Kinetics of the multitasking high-affinity Win binding site of WDR5 in restricted and unrestricted conditions.

Recent advances in quantitative proteomics show that WD40 proteins play a pivotal role in numerous cellular networks. Yet, they have been fairly unexplored and their physical associations with other proteins are ambiguous. A quantitative understanding of these interactions has wide-ranging significance. WD40 repeat protein 5 (WDR5) interacts with all members of human SET1/MLL methyltransferases, which regulate methylation of the histone 3 lysine 4 (H3K4). Here, using real-time binding measurements in a high-throughput setting, we identified the kinetic fingerprint of transient associations between WDR5 and 14-residue WDR5 interaction (Win) motif peptides of each SET1 protein (SET1Win). Our results reveal that the high-affinity WDR5-SET1Win interactions feature slow association kinetics. This finding is likely due to the requirement of SET1Win to insert into the narrow WDR5 cavity, also named the Win binding site. Furthermore, our explorations indicate fairly slow dissociation kinetics. This conclusion is in accordance with the primary role of WDR5 in maintaining the functional integrity of a large multisubunit complex, which regulates the histone methylation. Because the Win binding site is considered a key therapeutic target, the immediate outcomes of this study could form the basis for accelerated developments in medical biotechnology.

RPtag as an Orally Bioavailable, Hyperstable Epitope Tag and Generalizable Protein Binding Scaffold.

Antibodies are the most prolific biologics in research and clinical environments because of their ability to bind targets with high affinity and specificity. However, antibodies also carry liabilities. A significant portion of the life-science reproducibility crisis is driven by inconsistent performance of research-grade antibodies, and clinical antibodies are often unstable and require costly cold-chain management to reach their destinations in active form. In biotechnology, antibodies are also limited by difficulty integrating them in many recombinant systems due to their size and structural complexity. A switch to small, stable, sequence-verified binding scaffolds may overcome these barriers. Here we present such a scaffold, RPtag, based on a ribose-binding protein (RBP) from extremophile Caldanaerobacter subterraneus. RPtag binds an optimized peptide with pM affinity, is stable to extreme temperature, pH, and protease treatment, readily refolds after denaturation, is effective in common laboratory applications, was rationally engineered to bind bioactive PDGF-ß, and was formulated as a gut-stable orally bioavailable preparation.

Enantio- and Diastereoselective Synthesis of 1,2-Hydroxyboronates through Cu-Catalyzed Additions of Alkylboronates to Aldehydes.

The first catalytic enantio- and diastereoselective synthesis of 1,2-hydroxyboronates is reported. Reactions are promoted by a readily available chiral monodentate phosphoramidite-copper complex in the presence of an alkyl 1,1-diboron reagent. Products contain two contiguous stereogenic centers and are obtained in up to 91% yield, >98:2 d.r., and 98:2 e.r. The reaction is tolerant of aryl and vinyl aldehydes, and the 1,2-hydroxyboronate products can be transformed into versatile derivatives. Mechanistic experiments indicate control of absolute stereochemistry of the α-boryl component.

Silver(I)-Catalyzed Diastereoselective Synthesis of anti-1,2-Hydroxyboronates.

A catalytic protocol for the diastereoselective synthesis of anti-1,2-hydroxyboronates is described. The process provides access to secondary alkyl organoborons. The deborylative 1,2-addition reactions of alkyl 1,1-diborons proceed in the presence of a silver(I) salt with either KOtBu or nBuLi as an activator. The catalytic diastereoselective protocol can be extended to aryl, alkenyl, and alkyl aldehydes with up to 99:1 d.r.

Interplay of Affinity and Surface Tethering in Protein Recognition.

Surface-tethered ligand-receptor complexes are key components in biological signaling and adhesion. They also find increasing utility in single-molecule assays and biotechnological applications. Here, we study the real-time binding kinetics between various surface-immobilized peptide ligands and their unrestrained receptors. A long peptide tether increases the association of ligand-receptor complexes, experimentally proving the fly casting mechanism where the disorder accelerates protein recognition. On the other hand, a short peptide tether enhances the complex dissociation. Notably, the rate constants measured for the same receptor, but under different spatial constraints, are strongly correlated to one another. Furthermore, this correlation can be used to predict how surface tethering on a ligand-receptor complex alters its binding kinetics. Our results have immediate implications in the broad areas of biomolecular recognition, intrinsically disordered proteins, and biosensor technology.

Convergent Alterations of a Protein Hub Produce Divergent Effects within a Binding Site.

Progress in tumor sequencing and cancer databases has created an enormous amount of information that scientists struggle to sift through. While several research groups have created computational methods to analyze these databases, much work still remains in distinguishing key implications of pathogenic mutations. Here, we describe an approach to identify and evaluate somatic cancer mutations of WD40 repeat protein 5 (WDR5), a chromatin-associated protein hub. This multitasking protein maintains the functional integrity of large multi-subunit enzymatic complexes of the six human SET1 methyltransferases. Remarkably, the somatic cancer mutations of WDR5 preferentially distribute within and around an essential cavity, which hosts the WDR5 interaction (Win) binding site. Hence, we assessed the real-time binding kinetics of the interactions of key clustered WDR5 mutants with the Win motif peptide ligands of the SET1 family members (SET1Win). Our measurements highlight that this subset of mutants exhibits divergent perturbations in the kinetics and strength of interactions not only relative to those of the native WDR5 but also among various SET1Win ligands. These outcomes could form a fundamental basis for future drug discovery and other developments in medical biotechnology.

Fluorescence-Based Detection of Ferrous Iron in Senescent Cells.

A major limitation in aging research is the lack of reliable biomarkers to assess phenotypic changes with age or monitor response to antiaging interventions. This study investigates the role of intracellular ferrous iron (Fe2+) as a potential biomarker of senescence. Iron is known to accumulate in various tissues with age and recent studies have demonstrated that its level increases dramatically in senescent cells. The current techniques used to measure the accumulation of iron are cumbersome and only measure total iron not specific isotopes such as the redox reactive Fe2+. It is still to be determined whether the damaging form of iron (Fe2+) is specifically elevated in senescent cells. In this study, we assessed the potential use of a newly discovered Fe2+ reactive probe (SiRhoNox-1) for selective labeling of senescent cells in vitro. For this we have generated various senescent cell models and subjected them to SiRhoNox-1 labeling. Our results indicate that SiRhoNox-1 selectivity labels live senescent cells and was more specific and faster than current staining such as SA-ßGal or a derived fluorescent probe C12FDG. Together these findings suggest that SiRhoNox-1 may serve as a convenient tool to detect senescent cells based on their ferrous iron level.

C60 in olive oil causes light-dependent toxicity and does not extend lifespan in mice.

C60 is a potent antioxidant that has been reported to substantially extend the lifespan of rodents when formulated in olive oil (C60-OO) or extra virgin olive oil (C60-EVOO). Despite there being no regulated form of C60-OO, people have begun obtaining it from online sources and dosing it to themselves or their pets, presumably with the assumption of safety and efficacy. In this study, we obtain C60-OO from a sample of online vendors, and find marked discrepancies in appearance, impurity profile, concentration, and activity relative to pristine C60-OO formulated in-house. We additionally find that pristine C60-OO causes no acute toxicity in a rodent model but does form toxic species that can cause significant morbidity and mortality in mice in under 2 weeks when exposed to light levels consistent with ambient light. Intraperitoneal injections of C60-OO did not affect the lifespan of CB6F1 female mice. Finally, we conduct a lifespan and health span study in males and females C57BL/6 J mice comparing oral treatment with pristine C60-EVOO and EVOO alone versus untreated controls. We failed to observe significant lifespan and health span benefits of C60-EVOO or EVOO supplementation compared to untreated controls, both starting the treatment in adult or old age. Our results call into question the biological benefit of C60-OO in aging.

Recombinant Manganese Peroxidase Reduces A2E Burden in Age-Related and Stargardt's Macular Degeneration Models.

Macular degeneration is hallmarked by retinal accumulation of toxic retinoid species (e.g., A2E) for which there is no endogenous mechanism to eliminate it. This ultimately results in progressive dysfunction and loss of vision either in advanced age for genetically normal patients (age-related macular degeneration) or in adolescence for those with inherited genetic mutations (Stargardt's disease). In this article, we present a proof-of-concept study for an enzyme-based therapy to remove these retinoids, modeled on traditional enzyme replacement therapy. Recombinant manganese peroxidase (rMnP) is produced in Pichia pastoris. In vitro, we demonstrate that rMnP breaks down A2E and other lipofuscin fluorophores with limited cellular toxicity, and as this enzyme is mannosylated, it can be taken up into cells through mannose receptor-dependent endocytosis. In vivo, we demonstrate that rMnP can significantly reduce the A2E burden when administered by intravitreal injections. Together, these data provide encouraging results toward the development of an enzyme-based therapy for macular degeneration and indicate the need for additional work to characterize the molecular mechanism of A2E breakdown and to improve the pharmacological parameters of the enzyme.