Lenalidomide derivatives and proteolysis-targeting chimeras for controlling neosubstrate degradation.

Lenalidomide, an immunomodulatory drug (IMiD), is commonly used as a first-line therapy in many haematological cancers, such as multiple myeloma (MM) and 5q myelodysplastic syndromes (5q MDS), and it functions as a molecular glue for the protein degradation of neosubstrates by CRL4CRBN. Proteolysis-targeting chimeras (PROTACs) using IMiDs with a target protein binder also induce the degradation of target proteins. The targeted protein degradation (TPD) of neosubstrates is crucial for IMiD therapy. However, current IMiDs and IMiD-based PROTACs also break down neosubstrates involved in embryonic development and disease progression. Here, we show that 6-position modifications of lenalidomide are essential for controlling neosubstrate selectivity; 6-fluoro lenalidomide induced the selective degradation of IKZF1, IKZF3, and CK1α, which are involved in anti-haematological cancer activity, and showed stronger anti-proliferative effects on MM and 5q MDS cell lines than lenalidomide. PROTACs using these lenalidomide derivatives for BET proteins induce the selective degradation of BET proteins with the same neosubstrate selectivity. PROTACs also exert anti-proliferative effects in all examined cell lines. Thus, 6-position-modified lenalidomide is a key molecule for selective TPD using thalidomide derivatives and PROTACs.

Structural bases for aspartate recognition and polymerization efficiency of cyanobacterial cyanophycin synthetase.

Cyanophycin is a natural biopolymer consisting of equimolar amounts of aspartate and arginine as the backbone and branched sidechain, respectively. It is produced by a single enzyme, cyanophycin synthetase (CphA1), and accumulates as a nitrogen reservoir during N2 fixation by most cyanobacteria. A recent structural study showed that three constituent domains of CphA1 function as two distinct catalytic sites and an oligomerization interface in cyanophycin synthesis. However, it remains unclear how the ATP-dependent addition of aspartate to cyanophycin is initiated at the catalytic site of the glutathione synthetase-like domain. Here, we report the cryogenic electron microscopy structures of CphA1, including a complex with aspartate, cyanophycin primer peptide, and ATP analog. These structures reveal the aspartate binding mode and phosphate-binding loop movement to the active site required for the reaction. Furthermore, structural and mutational data show a potential role of protein dynamics in the catalytic efficiency of the arginine condensation reaction.

ß-elemene regulates M1-M2 macrophage balance through the ERK/JNK/P38 MAPK signaling pathway.

Macrophages are classified into classically activated M1 macrophages and alternatively activated M2 macrophages, and the two phenotypes of macrophages are present during the development of various chronic diseases, including obesity-induced inflammation. In the present study, ß-elemene, which is contained in various plant substances, is predicted to treat high-fat diet (HFD)-induced macrophage dysfunction based on the Gene Expression Omnibus (GEO) database and experimental validation. ß-elemene impacts the imbalance of M1-M2 macrophages by regulating pro-inflammatory cytokines in mouse white adipose tissue both in vitro and in vivo. In addition, the RAW 264 cell line, which are macrophages from mouse ascites, is used to identify the effects of ß-elemene on inhibiting bacterial endotoxin lipopolysaccharide (LPS)-induced phosphorylation of mitogen-activated protein kinase (MAPK) pathways. These pathways both induce and are activated by pro-inflammatory cytokines, and they also participate in the process of obesity-induced inflammation. The results highlight that ß-elemene may represent a possible macrophage-mediated therapeutic medicine.

Crystallization and melting properties studied by DSC and FTIR spectroscopy of goldenberry (Physalis peruviana) oil.

The chemical and thermal characteristics of goldenberry pomace oil (GPO) and goldenberry seed oil (GSO) were investigated. GPO and GSO contained high levels of unsaturated fatty acids (90.1% and 85.1%, respectively), and the major fatty acid was linoleic (62.0% and 72.8%, respectively). Additionally, GPO contained eleven triacylglycerol (TAG) species, three of which represented 82.7%, namely C54:6, C54:4 and C52:4, and trilinolein was the dominant one (35.5%). GSO contained nine TAG species, two of which represented 80.3%, namely C54:6 and C52:4, and trilinolein was dominant (53.3%). The DSC analysis of GPO and GSO revealed that three exothermal peaks were detected during cooling. Three endothermal peaks (one of which is exothermal for GSO) were detected during melting, and the most significant peaks occurred at low temperatures. FTIR spectra indicated that GPO and GSO did not contain peroxides or trans fatty acids, but they did contain low concentrations of free fatty acids.

Identification of the Effects of Chondroitin Sulfate on Inhibiting CDKs in Colorectal Cancer Based on Bioinformatic Analysis and Experimental Validation.

With a high occurrence rate and high mortality, the treatment of colorectal cancer (CRC) is increasingly attracting the attention of scholars. Hub genes that determine the phenotypes of CRC become essential for targeted therapy. In the present study, the importance of cyclin-dependent kinases (CDKs) on the occurrence of CRC was identified by data mining of The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO). The results showed that the gene expression levels of CDK1, CDK4, and CDK6 were obviously changed in different stages of CRC. Among the CDKs, CDK4 was suggested as an independent risk factor for CRC based on Cox analysis. Furthermore, chondroitin sulfate (CS), a kind of dietary supplement to treat osteoarthritis, was predicted to treat CRC based on its chemical structure and GEO datasets. Cell assay experiments with the human CRC cell line HCT-116 also verified this prediction. CS inhibited the gene and protein expression levels of CDKs and increased the ratios of apoptotic or dead HCT-116 cells by regulating mitogen-activated protein (MAP) kinase pathways. Our data highlight the essential roles of CDKs in CRC carcinogenesis and the effects of CS on treating CRC, both of which will contribute to the future CRC treatment.

Evaluation of spice and herb as phyto-derived selective modulators of human retinaldehyde dehydrogenases using a simple in vitro method.

Selective modulation of retinaldehyde dehydrogenases (RALDHs)-the main aldehyde dehydrogenase (ALDH) enzymes converting retinal into retinoic acid (RA), is very important not only in the RA signaling pathway but also for the potential regulatory effects on RALDH isozyme-specific processes and RALDH-related cancers. However, very few selective modulators for RALDHs have been identified, partly due to variable overexpression protocols of RALDHs and insensitive activity assay that needs to be addressed. In the present study, deletion of the N-terminal disordered regions is found to enable simple preparation of all RALDHs and their closest paralog ALDH2 using a single protocol. Fluorescence-based activity assay was employed for enzymatic activity investigation and screening for RALDH-specific modulators from extracts of various spices and herbs that are well-known for containing many phyto-derived anti-cancer constituents. Under the established conditions, spice and herb extracts exhibited differential regulatory effects on RALDHs/ALDH2 with several extracts showing potential selective inhibition of the activity of RALDHs. In addition, the presence of magnesium ions was shown to significantly increase the activity for the natural substrate retinal of RALDH3 but not the others, while His-tag cleavage considerably increased the activity of ALDH2 for the non-specific substrate retinal. Altogether we propose a readily reproducible workflow to find selective modulators for RALDHs and suggest potential sources of selective modulators from spices and herbs.

Antioxidant properties and inhibition of angiotensin-converting enzyme by highly active peptides from wheat gluten.

This study aimed to focus on the high-value utilization of raw wheat gluten by determining the potent antioxidant peptides and angiotensin I-converting enzyme (ACE) inhibitory peptides from wheat gluten oligopeptides (WOP). WOP were analyzed for in vitro antioxidant activity and inhibition of ACE, and the identification of active peptides was performed by reversed-phase high-performance liquid chromatography and mass spectrometry. Quantitative analysis was performed for highly active peptides. Five potent antioxidant peptides, Leu-Tyr, Pro-Tyr, Tyr-Gln, Ala-Pro-Ser-Tyr and Arg-Gly-Gly-Tyr (6.07 ± 0.38, 7.28 ± 0.29, 11.18 ± 1.02, 5.93 ± 0.20 and 9.04 ± 0.47 mmol 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox) equivalent/g sample, respectively), and five potent ACE inhibitory peptides, Leu-Tyr, Leu-Val-Ser, Tyr-Gln, Ala-Pro-Ser-Tyr and Arg-Gly-Gly-Tyr (half maximal inhibitory concentration (IC50) values = 0.31 ± 0.02, 0.60 ± 0.03, 2.00 ± 0.13, 1.47 ± 0.08 and 1.48 ± 0.11 mmol/L, respectively), were observed. The contents of Leu-Tyr, Pro-Tyr, Tyr-Gln, Ala-Pro-Ser-Tyr, Arg-Gly-Gly-Tyr, and Leu-Val-Ser were 155.04 ± 8.36, 2.08 ± 0.12, 1.95 ± 0.06, 22.70 ± 1.35, 0.25 ± 0.01, and 53.01 ± 2.73 µg/g, respectively, in the WOP. Pro-Tyr, Tyr-Gln, Ala-Pro-Ser-Tyr, Arg-Gly-Gly-Tyr, and Leu-Val-Ser are novel antioxidative/ACE inhibitory peptides that have not been previously reported. The results suggest that WOP could potentially be applied in the food industry as a functional additive.

Improved preparation of group-specific component (Gc) protein to derive macrophage activating factor.

Cancer immunotherapy has recently attracted attention as an approach for cancer treatment through the activation of the immune system. Group-specific component (Gc) protein is a precursor for macrophage activating factor (GcMAF), which has a promising immunomodulatory effect on the suppression of tumor growth and angiogenesis. In this study, we successfully purified Gc protein from human serum using anion-exchange chromatography combined with affinity chromatography using a 25-OH-D3-immobilized column. The purity of Gc protein reached 95.0% after anion-exchange chromatography. The known allelic variants of Gc protein are classified into three subtypes-Gc1F, Gc1S and Gc2. The fragment sequence of residues 412-424 determined according to their MS/MS spectra is available to evaluate the subtypes of Gc protein. The data showed that the Gc protein purified in this study consisted of the Gc1F and Gc2 subtypes. Our method improved the purity of Gc protein, which was not affected by the treatment to convert it into GcMAF using ß-galactosidase- or neuraminidase-immobilized resin, and will be useful for biological studies and/or advanced clinical uses of GcMAF, such as cancer immunotherapy.

GSTA4 mediates reduction of cisplatin ototoxicity in female mice.

Cisplatin is one of the most widely used chemotherapeutic drugs for the treatment of cancer. Unfortunately, one of its major side effects is permanent hearing loss. Here, we show that glutathione transferase α4 (GSTA4), a member of the Phase II detoxifying enzyme superfamily, mediates reduction of cisplatin ototoxicity by removing 4-hydroxynonenal (4-HNE) in the inner ears of female mice. Under cisplatin treatment, loss of Gsta4 results in more profound hearing loss in female mice compared to male mice. Cisplatin stimulates GSTA4 activity in the inner ear of female wild-type, but not male wild-type mice. In female Gsta4-/- mice, cisplatin treatment results in increased levels of 4-HNE in cochlear neurons compared to male Gsta4-/- mice. In CBA/CaJ mice, ovariectomy decreases mRNA expression of Gsta4, and the levels of GSTA4 protein in the inner ears. Thus, our findings suggest that GSTA4-dependent detoxification may play a role in estrogen-mediated neuroprotection.

Metabolic profiling of natural and cultured Cordyceps by NMR spectroscopy.

Cordyceps, a type of Chinese herbal medicine that exhibits anti-angiogenesis and tumor growth suppression effects, has recently gained increasing popularity. However, high-quality, natural Cordyceps, such as Ophiocordyceps sinensis, is very rare and difficult to obtain in large amounts. Cordyceps is cultured instead of harvested from natural sources, but the quality with respect to the ingredients has not been fully studied. In this study, we performed an NMR metabolic profiling of aqueous extracts of Cordyceps without any sample treatment to evaluate the proper species and medium and influence of two different disinfection methods. It was discovered that Cordyceps militaris fungus and silkworm chrysalis medium were suitable for cultivation of Cordyceps. Furthermore, cordycepin, a Cordyceps-specific functional compound, was produced at different growth stages during different cultivation processes, even at the mycelial stage, and was found at three times higher concentrations in cultured C. militaris compared to that in naturally occurring C. militaris.

Characterization of the Ca2+-coordination structures of L- and T-plastins in combination with their synthetic peptide analogs by FTIR spectroscopy.

FTIR spectroscopy was employed to characterize the coordination structures of divalent cations (M2+ = Ca2+ or Mg2+) bound by L- and T-plastins, which contain two EF-hand motifs. We focused on the N-terminal headpieces in the L- and T-plastins to analyze the regions of COO- stretching and amide-I in solution. The spectral profiles indicated that these headpieces have EF-hand calcium-binding sites because bands at 1551 cm-1 and 1555 cm-1 were observed for the bidentate coordination mode of Glu at the 12th position of the Ca2+-binding site of Ca2+-loaded L-plastin and T-plastin, respectively. The amide-I profile of the Mg2+-loaded L-plastin headpiece was identical with that of the apo L-plastin headpiece, meaning that L-plastin has a lower affinity for Mg2+. The amide-I profiles for apo, Mg2+-loaded and Ca2+-loaded T-plastin suggested that aggregation was generated in protein solution at a concentration of 1 mM. The implications of the FTIR spectral data for these plastin headpieces are discussed on the basis of data obtained for synthetic peptide analogs corresponding to the Ca2+-binding site.

Structure and Polymannuronate Specificity of a Eukaryotic Member of Polysaccharide Lyase Family 14.

Alginate is an abundant algal polysaccharide, composed of ß-d-mannuronate and its C5 epimer α-l-guluronate, that is a useful biomaterial in cell biology and tissue engineering, with applications in cancer and aging research. The alginate lyase (EC 4.2.2.3) from Aplysia kurodai, AkAly30, is a eukaryotic member of the polysaccharide lyase 14 (PL-14) family and degrades alginate by cleaving the glycosidic bond through a ß-elimination reaction. Here, we present the structural basis for the substrate specificity, with a preference for polymannuronate, of AkAly30. The crystal structure of AkAly30 at a 1.77 Å resolution and the putative substrate-binding model show that the enzyme adopts a ß-jelly roll fold at the core of the structure and that Lys-99, Tyr-140, and Tyr-142 form catalytic residues in the active site. Their arrangements allow the carboxyl group of mannuronate residues at subsite +1 to form ionic bonds with Lys-99. The coupled tyrosine forms a hydrogen bond network with the glycosidic bond, and the hydroxy group of Tyr-140 is located near the C5 atom of the mannuronate residue. These interactions could promote the ß-elimination of the mannuronate residue at subsite +1. More interestingly, Gly-118 and the disulfide bond formed by Cys-115 and Cys-124 control the conformation of an active-site loop, which makes the space suitable for substrate entry into subsite -1. The cleavage efficiency of AkAly30 is enhanced relative to that of mutants lacking either Gly-118 or the Cys-115-Cys-124 disulfide bond. The putative binding model and mutagenesis studies provide a novel substrate recognition mode explaining the polymannuronate specificity of PL-14 alginate lyases.

Development of an Azoreductase-based Reporter System with Synthetic Fluorogenic Substrates.

Enzyme/substrate pairs, such as ß-galactosidase with chromogenic x-gal substrate, are widely used as reporters to monitor biological events, but there is still a requirement for new reporter systems, which may be orthogonal to existing systems. Here, we focused on azoreductase (AzoR). We designed and synthesized a library of azo-rhodamine derivatives as candidate fluorogenic substrates. These derivatives were nonfluorescent, probably due to ultrafast conformational change around the N═N bond after photoexcitation. We found that AzoR-mediated reduction of the azo bond of derivatives bearing an electron-donating group on the azobenzene moiety was followed by nonenzymatic cleavage to afford highly fluorescent 2-methyl-rhodamine green (2-Me RG), which was well retained in cells. We show that the AzoR/compound 9 reporter system can detect azoreductase-expressing live cells at the single cell level.

Crystal Structure of Human Leukocyte Cell-derived Chemotaxin 2 (LECT2) Reveals a Mechanistic Basis of Functional Evolution in a Mammalian Protein with an M23 Metalloendopeptidase Fold.

Human leukocyte cell-derived chemotaxin 2 (LECT2), which is predominantly expressed in the liver, is a multifunctional protein. LECT2 is becoming a potential therapeutic target for several diseases of worldwide concern such as rheumatoid arthritis, hepatocellular carcinoma, and obesity. Here, we present the crystal structure of LECT2, the first mammalian protein whose structure contains an M23 metalloendopeptidase fold. The LECT2 structure adopts a conserved Zn(II) coordination configuration but lacks a proposed catalytic histidine residue, and its potential substrate-binding groove is blocked in the vicinity of the Zn(II)-binding site by an additional intrachain loop at the N terminus. Consistent with these structural features, LECT2 was found to be catalytically inactive as a metalloendopeptidase against various types of peptide sequences, including pentaglycine. In addition, a surface plasmon resonance analysis demonstrated that LECT2 bound to the c-Met receptor with micromolar affinity. These results indicate that LECT2 likely plays its critical roles by acting as a ligand for the corresponding protein receptors rather than as an enzymatically active peptidase. The intrachain loop together with the pseudo-active site groove in LECT2 structure may be specific for interactions between LECT2 and receptors. Our study reveals a mechanistic basis for the functional evolution of a mammalian protein with an M23 metalloendopeptidase fold and potentially broadens the implications for the biological importance of noncatalytic peptidases in the M23 family.

Electron microscopic recording of myosin head power stroke in hydrated myosin filaments.

Muscle contraction results from cyclic attachment and detachment between myosin heads and actin filaments, coupled with ATP hydrolysis. Despite extensive studies, however, the amplitude of myosin head power stroke still remains to be a mystery. Using the gas environmental chamber, we have succeeded in recording the power stroke of position-marked myosin heads in hydrated mixture of actin and myosin filaments in a nearly isometric condition, in which myosin heads do not produce gross myofilament sliding, but only stretch adjacent elastic structures. On application of ATP, individual myosin heads move by ~3.3 nm at the distal region, and by ~2.5 nm at the proximal region of myosin head catalytic domain. After exhaustion of applied ATP, individual myosin heads return towards their initial position. At low ionic strength, the amplitude of myosin head power stroke increases to >4 nm at both distal and proximal regions of myosin heads catalytic domain, being consistent with the report that the force generated by individual myosin heads in muscle fibers is enhanced at low ionic strength. The advantages of the present study over other in vitro motility assay systems, using myosin heads detached from myosin filaments, are discussed.

A secreted protein with plant-specific cysteine-rich motif functions as a mannose-binding lectin that exhibits antifungal activity.

Plants have a variety of mechanisms for defending against plant pathogens and tolerating environmental stresses such as drought and high salinity. Ginkbilobin2 (Gnk2) is a seed storage protein in gymnosperm that possesses antifungal activity and a plant-specific cysteine-rich motif (domain of unknown function26 [DUF26]). The Gnk2-homologous sequence is also observed in an extracellular region of cysteine-rich repeat receptor-like kinases that function in response to biotic and abiotic stresses. Here, we report the lectin-like molecular function of Gnk2 and the structural basis of its monosaccharide recognition. Nuclear magnetic resonance experiments showed that mannan was the only yeast (Saccharomyces cerevisiae) cell wall polysaccharide that interacted with Gnk2. Gnk2 also interacted with mannose, a building block of mannan, with a specificity that was similar to those of mannose-binding legume lectins, by strictly recognizing the configuration of the hydroxy group at the C4 position of the monosaccharide. The crystal structure of Gnk2 in complex with mannose revealed that three residues (asparagine-11, arginine-93, and glutamate-104) recognized mannose by hydrogen bonds, which defined the carbohydrate-binding specificity. These interactions were directly related to the ability of Gnk2 to inhibit the growth of fungi, including the plant pathogenic Fusarium spp., which were disrupted by mutation of arginine-93 or the presence of yeast mannan in the assay system. In addition, Gnk2 did not inhibit the growth of a yeast mutant strain lacking the α1,2-linked mannose moiety. These results provide insights into the molecular basis of the DUF26 protein family.

Definite differences between in vitro actin-myosin sliding and muscle contraction as revealed using antibodies to myosin head.

Muscle contraction results from attachment-detachment cycles between myosin heads extending from myosin filaments and actin filaments. It is generally believed that a myosin head first attaches to actin, undergoes conformational changes to produce force and motion in muscle, and then detaches from actin. Despite extensive studies, the molecular mechanism of myosin head conformational changes still remains to be a matter for debate and speculation. The myosin head consists of catalytic (CAD), converter (CVD) and lever arm (LD) domains. To give information about the role of these domains in the myosin head performance, we have examined the effect of three site-directed antibodies to the myosin head on in vitro ATP-dependent actin-myosin sliding and Ca2+-activated contraction of muscle fibers. Antibody 1, attaching to junctional peptide between 50K and 20K heavy chain segments in the CAD, exhibited appreciable effects neither on in vitro actin-myosin sliding nor muscle fiber contraction. Since antibody 1 covers actin-binding sites of the CAD, one interpretation of this result is that rigor actin-myosin linkage is absent or at most a transient intermediate in physiological actin-myosin cycling. Antibody 2, attaching to reactive lysine residue in the CVD, showed a marked inhibitory effect on in vitro actin-myosin sliding without changing actin-activated myosin head (S1) ATPase activity, while it showed no appreciable effect on muscle contraction. Antibody 3, attaching to two peptides of regulatory light chains in the LD, had no significant effect on in vitro actin-myosin sliding, while it reduced force development in muscle fibers without changing MgATPase activity. The above definite differences in the effect of antibodies 2 and 3 between in vitro actin-myosin sliding and muscle contraction can be explained by difference in experimental conditions; in the former, myosin heads are randomly oriented on a glass surface, while in the latter myosin heads are regularly arranged within filament-lattice structures.

Splenic stromal cells from aged mice produce higher levels of IL-6 compared to young mice.

Inflamm-aging indicates the chronic inflammatory state resulting from increased secretion of proinflammatory cytokines and mediators such as IL-6 in the elderly. Our principle objective was to identify cell types that were affected with aging concerning IL-6 secretion in the murine model. We compared IL-6 production in spleen cells from both young and aged mice and isolated several types of cells from spleen and investigated IL-6 mRNA expression and protein production. IL-6 protein productions in cultured stromal cells from aged mice spleen were significantly high compared to young mice upon LPS stimulation. IL-6 mRNA expression level of freshly isolated stromal cells from aged mice was high compared to young mice. Furthermore, stromal cells of aged mice highly expressed IL-6 mRNA after LPS injection in vivo. These results suggest that stromal cells play a role in producing IL-6 in aged mice and imply that they contribute to the chronic inflammatory condition in the elderly.

Structure of conjugated polyketone reductase from Candida parapsilosis IFO 0708 reveals conformational changes for substrate recognition upon NADPH binding.

Conjugated polyketone reductase C2 (CPR-C2) from Candida parapsilosis IFO 0708, identified as a nicotinamide adenine dinucleotide phosphate (NADPH)-dependent ketopantoyl lactone reductase, belongs to the aldo-keto reductase superfamily. This enzyme reduces ketopantoyl lactone to D-pantoyl lactone in a strictly stereospecific manner. To elucidate the structural basis of the substrate specificity, we determined the crystal structures of the apo CPR-C2 and CPR-C2/NADPH complex at 1.70 and 1.80 Å resolutions, respectively. CPR-C2 adopted a triose-phosphate isomerase barrel fold at the core of the structure. Binding with the cofactor NADPH induced conformational changes in which Thr27 and Lys28 moved 15 and 5.0 Å, respectively, in the close vicinity of the adenosine 2'-phosphate group of NADPH to form hydrogen bonds. Based on the comparison of the CPR-C2/NADPH structure with 3-α-hydroxysteroid dehydrogenase and mutation analyses, we constructed substrate binding models with ketopantoyl lactone, which provided insight into the substrate specificity by the cofactor-induced structure. The results will be useful for the rational design of CPR-C2 mutants targeted for use in the industrial manufacture of ketopantoyl lactone.

Crystal structure of conjugated polyketone reductase (CPR-C1) from Candida parapsilosis IFO 0708 complexed with NADPH.

Conjugated polyketone reductase (CPR-C1) from Candida parapsilosis IFO 0708 is a member of the aldo-keto reductase (AKR) superfamily and reduces ketopantoyl lactone to d-pantoyl lactone in a NADPH-dependent and stereospecific manner. We determined the crystal structure of CPR-C1.NADPH complex at 2.20 Å resolution. CPR-C1 adopted a triose-phosphate isomerase (TIM) barrel fold at the core of the structure in which Thr25 and Lys26 of the GXGTX motif bind uniquely to the adenosine 2'-phosphate group of NADPH. This finding provides a novel structural basis for NADPH binding of the AKR superfamily.