Chemical Biology: Learning through Case Studies . By Herbert Waldmann and Petra Janning .

No AbstractPeer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/64308/1/2540_ftp.pd

Competing protein-protein interactions regulate binding of Hsp27 to its client protein tau.

Small heat shock proteins (sHSPs) are a class of oligomeric molecular chaperones that limit protein aggregation. However, it is often not clear where sHSPs bind on their client proteins or how these protein-protein interactions (PPIs) are regulated. Here, we map the PPIs between human Hsp27 and the microtubule-associated protein tau (MAPT/tau). We find that Hsp27 selectively recognizes two aggregation-prone regions of tau, using the conserved β4-β8 cleft of its alpha-crystallin domain. The β4-β8 region is also the site of Hsp27-Hsp27 interactions, suggesting that competitive PPIs may be an important regulatory paradigm. Indeed, we find that each of the individual PPIs are relatively weak and that competition for shared sites seems to control both client binding and Hsp27 oligomerization. These findings highlight the importance of multiple, competitive PPIs in the function of Hsp27 and suggest that the β4-β8 groove acts as a tunable sensor for clients

Exported plasmodial J domain protein, PFE0055c, and PfHsp70-x form a specific co-chaperone-chaperone partnership

Plasmodium falciparum is a unicellular protozoan parasite and causative agent of a severe form of malaria in humans, accounting for very high worldwide fatality rates. At the molecular level, survival of the parasite within the human host is mediated by P. falciparum heat shock proteins (PfHsps) that provide protection during febrile episodes. The ATP-dependent chaperone activity of Hsp70 relies on the co-chaperone J domain protein (JDP), with which it forms a chaperone-co-chaperone complex. The exported P. falciparum JDP (PfJDP), PFA0660w, has been shown to stimulate the ATPase activity of the exported chaperone, PfHsp70-x. Furthermore, PFA0660w has been shown to associate with another exported PfJDP, PFE0055c, and PfHsp70-x in J-dots, highly mobile structures found in the infected erythrocyte cytosol. Therefore, the present study aims to conduct a structural and functional characterization of the full-length exported PfJDP, PFE0055c. Recombinant PFE0055c was successfully expressed and purified and found to stimulate the basal ATPase activity of PfHsp70-x to a greater extent than PFA0660w but, like PFA0660w, did not significantly stimulate the basal ATPase activity of human Hsp70. Small-molecule inhibition assays were conducted to determine the effect of known inhibitors of JDPs (chalcone, C86) and Hsp70 (benzothiazole rhodacyanines, JG231 and JG98) on the basal and PFE0055c-stimulated ATPase activity of PfHsp70-x. In this study, JG231 and JG98 were found to inhibit both the basal and PFE0055c-stimulated ATPase activity of PfHsp70-x. C86 only inhibited the PFE0055c-stimulated ATPase activity of PfHsp70-x, consistent with PFE0055c binding to PfHsp70-x through its J domain. This research has provided further insight into the molecular basis of the interaction between these exported plasmodial chaperones, which could inform future antimalarial drug discovery studies

Mechanism of action of VP1-001 in cryAB(R120G)-associated and age-related cataracts

PurposeWe previously identified an oxysterol, VP1-001 (also known as compound 29), that partially restores the transparency of lenses with cataracts. To understand the mechanism of VP1-001, we tested the ability of its enantiomer, ent-VP1-001, to bind and stabilize αB-crystallin (cryAB) in vitro and to produce a similar therapeutic effect in cryAB(R120G) mutant and aged wild-type mice with cataracts. VP1-001 and ent-VP1-001 have identical physicochemical properties. These experiments are designed to critically evaluate whether stereoselective binding to cryAB is required for activity.MethodsWe compared the binding of VP1-001 and ent-VP1-001 to cryAB using in silico docking, differential scanning fluorimetry (DSF), and microscale thermophoresis (MST). Compounds were delivered by six topical administrations to mouse eyes over 2 weeks, and the effects on cataracts and lens refractive measures in vivo were examined. Additionally, lens epithelial and fiber cell morphologies were assessed via transmission electron microscopy.ResultsDocking studies suggested greater binding of VP1-001 into a deep groove in the cryAB dimer compared with ent-VP1-001. Consistent with this prediction, DSF and MST experiments showed that VP1-001 bound cryAB, whereas ent-VP1-001 did not. Accordingly, topical treatment of lenses with ent-VP1-001 had no effect, whereas VP1-001 produced a statistically significant improvement in lens clarity and favorable changes in lens morphology.ConclusionsThe ability of VP1-001 to bind native cryAB dimers is important for its ability to reverse lens opacity in mouse models of cataracts

Promoting tau secretion and propagation by hyperactive p300/CBP via autophagy-lysosomal pathway in tauopathy.

BackgroundThe trans-neuronal propagation of tau has been implicated in the progression of tau-mediated neurodegeneration. There is critical knowledge gap in understanding how tau is released and transmitted, and how that is dysregulated in diseases. Previously, we reported that lysine acetyltransferase p300/CBP acetylates tau and regulates its degradation and toxicity. However, whether p300/CBP is involved in regulation of tau secretion and propagation is unknown.MethodWe investigated the relationship between p300/CBP activity, the autophagy-lysosomal pathway (ALP) and tau secretion in mouse models of tauopathy and in cultured rodent and human neurons. Through a high-through-put compound screen, we identified a new p300 inhibitor that promotes autophagic flux and reduces tau secretion. Using fibril-induced tau spreading models in vitro and in vivo, we examined how p300/CBP regulates tau propagation.ResultsIncreased p300/CBP activity was associated with aberrant accumulation of ALP markers in a tau transgenic mouse model. p300/CBP hyperactivation blocked autophagic flux and increased tau secretion in neurons. Conversely, inhibiting p300/CBP promoted autophagic flux, reduced tau secretion, and reduced tau propagation in fibril-induced tau spreading models in vitro and in vivo.ConclusionsWe report that p300/CBP, a lysine acetyltransferase aberrantly activated in tauopathies, causes impairment in ALP, leading to excess tau secretion. This effect, together with increased intracellular tau accumulation, contributes to enhanced spreading of tau. Our findings suggest that inhibition of p300/CBP as a novel approach to correct ALP dysfunction and block disease progression in tauopathy

Making Community Engaged Games (Fall 2021)

The students in this immersive learning experience were enrolled in CS215: Introduction to Game Design. This course provides a practical introduction to game design using a systems thinking approach, emphasizing the iterative and creative processes used to create engaging games. The first half of the semester was spent exploring fundamental theories of game design, including an introduction to educational game design. The students devoted their second half of the semester to iterative development of an original game based on the core stories of Minnetrista. George Buss, Jr, Vice President of Visitor Experience at Minnetrista, made several visits to the class to talk about Minnetrista's role in the community and give students feedback on their design ideas. Contained in this showcase are near-final drafts of many students' final projects, all of which are print-and-play games. That means you can download the files, print them up, and try the games for yourself! The authors' contact information should be provided in each packet in case you would like to send them feedback on their designs. Note that the students' projects will be finalized after the deadline for submission to this online showcase, and so the versions submitted to Minnetrista may vary slightly from what is available here. The games created by this semester's students will provide the input into next semester's continuation of this immersive project. The Spring team will work with Minnetrista and their faculty mentor to choose the designs most amenable to digital production, and then we will work together to produce an original video game based on these designs

A poke in the eye: Inhibiting HIV-1 protease through its flap-recognition pocket

A novel mechanism of inhibiting HIV-1 protease (HIVp) is presented. Using computational solvent mapping to identify complementary interactions and the Multiple Protein Structure method to incorporate protein flexibility, we generated a receptor-based pharmacophore model of the flexible flap region of the semiopen, apo state of HIVp. Complementary interactions were consistently observed at the base of the flap, only within a cleft with a specific structural role. In the closed, bound state of HIVp, each flap tip docks against the opposite monomer, occupying this cleft. This flap-recognition site is filled by the protein and cannot be identified using traditional approaches based on bound, closed structures. Virtual screening and dynamics simulations show how small molecules can be identified to complement this cleft. Subsequent experimental testing confirms inhibitory activity of this new class of inhibitor. This may be the first new inhibitor class for HIVp since dimerization inhibitors were introduced 17 years ago. © 2008 Wiley Periodicals, Inc. Biopolymers 89: 643–652, 2008. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at [email protected] Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/58588/1/20993_ftp.pd