Contour length and refolding rate of a small protein controlled by engineered disulfide bonds

The introduction of disulfide bonds into proteins creates additional mechanical barriers and limits the unfolded contour length (i.e., the maximal extension) measured by single-molecule force spectroscopy. Here, we engineer single disulfide bonds into four different locations of the human cardiac titin module (I27) to control the contour length while keeping the distance to the transition state unchanged. This enables the study of several biologically important parameters. First, we are able to precisely determine the end-to-end length of the transition state before unfolding (53 Angstrom), which is longer than the end-to-end length of the protein obtained from NMR spectroscopy (43 Angstrom). Second, the measured contour length per amino acid from five different methods (4.0 +/- 0.2 Angstrom) is longer than the end-to-end length obtained from the crystal structure (3.6 Angstrom). Our measurement of the contour length takes into account all the internal degrees of freedom of the polypeptide chain, whereas crystallography measures the end-to-end length within the "frozen" protein structure. Furthermore, the control of contour length and therefore the number of amino acids unraveled before reaching the disulfide bond (n) facilitates the test of the chain length dependence on the folding time (tau(F)). We find that both a power law scaling tau(F) lambda n(lambda) with lambda = 4.4, and an exponential scaling with n(0.6) fit the data range, in support of different protein-folding scenarios.     

Complex, unusual conformational changes in kidney betaine aldehyde dehydrogenase suggested by chemical modification with disulfiram

The NAD⁺-dependent animal betaine aldehyde dehydrogenases participate in the biosynthesis of glycine betaine and carnitine, as well as in polyamines catabolism. We studied the kinetics of inactivation of the porcine kidney enzyme (pkBADH) by the drug disulfiram, a thiol-reagent, with the double aim of exploring the enzyme dynamics and investigating whether it could be an in vivo target of disulfiram. Both inactivation by disulfiram and reactivation by reductants were biphasic processes with equal limiting amplitudes. Under certain conditions half of the enzyme activity became resistant to disulfiram inactivation. NAD⁺ protected almost 100% at 10 μM but only 50% at 5 mM, and vice versa if the enzyme was pre-incubated with NAD⁺ before the chemical modification. NADH, betaine aldehyde, and glycine betaine also afforded greater protection after pre-incubation with the enzyme than without pre-incubation. Together, these findings suggest two kinds of active sites in this seemingly homotetrameric enzyme, and complex, unusual ligand-induced conformational changes. In addition, they indicate that, in vivo, pkBADH is most likely protected against disulfiram inactivation.     

Phenylpyruvate tautomerase activity of trans-3-chloroacrylic acid dehalogenase: evidence for an enol intermediate in the dehalogenase reaction?

The enzymatic conversion of cis- or trans-3-chloroacrylic acid to malonate semialdehyde is a key step in the bacterial degradation of the nematocide 1,3-dichloropropene. Two mechanisms have been proposed for the isomer-specific hydrolytic dehalogenases, cis- and trans-3-chloroacrylic acid dehalogenase (cis-CaaD and CaaD, respectively), responsible for this step. In one mechanism, the enol isomer of malonate semialdehyde is produced by the alpha,beta-elimination of HCl from an initial halohydrin species. Phenylenolpyruvate has now been found to be a substrate for CaaD with a kcat/Km value that approaches the one determined for the CaaD reaction using trans-3-chloroacrylate. Moreover, the reaction is stereoselective, generating the 3S isomer of [3-2H]phenylpyruvate in a 1.8:1 ratio in 2H2O. These two observations and a kinetic analysis of active site mutants of CaaD suggest that the active site of CaaD is responsible for the phenylpyruvate tautomerase (PPT) activity. The activity is a striking example of catalytic promiscuity and could reflect the presence of an enol intermediate in CaaD-mediated dehalogenation of trans-3-chloroacrylate. CaaD and cis-CaaD represent different families in the tautomerase superfamily, a group of structurally homologous proteins characterized by a core beta-alpha-beta building block and a catalytic Pro-1. The eukaryotic immunoregulatory protein known as macrophage migration inhibitory factor (MIF), also a tautomerase superfamily member, exhibits a PPT activity, but the biological relevance is unknown. In addition to the mechanistic implications, these results establish a functional link between CaaD and the superfamily tautomerases, highlight the catalytic and binding promiscuity of the beta-alpha-beta scaffold, and suggest that the PPT activity of MIF could reflect a partial reaction in an unknown MIF-catalyzed reaction.     

Doxorubicin-induced cardiotoxicity: direct correlation of cardiac fibroblast and H9c2 cell survival and aconitase activity with heat shock protein 27

The use of doxorubicin (Dox) and its derivatives as chemotherapeutic drugs to treat patients with cancer causes dilated cardiomyopathy and congestive heart failure due to Dox-induced cardiotoxicity. In this work, using heat shock factor-1 wild-type (HSF-1(+/+)) and HSF-1 knockout (HSF-1(-/-)) mouse fibroblasts and embryonic rat heart-derived cardiac H9c2 cells, we show that the magnitude of protection from Dox-induced toxicity directly correlates with the level of the heat shock protein 27 (HSP27). Western blot analysis of normal and heat-shocked cells showed the maximum expression of HSP27 in heat-shocked cardiac H9c2 cells and no HSP27 in HSF-1(-/-) cells (normal or heat-shocked). Correspondingly, the cell viability, measured [with (3,4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay] after treatment with various concentrations of Dox, was the highest in heat-shocked H9c2 cells and the lowest in HSF-1(-/-) cells. Depleting HSP27 in cardiac H9c2 cells by small interfering (si)RNA also reduced the viability against Dox, confirming that HSP27 does protect cardiac cells against the Dox-induced toxicity. The cells that have lower HSP27 levels such as HSF-1(-/-), were found to be more susceptible for aconitase inactivation. Based on these results we propose a novel mechanism that HSP27 plays an important role in protecting aconitase from Dox-generated O(2)*(-), by increasing SOD activity. Such a protection of aconitase by HSP27 eliminates the catalytic recycling of aconitase released Fe(II) and its deleterious effects in cardiac cells.     

An analog of 1α,25-dihydroxy-19-norvitamin D3 with the 1α-hydroxy group fixed in the axial position lacks biological activity in vitro

The relationship between the A-ring chair conformation of vitamin D compounds and their ability to bind the vitamin D receptor (VDR) has long attracted the attention of many researchers. It was established that in the crystalline complexes of hVDRmt with the natural hormone, 1α,25-dihydroxyvitamin D₃ (1), and its side-chain analogs the vitamins exist in β-chair form with an equatorial orientation of 1α-OH. However, with all these ligands the interconversion between both A-ring forms would be possible in solution. In an attempt to verify the conformation of vitamin D compounds required for binding the VDR we prepared analog 4, characterized by the presence of an axial 1α-hydroxy group. Since the additional ring connecting 3β-oxygen and C-2 prevents A-ring conformational flexibility, the synthesized vitamin 4 can exist exclusively in the α-chair form. The geometrical isomer 5 with a free 3β-OH group was also obtained. The analog 5 binds very poorly to VDR, whereas the vitamin 4 is practically devoid of binding ability. Both compounds also show very low HL-60-differentiating activity. When tested in vivo in mice the analogs 4 and 5 exhibit significant calcemic responses with analog 4 showing more activity than analog 5.     

Study of recombinant antibody fragments and PAI-1 complexes combining protein-protein docking and results from site-directed mutagenesis

Elevated plasma levels of plasminogen activator inhibitor-1 (PAI-1) have been correlated with cardiovascular diseases such as myocardial infarction and venous thrombosis. PAI-1 has also been shown to play an important role in tumor development, diabetes, and obesitas. Monoclonal antibodies MA-8H9D4 and MA-56A7C10, and their single-chain variable fragments (scFv), exhibit PAI-1-neutralizing properties. In this study, a rigid-body docking approach is used to predict the binding geometry of two distinct conformations of PAI-1 (active and latent) in complex with these antibody fragments. Resulting models were initially refined by using the dead-end elimination algorithm. Different filtering criteria based on the mutagenesis studies and structural considerations were applied to select the final models. These were refined by using the slow-cooling torsion-angle dynamic annealing protocol. The docked structures reveal the respective epitopes and paratopes and their potential interactions. This study provides crucial information that is necessary for the rational development of low-molecular weight PAI-1 inhibitors.     

Artificial neural networks for qualitative and quantitative analysis of target proteins with polymerized liposome vesicles

We investigate the feasibility of using the luminescence response of polymerized liposomes incorporating ethylenediaminetetraacetate europium(III) (EDTA-Eu(3+)) for monitoring protein concentrations in aqueous media. Quantitative analysis is based on the linear relationship between the luminescence enhancement of the lanthanide ion and protein concentration. Analytical figures of merit are presented for carbonic anhydrase, human serum albumin, gamma-globulins, and thermolysin. Qualitative analysis is based on the luminescence lifetime of the liposome sensor. This parameter, which follows well-behaved single exponential decays and provides characteristic values for each of the four studied proteins, demonstrates the selective potential for protein identification. Then partial least squares-1 and artificial neural networks are compared toward the quantitative and qualitative analysis of human serum albumin and carbonic anhydrase in binary mixtures without previous separation at the concentration levels found in aqueous humor.     

Low Sugar Is Not Always Good: Impact of Specific O-Glycan Defects on Tip Growth in Arabidopsis

Mutants of the O-glycosylation pathway of extensins as well as molecular dynamics simulations uncover the effects of the O-glycosylation machinery on root hair tip growth.The hydroxyproline-rich O-glycoproteins (HRGPs) comprise several groups of O-glycoproteins, including extensins (EXTs), ultimately secreted into plant cell walls. The latter are shaped by several posttranslational modifications, mainly hydroxylation of Pro residues into Hyp and further O-glycosylation on Hyp and Ser (Supplemental Fig. S1A). EXTs contain several Ser-(Hyp)₄ repeats, usually O-glycosylated with chains of up to four or five linear arabinosyl units on each Hyp (Velasquez et al., 2011; Ogawa-Ohnishi et al., 2013) and monogalactosylated on Ser residues (Saito et al., 2014). O-Glycosylated Ser-(Hyp)₄ repeats are not only present in EXTs but can potentially decorate several other EXT-like chimeras and hybrid EXT glycoproteins that contain other domains, such as arabinogalactan protein-EXTs, Pro-rich protein-EXTs, Leu-rich repeat-EXTs, Pro-rich kinases, and formins with an extracellular EXT domain, etc. In addition, Hyp-O-arabinosylation also occurs in single Hyp units in the small secreted glycopeptide hormones (e.g. CLAVATA3 [CLV3]) with up to three arabinosyl units (Ohyama et al., 2009; Matsubayashi, 2010; Shinohara and Matsubayashi, 2013). In this context, three groups of arabinosyltransferases (AraTs), hydroxyproline O-arabinosyltransferase1 (HPAT1) to HPAT3 (classified as GT8 in the Carbohydrate Active Enzymes database), Reduced Residual Arabinose1 (RRA1) to RRA3, and Xyloglucanase113 (XEG113; GT77 family), have recently been implicated in the sequential addition of the innermost three l-arabinosyl residues (Egelund et al., 2007; Ogawa-Ohnishi et al., 2013; Supplemental Table S1). In addition, one novel peptidyl-Ser galactosyltransferase named SERGT1 has been reported to add a single α-galactopyranose residue to each Ser residue in Ser-(Hyp)₄ motifs of EXTs, thus belonging to the GT96 family within the Carbohydrate Active Enzymes database (Supplemental Table S1). Finally, glycosylated EXTs are possibly cross-linked by putative type III peroxidases at the Tyr residues, forming EXT linkages (Cannon et al., 2008) able to build a three-dimensional network likely to interact with other cell wall components like pectins (Cannon et al., 2008). EXT assembly into a putative glycoprotein network seems to be crucial for cell expansion of root hair, and several EXT and EXT-related mutants (e.g. ext6-7, ext10-12, and Leu-rich repeat extensin1, etc.) were previously isolated with abnormal root hair cell expansion phenotypes (Ringli, 2010; Velasquez et al., 2011).Here, by using mutants of several known enzymes of the O-glycosylation pathway of HRGPs, we addressed to what extent each specific defect on the O-glycosylation machinery impacts root hair tip growth. In addition, we refer only to Hyp-O-arabinosylation and Ser-O-galactosylation modifications of EXT and EXT-related proteins, while we have excluded Hyp O-(arabino)galactosylation, commonly present in other types of HRGP like arabinogalactan proteins, from our analysis. Finally, by molecular dynamic simulations, we propose a possible model to explore how these two specific types of O-glycan defects would affect EXT self-assembly and, ultimately, their impact on the polarized cell expansion. We use a classical EXT repetitive sequence to begin to explore how O-glycosylation might affect glycoprotein conformation and possible self-interactions in the context of polarized growth, but we are aware of the complexity and diversity of EXT and EXT-related proteins that offers several other possible scenarios.