     

Serine/threonine kinase activity in the putative histidine kinase-like ethylene receptor NTHK1 from tobacco

A histidine kinase-based signaling system has been proposed to function in ethylene signal transduction pathway of plants and one ethylene receptor has been found to possess His kinase activity. Here we demonstrate that a His kinase-like ethylene receptor homologue NTHK1 from tobacco has serine/threonine (Ser/Thr) kinase activity, but no His kinase activity. Evidence obtained by analyzing acid/base stability, phosphoamino acid and substrate specificity of the phosphorylated kinase domain, supports this conclusion. In addition, mutation of the presumptive phosphorylation site His (H378) to Gln did not affect the kinase activity whereas deletion of the ATP-binding domain eliminated it, indicating that the conserved His (H378) is not required for the kinase activity and this activity is intrinsic to the NTHK1-KD. Moreover, confocal analysis of NTHK1 expression in insect cells and plant cells suggested the plasma membrane localization of the NTHK1 protein. Thus, NTHK1 may represent a distinct Ser/Thr kinase-type ethylene receptor and function in an alternative mechanism for ethylene signal transduction.     

Post-translational modification of proteins and the discovery of new medicine

Post-translational modifications are fundamental to processes controlling behaviour, including cellular signaling, growth and transformation. As the molecular basis of protein modifications in normal and disease processes are becoming better defined, so new strategies for designing therapeutic entities to control complex disease processes are emerging.     

Stability enhancement and circular dichroism spectral anomaly as an indication of non-covalent interactions in histidine- and tyrosine-containing ternary copper(II)—amino acid systems

Visible absorption and CD spectral and potentiometric studies on the His- and Tyr-containing ternary copper(II) complexes Cu(A)(L-B), where A refers to L-His, D-His, or L-Tyr and B to Lys, Tyr, Trp, Phe, Ala, Val, Arg, Glu, Asn, Gln, Ser, or Thr, were made to study ligand-ligand interactions in the complexes. While the CD spectral magnitudes in the d—d region are additive in the absence of side chain interactions and can be estimated from the magnitudes for the ternary systems involving DL-A or DL-B, deviation from the additivity was observed for Cu(L-His)(L-B) (B = LysH, Tyr, Trp, or Phe) and Cu(L-Tyr)(L-Trp). From the stability constants determined at 25 °C and I = 0.1 M (KNO₃), the equilibrium constants, K, for the following hypothetical equilibria were calculated to be large (0.14–0.60) for formation of Cu(L-/D-His)(L-B)(B = Tyr or Trp) and Cu(D-His)(L-Phe) with Cu(en)(L-Ala) as standard: $Cu(A)(L?Ala)+Cu(en)(L?b)\stackrel{K}{?}Cu(A)(L?B)+Cu(en)(L?Ala)$ The positive values indicate the stabilization due to the stacking between the imidazole ring of His and the aromatic side chain of L-B. Solvent dependence of the CD spectra for Cu(L-His)(L-LysH) and Cu(L-His) L-Trp) further supported the existence of the intramolecular electrostatic and hydrophobic interactions.     

Postnatal exposure to di-(2-ethylhexyl)phthalate alters cardiac insulin signaling molecules and GLUT4Ser488 phosphorylation in male rat offspring

Di-(2-ethylhexyl)phthalate (DEHP), a distinctive endocrine-disrupting chemical, is widely used as a plasticizer in a variety of consumer products. It can easily cross the placenta and enter breast milk and then it is rapidly absorbed by offspring. Since it is generally accepted that individuals are more sensitive to chemical exposure during vital developmental periods, we investigated whether DEHP exposure during lactation affects cardiac insulin signaling and glucose homeostasis in the F₁ male rat offspring at postnatal day 22 (PND22). Lactating Wistar rats were administered with DEHP (1, 10, and 100 mg/kg/d) or olive oil from lactation day 1 to 21 by oral gavage. All the male pups were perfused and killed on PND22. On the day before the killing, they were kept for fasting overnight and blood was collected. The cardiac muscle was dissected out, washed in ice-cold physiological saline repeatedly and used for the assay of various parameters. DEHP-exposed offspring had significantly lower body weight than the control. DEHP-exposed offspring showed elevated blood glucose, decreased ¹⁴C-2-deoxyglucose uptake and ¹⁴C-glucose oxidation in cardiac muscle at PND22. The concentration of upstream insulin signaling molecules such as insulin receptor subunit β (InsRβ) and insulin receptor substrate 1 (IRS1) were downregulated in DEHP-exposed offspring. However, no significant alterations were observed in protein kinase B (Akt) and Akt substrate of 160 kDa (AS160). Surprisingly, phosphorylation of IRS1 ^Tyr632 and Akt ^Ser473 were diminished. Low levels of glucose transporter type 4 (GLUT4) protein and increased GLUT4 ^Ser488 phosphorylation which decreases its intrinsic activity and translocation towards plasma membrane were also recorded. Lactational DEHP exposure predisposes F ₁ male offspring to cardiac glucometabolic disorders at PND22, which may impair cardiac function.     

A loop of coagulation factor VIIa influencing macromolecular substrate specificity

Coagulation factor VIIa (FVIIa) belongs to a family of proteases being part of the stepwise, self-amplifying blood coagulation cascade. To investigate the impact of the mutation Met(298{156})Lys in FVIIa, we replaced the Gly(283{140})-Met(298{156}) loop with the corresponding loop of factor Xa. The resulting variant exhibited increased intrinsic activity, concurrent with maturation of the active site, a less accessible N-terminus, and, interestingly, an altered macromolecular substrate specificity reflected in an increased ability to cleave factor IX (FIX) and a decreased rate of FX activation compared to that of wild-type FVIIa. In complex with tissue factor, activation of FIX, but not of FX, returned to normal. Deconvolution of the loop graft in order to identify important side chain substitutions resulted in the mutant Val(158{21})Asp/Leu(287{144})Thr/Ala(294{152})Ser/Glu(296{154}) Ile/Met(298{156})Lys-FVIIa with almost the same activity and specificity profile. We conclude that a lysine residue in position 298{156} of FVIIa requires a hydrophilic environment to be fully accommodated. This position appears critical for substrate specificity among the proteases of the blood coagulation cascade due to its prominent position in the macromolecular exosite and possibly via its interaction with the corresponding position in the substrate (i.e. FIX or FX).     

The structure of PknB in complex with mitoxantrone, an ATP-competitive inhibitor, suggests a mode of protein kinase regulation in mycobacteria

Mycobacterium tuberculosis PknB is an essential receptor-like protein kinase involved in cell growth control. Here, we demonstrate that mitoxantrone, an anthraquinone derivative used in cancer therapy, is a PknB inhibitor capable of preventing mycobacterial growth. The structure of the complex reveals that mitoxantrone partially occupies the adenine-binding pocket in PknB, providing a framework for the design of compounds with potential therapeutic applications. PknB crystallizes as a 'back-to-back' homodimer identical to those observed in other structures of PknB in complex with ATP analogs. This organization resembles that of the RNA-dependent protein kinase PKR, suggesting a mechanism for kinase activation in mycobacteria.     

Protein phosphatase 2A holoenzyme and its subunits from Medicago sativa

We detected an about 200 kDa holoenzyme of protein phosphatase 2A (PP2A) in the crude extract of Medicago sativa microcallus cells by gel permeation chromatography. By polymerase chain reaction (PCR) we isolated two M. sativa cDNA fragments corresponding to the catalytic (C) subunit, and one each coding for the A and the B regulatory subunits of PP2A. The C subunit sequences were different from that published previously, indicating the existence of at least three different isoforms in M. sativa. Using the PCR fragments as probes, we obtained two distinct full-length clones for both the A and B subunits from an alfalfa cDNA library. Our results demonstrate that the components of the PP2A holoenzyme, namely the catalytic and regulatory subunits, are present in alfalfa in several isoforms and that their sequences are highly similar to their plant, yeast and animal counterparts. The distinct regulatory subunit genes are constitutively expressed during the cell cycle. Interestingly, two A-B subunit pairs had parallel mRNA steady-state levels in different plant tissues suggesting that not all of the possible isoform combinations are present in all tissues. The expression of the MsPP2A B subunit form was induced by abscisic acid indicating a specific function for this protein in the stress response.     

酿酒酵母丝氨酸/苏氨酸蛋白磷酸酯酶的功能与调控

从酿酒酵母蛋白磷酸酯酶的分类和结构特征入手,阐述了该蛋白家族中的亚家族成员丝氨酸/苏氨酸蛋白磷酸酯酶的功能和表达调控.深入研究酿酒酵母丝氨酸/苏氨酸蛋白磷酸酯酶,特别是PP2C蛋白磷酸酯酶的细胞功能及其调控,将对新药研发和疾病干预治疗提供重要基础.