Ubiquitin — protein conjugates

Summary The data available at present indicates there are three distinct functions of ubiquitin, two of which are related to protein conjugation. The first of these has been extensively studied by our laboratory and others interested in nucleosomes and changes in chromatin states. The ubiquitin-histone (Ub-2A, Ub-2B) conjugation reaction now appears to be a very dynamic process. In the deconjugation (lyase) reaction, both the histone 2A and the ubiquitin are left intact and in a form which makes possible ready reconjugation. Accordingly, this may be a mechanism for moment-to-moment control of the genome.The second function in which ubiquitin is conjugated involves proteolytic activity. This activity is correlated with protein turnover. In this process, the ubiquitin-protein conjugate apparently serves as a signal for the protease cleavage of the protein. The released ubiquitin is also intact and is probably available for reconjugation.In the third function, ubiquitin was suggested to serve as hormone. The studies thus far have been carried out primarily on induction of T- and B-lymphocytes, reduction or delay of Coombs' positivity and reduction of spleen weight. The precise physiological role of this reported function is still unclear, particularly because the ubiquitin used was probably not the physiologically active form.     

Well-defined protein–polymer conjugates—synthesis and potential applications

During the last decades, numerous studies have focused on combining the unique catalytic/functional properties and structural characteristics of proteins and enzymes with those of synthetic molecules and macromolecules. The aim of such multidisciplinary studies is to improve the properties of the natural component, combine them with those of the synthetic, and create novel biomaterials in the nanometer scale. The specific coupling of polymers onto the protein structures has proved to be one of the most straightforward and applicable approaches in that sense. In this article, we focus on the synthetic pathways that have or can be utilized to specifically couple proteins to polymers. The different categories of well-defined protein–polymer conjugates and the effect of the polymer on the protein function are discussed. Studies have shown that the specific conjugation of a synthetic polymer to a protein conveys its physico-chemical properties and, therefore, modifies the biodistribution and solubility of the protein, making it in certain cases soluble and active in organic solvents. An overview of the applications derived from such bioconjugates in the pharmaceutical industry, biocatalysis, and supramolecular nanobiotechnology is presented at the final part of the article.     

IAA conjugates与bound IAA

IAA conjugates(或称conjugated IAA)的化学本质是IAA与葡萄糖、肌醇、天冬氨酸等以糖苷键、酯键、酰胺键等共价键结合形成的复合物。是1935年由Cholodny NG从禾本科植物种子的胚乳中发现的(Planta 1935,23:289)。1972年Sembdner等[In Carr DJ(ed). Plant Growth Substances 1970. Berlin: Springer-Verlag, 1972. 143]第一次正式提出将这一过程,即植物激素与醇、氨基酸以及低分子量的糖等小分子以共价键结     

Lectin — digoxigenin conjugates: a new hapten system for glycoconjugate cytochemistry

Summary We report the use of a novel hapten system for lectin cytochemistry. Various lectins conjugated to the steroid hapten digoxigenin (DIG) and monospecific anti-digoxigenin antibodies were applied for the light and electron microscopic detection of glycoconjugates in tissue sections. Both IgG and Fab' anti-DIG antibodies were complexed to particles of colloidal gold and compared to commercially available alkaline phosphatase and horseradish peroxidase conjugated Fab' as general second step reagents. The three different markers performed equally well on paraffin sections whereas the gold-labeled antibodies were superior reagents for semithin and ultrathin sections of Lowicryl K4M embedded tissues. In conjunction with the latter marker, no pretreatment to abolish endogenous enzyme activity was necessary. At the light microscope level, gold signal amplification by the photochemical silver reaction was required. DIG, in contrast to biotin, does not occur in animal tissues thus eliminating the need for blocking reactions prior to lectin incubation. Compared to affinity techniques using glycoprotein-gold complexes as second step reagent the DIG hapten system required smaller amounts of lectins. The staining patterns were indistinguishable from those obtained in other lectin-gold techniques and the specificity of the labeling could be demonstrated in sugar inhibition tests.     

Synthesis of citrate–ciprofloxacin conjugates

Two regioisomeric citrate-functionalized ciprofloxacin conjugates have been synthesized and their antimicrobial activities against a panel of clinically-relevant bacteria have been determined. Cellular uptake mechanisms were investigated using wild-type and ompF deletion strains of Escherichia coli K-12.     

Activation of α-secretase by curcumin-aminoacid conjugates

The extracellular senile plaques observed in Alzheimer's disease (AD) patients are mainly composed of amyloid peptides produced from the β-amyloid precursor protein (βAPP) by β- and γ-secretases. A third non-amyloidogenic α-secretase activity performed by the disintegrins ADAM10 and ADAM17 occurs in the middle of the amyloid-β peptide Aβ and liberates the large sAPPα neuroprotective fragment. Since the activation of α-secretase recently emerged as a promising therapeutic approach to treat AD, the identification of natural compounds able to trigger this cleavage is highly required. Here we describe new curcumin-based modified compounds as α-secretase activators. We established that the aminoacid conjugates curcumin-isoleucine, curcumin-phenylalanine and curcumin-valine promote the constitutive α-secretase activity and increase ADAM10 immunoreactivity. Strickingly, experiments carried out under conditions mimicking the PKC/muscarinic receptor-regulated pathway display different patterns of activation by these compounds. Altogether, our data identified new lead natural compounds for the future development of powerful and stable α-secretase activators and established that some of these molecules are able to discriminate between the constitutive and regulated α-secretase pathways.     

DNA photocleavage by porphyrin–polyamine conjugates

A series of polyamine–porphyrin conjugates bearing two (cis or trans position) or four units of spermidine or spermine was synthesized. We studied the binding of these cationic porphyrins to calf thymus DNA by the means of UV–vis spectroscopy and we investigated their ability to cleave plasmid DNA in the presence of light. DNA binding and DNA photocleavage abilities were found to depend on structural characteristics as (a) the relative positions of the side chains on the porphyrin ring and (b) the nature of the attached side chains (spermidine or spermine). DNA cleavage was also studied in the presence of a singlet oxygen quencher (NaN₃) and in the presence of a hydroxyl radical scavenger (mannitol). Singlet oxygen was the major species responsible for the cleavage of DNA previously observed. Collectively, these data show that polyamine–porphyrin conjugates could be promising phototherapeutic agents.     

Antibody–drug conjugates as novel anti-cancer chemotherapeutics

Over the past couple of decades, antibody–drug conjugates (ADCs) have revolutionized the field of cancer chemotherapy. Unlike conventional treatments that damage healthy tissues upon dose escalation, ADCs utilize monoclonal antibodies (mAbs) to specifically bind tumour-associated target antigens and deliver a highly potent cytotoxic agent. The synergistic combination of mAbs conjugated to small-molecule chemotherapeutics, via a stable linker, has given rise to an extremely efficacious class of anti-cancer drugs with an already large and rapidly growing clinical pipeline. The primary objective of this paper is to review current knowledge and latest developments in the field of ADCs. Upon intravenous administration, ADCs bind to their target antigens and are internalized through receptor-mediated endocytosis. This facilitates the subsequent release of the cytotoxin, which eventually leads to apoptotic cell death of the cancer cell. The three components of ADCs (mAb, linker and cytotoxin) affect the efficacy and toxicity of the conjugate. Optimizing each one, while enhancing the functionality of the ADC as a whole, has been one of the major considerations of ADC design and development. In addition to these, the choice of clinically relevant targets and the position and number of linkages have also been the key determinants of ADC efficacy. The only marketed ADCs, brentuximab vedotin and trastuzumab emtansine (T-DM1), have demonstrated their use against both haematological and solid malignancies respectively. The success of future ADCs relies on improving target selection, increasing cytotoxin potency, developing innovative linkers and overcoming drug resistance. As more research is conducted to tackle these issues, ADCs are likely to become part of the future of targeted cancer therapeutics.     

Dead end for auxin conjugates in Physcomitrella?

For proper development of plants auxin levels need to be tightly controlled. For this, several routes have evolved and it is plausible that different organisms use these differently. To determine whether members of the family of GH3 proteins, which partially act as auxin conjugate synthetases in Arabidopsis thaliana, have similar roles in the moss Physcomitrella patens, we have investigated the in vitro activity of the two GH3 members in moss. We showed that both proteins can form amino acid conjugates with indole-3-acetic acid (IAA) but also with jasmonic acid (JA). Confirming these findings, single and double knockout-mutants showed lower levels of IAA conjugates than wild type. We discuss the results in light of the possible functions of IAA conjugate formation in lower land plants.Key words: Arabidopsis thaliana, auxin metabolism, jasmonic acid, GH3 genes, moss, Physcomitrella patensAuxins play diverse roles in many aspects of plant growth and development. Their activity is relying on the correct concentration in a given tissue and developmental stage.¹ If higher levels of indole-3-acetic acid (IAA) are present, the hormone can also have an inhibitory effect on growth processes.² Therefore, the tight control of IAA concentrations is absolutely necessary. To this end plants have evolved different mechanisms.³ First, biosynthesis is contributing to increasing IAA concentrations, mostly in young tissues such as meristems. Second, IAA can be transported in a polar way, depending on transport molecules, from cell to cell, away from the site of synthesis, thereby forming an auxin gradient along the plant axis. Third, IAA can be degraded, and fourth, IAA can be reversibly inactivated by conjugation to small molecules such as amino acids or sugars, but also be linked to larger molecules such as peptides or proteins.⁴ The inactive IAA conjugates can be hydrolyzed to yield free (i.e., active) IAA if needed. In higher plants the levels of free IAA constitutes between 5 and 20% depending on the tissue or age of the plant, whereas the conjugated form constitutes the major part.⁴ However, it is not yet clear in which way auxin homeostasis has evolved. The hypothesis that auxin has to be present during the evolution of a body plan has been tested by using different lower land plants which were compared in their mechanism to control auxin homeostasis. In algae, e.g., charophytes, the major metabolic way of controlling IAA is via biosynthesis. In bryophytes, the formation of IAA conjugates has been shown, although the amount was lower than for example in seed plants such as Arabidopsis thaliana.^5,6 Since the molecular biology of auxin homeostasis in Arabidopsis is most advanced, we will use this model plant to compare the knowledge on seed plants with that in the moss Physcomitrella patens. The recent publication of the Physcomitrella genome⁷ gives the possibility to investigate components of the machinery controlling IAA levels in a lower land plant.In general, there seem to be high levels of redundancy involved in the pathways leading to decrease or increase of IAA, respectively. In Figure 1 we compare the current knowledge about genes related to IAA concentrations in Physcomitrella with Arabidopsis. While in Arabidopsis many different biosynthetic routes leading to IAA were identified,⁸ in the Physcomitrella genome homologs of the YUCCA genes have been detected.⁷ The presence of auxin conjugate synthetases has been experimentally verified in the moss (see below) and additional evidence for ester conjugate synthesis comes from sequence homology to UDP-glucosyl transferases.⁷ There is also the possibility of degradation of either IAA or an amino acid conjugate with IAA^9,10 as discussed below.Open in a separate windowFigure 1Comparison of possibilities to regulate auxin homeostasis in Physcomitrella (solid lines) and Arabidopsis (dotted lines). Biosynthesis—AO, aldehyde oxidase; AMI1, amidase; CYP, cytochrome P450; NIT, nitrilase; TAA1, tryptophan aminotransferase; YUCCA, flavin monooxygenase; transport—AUX/LAX, auxin influx facilitator family; PIN, auxin efflux carrier family; PGP, ABC transporter type auxin efflux carrier family; conjugation/hydrolysis—UGT, UDP-glucosyl transferase; GH3, auxin conjugate synthetase family; ILR/IAR, auxin conjugate hydrolase family; Ox-IAA, oxindole-3-acetic acid; Ox-IAAsp, oxindole-3-aspartic acid.So far our work has focussed on the characterization of two members of the so called GH3 family, of which several from Arabidopsis can form conjugates of IAA with a variety of amino acids.¹¹ While 19 members of this family have been described in Arabidopsis, only two are present in Physcomitrella.¹² The Arabidopsis family clusters in three groups: group I containing the jasmonic acid conjugate synthetase JAR1 and a few others with as yet unkown function, group II the auxin conjugate synthetases, and group III with mostly as yet uncharacterized members.^11,13 Sequence similarity of the GH3 genes from Physcomitrella showed that both cluster within the JAR1 group.¹² Therefore, we analyzed the enzymatic activity of the two Physcomitrella GH3 proteins (PpGH3-1 and PpGH3-2) in vitro¹⁴ and found that both were active on jasmonic acid and a variety of different amino acids, whereas PpGH3-2 was active mostly with IAA. PpGH3-1 showed only weak activity with IAA and only two amino acids. For this reason, it could be assumed that the two Physcomitrella genes evolved by gene duplication, from which the initial activities would be for IAA and jasmonic acid. One of these genes might have evolved into a jasmonate conjugate synthetase (maybe AtJAR1),¹³ thereby loosing its activity on IAA. The second may have given rise to the auxin conjugate synthetase family in Arabidopsis,¹¹ but the conjugate synthetases of Physcomitrella have still activity with both hormones. Interestingly, there is no evidence as yet that jasmonic acid itself has a role during Physcomitrella development, although a possible function of JA-conjugates has not been closely investigated. Since in Arabidopsis the JA conjugate with isoleucine is the active compound to be recognized by the COI1 receptor protein,¹⁵ it could be the case that JA itself has no effect in Physcomitrella. However, in our growth experiments a small growth promoting effect of JA, independently on the presence of GH3 genes was found. Similar observations were made with gibberellins in Physcomitrella.¹⁶Further characterization of single and double KO mutants in each of the PpGH3 genes has led to the hypothesis that GH3 proteins are indeed involved in regulating the auxin homeostasis in Physcomitrella.¹⁴ Both single KO mutants were more sensitive to increasing IAA concentrations in the medium than the wild type. Furthermore, the levels of free IAA were higher and the levels of conjugated IAA concomitantly dropped. A double KO mutant had almost no IAA conjugates when compared to the wild type. However, this mutant was still able to synthesize ester conjugates with IAA. Interestingly, the role of GH3 proteins in auxin conjugation seemed to be only important in the gametophore stage, whereas protonema cultures of GH3 KO mutants did not show any changes in auxin homeostasis. Therefore, we hypothesize that the role of GH3 proteins is dependent on a certain developmental stage of the moss. Additionally, we propose other detoxification mechanisms for example, export or degradation, in protonema.In higher plants the ester conjugate formation of IAA has been shown to be dependent on UDP-glucosyl transferases (AtUGT84B1 for Arabidopsis¹⁷ and ZmIAGLU for maize¹⁸). In the genome of Physcomitrella we could detect candidate sequence(s) for these genes, indicating that Physcomitrella has indeed the potential to synthesise the ester conjugates found in the gametophores in addition to amide conjugates. However, in the Physcomitrella genome, no homolog for an auxin conjugate hydrolase was found. In higher plants, auxin conjugate hydrolysis is thought to contribute to free IAA and depending on the plant species, large gene families with overlapping but distinct substrate preferences for individual amino acid conjugates with IAA are present.^19,20 Since this is not the case for Physcomitrella, one has to ask the question whether the conjugation of auxin is a one-way road for inactivation of excess auxin and whether auxin conjugate hydrolysis has evolved later during plant evolution.In the Selaginella moellendorffii genome (http://genome.jgi-psf.org/Selmo1/Selmo1.home.html), an auxin conjugate hydrolase sequence related to higher plant ones, has been found based on homology searches, but the completion of the genome has to be awaited to draw final conclusions. Likewise, it is not clear, if this effect is specific for Physcomitrella, or found in bryophytes in general. Therefore, additional sequenced bryophyte genomes are needed.²¹Since in Arabidopsis the degradation of the IAA-Aspartate conjugate to Ox-IAA-Asp (see Fig. 1) has been described,^9,10 a similar scenario could be suggested to occur in Physcomitrella with the amino acid conjugates formed. Alternatively, the hydrolysis of IAA conjugates by members of the M20 dipeptidase family can be envisioned. However, this would need the activity of enzymes with very low sequence conservation to auxin conjugate hydrolases. These questions will be addressed in future research by studying the metabolism of IAA and IAA conjugates of Physcomitrella in more detail.     

Probing linker design in citric acid–ciprofloxacin conjugates

A series of structurally related citric acid–ciprofloxacin conjugates was synthesised to investigate the influence of the linker between citric acid and ciprofloxacin on antibacterial activities. Minimum inhibitory concentrations (MICs) were determined against a panel of reference strains and clinical isolates of bacteria associated with infection in humans and correlated with the DNA gyrase inhibitory activity. The observed trend was rationalised by computational modelling.     

Synthesis and antibacterial activity of catecholate–ciprofloxacin conjugates

The development of an efficient route to obtain artificial siderophore–antibiotic conjugates active against Gram-negative bacteria is crucial. Herein, a practical access to triscatecholate enterobactin analogues linked to the ciprofloxacin along with their antibacterial evaluation are described. Two series of conjugates were obtained with and without a piperazine linker which is known to improve the pharmacokinetics profile of a drug. A monocatecholate–ciprofloxacin conjugate was also synthesized and evaluated. The antibacterial activities against Pseudomonas aeruginosa for some conjugates are related to the iron concentration in the culture medium and seem to depend on the bacterial iron uptake systems.     

Recognition of HIV-TAR RNA using neomycin–benzimidazole conjugates

Synthesis of a novel class of compounds and their biophysical studies with TAR-RNA are presented. The synthesis of these compounds was achieved by conjugating neomycin, an aminoglycoside, with benzimidazoles modeled from a B-DNA minor groove binder, Hoechst 33258. The neomycin–benzimidazole conjugates have varying linkers that connect the benzimidazole and neomycin units. The linkers of varying length (5–23 atoms) in these conjugates contain one to three triazole units. The UV thermal denaturation experiments showed that the conjugates resulted in greater stabilization of the TAR-RNA than either neomycin or benzimidazole used in the synthesis of conjugates. These results were corroborated by the FID displacement and tat-TAR inhibition assays. The binding of ligands to the TAR-RNA is affected by the length and composition of the linker. Our results show that increasing the number of triazole groups and the linker length in these compounds have diminishing effect on the binding to TAR-RNA. Compounds that have shorter linker length and fewer triazole units in the linker displayed increased affinity towards the TAR RNA.     

Kojic acid–amino acid conjugates as tyrosinase inhibitors

Kojic acid (KA), a well known tyrosinase inhibitor, has insufficient inhibitory activity and stability. We modified KA with amino acids and screened their tyrosinase inhibitory activity. Among them, kojic acid–phenylalanine amide (KA-F-NH₂) showed the strongest inhibitory activity, which was maintained for over 3 months at 50 °C, and acted as a noncompetitive inhibitor as determined by kinetic analysis. It also exhibited dopachrome reducing activity. We also propose a new tyrosinase inhibition mechanism based on the docking simulation data.     

Laccase-catalysed protein–flavonoid conjugates for flax fibre modification

The introduction of flavonoid compounds into proteins can improve the natural properties of proteins, being promising products which essentially require antioxidant property. The oxidative conjugation of protein–flavonoids was processed by laccase catalysis resulting in the synthesis of biologically functional polymers. The new reaction products were detected in terms of sodium dodecyl sulfate polyacrylamide gel electrophoresis and matrix-assisted laser desorption/ionisation-time of flight mass spectra, showing a greater molecular weight formation. Their characterisations were further carried out in terms of UV–Vis spectroscopy, photon correlation spectroscopy, differential scanning calorimetry and Fourier transform infrared (FT-IR) spectroscopy analysis. In addition, their application of protein–flavonoid conjugates onto flax fibres was exploited to supplement a suitable microorganism environment of protein-possessed fibres. The anchoring of conjugates onto cationised fibres was successfully performed by ionic interaction with negatively charged proteins. The level of anchoring efficiency was quantified in terms of measuring colour strength (k/s) and fluorescence microscopy analysis. The conjugates onto fibres presented acceptable durability in terms of washing resistance and the surface became hydrophilic when α-casein–catechin was applied (lower contact angle 48°). By the anchoring of protein–flavonoid conjugates onto flax fibres, the final products with new colour generation and antioxidant activity (>93%) were obtained.     

Synthesis and preliminary evaluation steroidal antiestrogen–geldanamycin conjugates

Three novel steroidal antiestrogen–geldanamycin conjugates were prepared using a convergent strategy. The antiestrogenic component utilized the 11β-(4-functionalized-oxyphenyl) estradiol scaffold, while the geldanamycin component was derived by replacement of the 17-methoxy group with an appropriately functionalized amine. Ligation was achieved in high yield using azide alkyne cyclization reactions. Evaluation of the products against two breast cancer cell lines indicated that the conjugates retained significant antiproliferative activity.     

Synthesis and radical scavenging activity of phenol–imidazole conjugates

Novel hydroxylated benzylideneamino imidazole derivatives were synthesized and their radical scavenging activity was assessed against DPPH and hydroxyl radicals. In the DPPH assay, most of the synthesized compounds showed an IC₅₀ in the range 3.2 μM ⩽ IC₅₀ ⩽ 8.4 μM, lower than the reference compound trolox (IC₅₀ = 9.5 μM) or the parent aldehydes (5.4 μM ⩽ IC₅₀ ⩽ 11.6 μM). The activity depends mainly on the phenolic subunit (number and position of the hydroxyl groups) and the extent of conjugation with the imidazole ring. In the deoxyribose assay, all the compounds, including parent imidazoles and aldehydes, showed high activity against the hydroxyl radical and the ability to chelate iron ions. At 5 μM concentration, the compounds protected the deoxyribose from degradation by hydroxyl radical between 62% and 38%.     

Synthesis and biological evaluation of novel biotin–iminoalditol conjugates

Biotin–iminosugar conjugates of different configuration such as d-gluco, d-galacto, l-ido as well as a furanoid representative in the d-manno configuration have been synthesised and exhibit powerful inhibition of β-glucosidase from Agrobacterium sp. with K_i values in the range of the respective parent compounds. Such molecular probes have potential for activity-based protein profiling taking advantage of the biotin–(strept)avidin interaction.     

Design and biological activity of β-sheet breaker peptide conjugates

The sequence LPFFD (iAβ₅) prevents amyloid-β peptide (Aβ) fibrillogenesis and neurotoxicity, hallmarks of Alzheimer’s disease (AD), as previously demonstrated. In this study iAβ₅ was covalently linked to poly(ethylene glycol) (PEG) and the activity of conjugates was assessed and compared to the activity of the peptide alone by in vitro studies. The conjugates were characterized by MALDI-TOF. Competition binding assays established that conjugates retained the ability to bind Aβ with similar strength as iAβ₅. Transmission electron microscopy analysis showed that iAβ₅ conjugates inhibited amyloid fibril formation, which is in agreement with binding properties observed for the conjugates towards Aβ. The conjugates were also able to prevent amyloid-induced cell death, as evaluated by activation of caspase 3. These results demonstrated that the biological activity of iAβ₅ is not affected by the pegylation process.     

Enzymatic activity and thermal stability of PEG-α-chymotrypsin conjugates

α-Chymotrypsin was chemically modified with methoxypoly(ethylene glycol) (PEG) of different molecular weights (700, 2,000, and 5,000 Da) and the amount of polymer attached to the enzyme was varied systematically from 1 to 9 PEG molecules per enzyme molecule. Upon PEG conjugation, enzyme catalytic turnover (k _cat) decreased by 50% and substrate affinity was lowered as evidenced by an increase in the K _M from 0.05 to 0.19 mM. These effects were dependent on the amount of PEG bound to the enzyme but were independent of the PEG size. In contrast, stabilization toward thermal inactivation depended on the PEG molecular weight with conjugates with the larger PEGs being more stable.     

Synthesis of guanidino sugar conjugates as GlcβArg analogs

The β-glucosyl linkage to the guanidine group of arginine (Arg) is found in amylogenin, a glycoprotein from sweet corn. Such a linkage is formed by a rare N-glycosylation of proteins. Synthesis of analogs of the unusual N-glycosidic linkage (GlcβArg) with an acetamido or triazole spacer between the glycosyl residue and the guanidine moiety was accomplished by the reaction of fully acetylated sugar unit containing a free amino group with bis-Boc-thiourea. Synthesis of N-glucosylarginine with an amido linker was also achieved during the present study. This methodology was also extended to the synthesis of cationic glucolipid.