Machha, Venkata R.

Abstract The A1 domain in Von Willebrand Factor (VWF) initiates coagulation through binding to platelet glycoprotein GPIbα receptors. Von Willebrand Disease (VWD)-Mutations in A1 that either impair (type 2M) or enhance (type 2B) platelet adhesion to VWF can locally destabilize and even misfold the domain. We leveraged misfolding in the gain-of-function type 2B VWD phenotype as a target, distinct from the normal conformation. Two nuclease-resistant 2′-fluoropyrimidine RNA aptamers were selected to discriminate normal A1 domains from a type 2B V1314D A1 variant in a glycosylated A1A2A3 tri-domain VWF-fragment. Two aptamers, W9 and V1, were isolated that selectively recognize, bind, and inhibit the A1–GPIbα interaction with WT A1A2A3 and V1314D A1A2A3, respectively. These aptamers were tested against their respective recombinant targets, plasma VWF, VWF concentrates, and patient plasma with the heterozygous type 2B VWD R1306W variant using clinical assays, surface plasmon resonance and inhibition assays of platelet adhesion to recombinant A1 and A1A2A3 domains under shear stress. The specificity of W9 and V1 aptamers confirms that pathological conformations of VWD Type 2B proteins are different from normal VWF. The availability of aptamers that distinguish normal plasma-derived VWF from VWD suggests potential applicability in clinical diagnosis of severe gain-of-function phenotypes.

Significance This study demonstrates that underexpression of succinate dehydrogenase (SDH) subunits resulting in accumulation of oncogenic succinate is a common, adverse, epigenetic modulating feature in clear cell renal cell carcinoma (ccRCC), during pathogenesis and progression. The study sheds light on the mechanisms of down-regulation of SDH subunits in ccRCC and deciphers the consequent oncogenic effects. It shows that functional SDH deficiency is a common feature of ccRCC (∼80% of all kidney cancers), and not just limited to the 0.05 to 0.5% of kidney cancers with germline SDH mutations.

AbstractProtein phase diagrams have a unique potential to identify the presence of additional thermodynamic states even when non‐2‐state character is not readily apparent from the experimental observables used to follow protein unfolding transitions. Two‐state analysis of the von Willebrand factor A3 domain has previously revealed a discrepancy in the calorimetric enthalpy obtained from thermal unfolding transitions as compared with Gibbs‐Helmholtz analysis of free energies obtained from the Linear Extrapolation Method (Tischer and Auton, Prot Sci 2013; 22(9):1147‐60). We resolve this thermodynamic conundrum using a Clausius‐Clapeyron analysis of the urea‐temperature phase diagram that defines how and the urea m‐value interconvert through the slope of cm versus T, . This relationship permits the calculation of at low temperature from m‐values obtained through iso‐thermal urea denaturation and high temperature m‐values from obtained through iso‐urea thermal denaturation. Application of this equation uncovers sigmoid transitions in both cooperativity parameters as temperature is increased. Such residual thermal cooperativity of and the m‐value confirms the presence of an additional state which is verified to result from a cooperative phase transition between urea‐expanded and thermally‐compact denatured states. Comparison of the equilibria between expanded and compact denatured ensembles of disulfide‐intact and carboxyamidated A3 domains reveals that introducing a single disulfide crosslink does not affect the presence of the additional denatured state. It does, however, make a small thermodynamically favorable free energy (∼–13 ± 1 kJ/mol) contribution to the cooperative denatured state collapse transition as temperature is raised and urea concentration is lowered. The thermodynamics of this “cooperative collapse” of the denatured state retain significant compensations between the enthalpy and entropy contributions to the overall free energy.

Isothermal titration calorimetry (ITC) and circular dichroism (CD) were used to study the thermodynamics of RPC·G‐quadruplex DNA (G4) complex formation. The ruthenium polypyridyl complexes (RPCs) were [Ru(phen)3]2+ (12+), [Ru(phen)2(dppz)]2+ (22+), [Ru(phen)2(tatpp)]2+ (32+), and [Ru(phen)2(tatpp)(phen)2Ru]4+ (44+), and target DNAs were c‐MYC NHE‐III1 promoter sequence mutants forming 1‐2‐1 and 1‐6‐1 G‐quadruplexes. Formation of the 2:1 RPC·G4 complexes is characterized by entropy driven RPC binding to the top and bottom of G‐tetrad faces. 12+ appears to bind very weakly or not at all to G4 DNA. 22+ having a dipyridophenazine group to stack on the top and bottom of the G4 core, exhibits an average Ka = 6.7 × 104 m–1. 32+, with a larger G4 interactive tetraazatetrapyridopentacene group, binds with significantly higher affinity, Ka = 1.1 × 106 m–1. 22+ and 32+ appear to bind independently of G4 folding topology and RPC conformation. The thermograms for the titration of G4 DNA with rac‐44+ are characterized by two binding modes exhibiting higher and lower affinity (Ka,1 = 3.6 × 107 m–1 and Ka,2 = 3.2 × 105 m–1). The two binding modes are attributed to preferential binding of one of the 44+ enantiomers (e.g. ΛΛ) over the other isomers (e.g. ΔΔ or ΔΛ). Tighter binding of the preferred 44+ enantiomer, in comparison to 32+, is due to additional favorable entropy for locating a second [(phen)2Ru–]2+ moiety in a G4 groove. Weaker binding of the disfavored 44+ isomers must be due to a poorer fit of these isomers with the G4 faces.

AbstractInvited for the cover of this issue is the group of Edwin Lewis, Mississippi State University, in collaboration with Frederick MacDonnell, University of Texas at Arlington, USA. The cover image shows Ru(phen)2(tatpp)]2+ binding to DNA.

The ruthenium(II) polypyridyl complexes (RPCs), [(phen)2Ru(tatpp)]Cl2 (3Cl2) and [(phen)2Ru (tatpp)Ru(phen)2]Cl4 (4Cl4), containing the large planar and redox‐active tetraazatetrapyrido‐pentacene (tatpp) ligand, cleave DNA in the presence of reducing agents in cell‐free assays and show significant tumor regression in mouse tumor models with human non‐small cell lung carcinoma xenografts. ITC, CD, and ESI‐MS techniques were used to study the thermodynamics of RPC·DNA complex formation and the complex structure for binding three different RPCs to duplex DNA. The specific RPCs were [Ru(phen)3]2+ (12+), [Ru(phen)2(dppz)]2+ (22+), and [Ru(phen)2(tatpp)]2+ (32+). We examined the enatiomerically pure Δ‐RPC and Λ‐RPC isomers as well as the racemic mixture in terms of their binding to B‐DNA. B‐DNA binding of the three RPCs is characterized by a combination of groove binding (including electrostatic effects) and intercalation, with the thermodynamics being very similar for binding both the enantiomerically pure compounds and the racemic mixture. 12+ is the weakest DNA binder, exhibiting a Ka = 1.3 × 104 m–1 while 22+ binds with significantly higher affinity, Ka,1 = 1.4 × 106 m–1, and 32+ exhibits the tightest binding, Ka = 4.7 × 106 m–1. The trend is to increase the affinity as longer bridging ligands engage in additional π‐bonding with the DNA base pairs via an intercalative binding mode. A second binding mode, two orders of magnitude weaker was also seen for 22+. The ITC values for binding the racemic mixtures exhibit ΔGs ranging from –5.6 to –9.1 kcal mol–1 (12+·DNA and 32+·DNA respectively), while the ITC values for ΔH range from +4.9 to –5.0 kcal mol–1 (32+·DNA and mode 2 binding for 22+·DNA respectively). All of the primary complexes exhibit very negative values for –TΔS ranging from –7.3 to –14.0 kcal mol–1 (mode 1 binding for 22+·DNA and 32+·DNA respectively). To further understand the intercalation vs. groove binding contributions to the overall binding energy we compared the thermodynamics for formation of the 12+·DNA, 22+·DNA, and 32+·DNA complexes to the thermodynamics for formation of the 44+·DNA complex. The RPC binding affinities to duplex DNA follow the trend: 12+ < 44+ < 22+ < 32+. Differences in the affinity for binding 12+ vs. 22+ or 32+ are almost entirely due to the size of the intercalating moiety, e.g. phen which can only be partially intercalated in comparison to dppz and tatpp which can be completely intercalated. The lower affinity for the dinuclear ruthenium 44+ is due to the solvation penalty for the second Ru core complex that extends out from the major groove. Comparing these results, we have begun to develop the structure function relationships for the interaction of RPCs with B‐DNA.