Proteolysis targeting chimeras (PROTACs) are heterobifunctional small molecules that simultaneously bind to a target protein and an E3 ligase, thereby leading to ubiquitination and subsequent degradation of the target. They present an exciting opportunity to modulate proteins in a manner independent of enzymatic or signaling activity. As such, they have recently emerged as an attractive mechanism to explore previously "undruggable" targets. Despite this interest, fundamental questions remain regarding the parameters most critical for achieving potency and selectivity. Here we employ a series of biochemical and cellular techniques to investigate requirements for efficient knockdown of Bruton's tyrosine kinase (BTK), a nonreceptor tyrosine kinase essential for B cell maturation. Members of an 11-compound PROTAC library were investigated for their ability to form binary and ternary complexes with BTK and cereblon (CRBN, an E3 ligase component). Results were extended to measure effects on BTK-CRBN cooperative interactions as well as in vitro and in vivo BTK degradation. Our data show that alleviation of steric clashes between BTK and CRBN by modulating PROTAC linker length within this chemical series allows potent BTK degradation in the absence of thermodynamic cooperativity.

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Background: Switch Regions of G protein G␣ subunits activate downstream cell signaling events. Results: G␣ 13 Switch Region 2 forms a calcium-dependent bi-molecular complex with the head domain of talin. Conclusion: Binding of the G␣ 13 to the talin head domain promotes ␣IIb␤3 integrin activation. Significance: These results provide a new paradigm for inside-out signaling and ␣IIb␤3 integrin activation in platelets.

Malaria remains a major global threat to human health and economic development. Microvascular lesions caused by Plasmodium falciparum–infected human erythrocytes/red blood cells are hallmarks of severe pathogenesis contributing to high mortality, particularly in children from sub-Saharan Africa. In this study, we used a phage display complementary DNA library screening strategy to identify P falciparum glutamic acid–rich protein (PfGARP) as a secreted ligand that recognizes an ectodomain of human erythrocyte anion-exchanger, band 3/AE1, as a host receptor. Domain mapping of PfGARP revealed distinct nonoverlapping repeats encoding the immune response epitopes and core erythrocyte-binding activity. Synthetic peptides derived from the erythrocyte-binding repeats of PfGARP induced erythrocyte aggregation reminiscent of the rosetting phenomenon. Using peptides derived from the immunogenic repeats, a quantitative immunoassay was developed to detect a selective immune response against PfGARP in human plasma samples obtained from patients in rural Mali, suggesting the feasibility of PfGARP as a potential biomarker of disease progression. Collectively, our results suggest that PfGARP may play a functional role in enhancing the adhesive properties of human erythrocytes by engaging band 3 as a host receptor. We propose that immunological and pharmacological inhibition of PfGARP may unveil new therapeutic options for mitigating lesions in cerebral and pregnancy-associated malaria.

Edited by Henrik DohlmanIntegrins function as bi-directional signaling transducers that regulate cell-cell and cell-matrix signals across the membrane. A key modulator of integrin activation is talin, a large cytoskeletal protein that exists in an autoinhibited state in quiescent cells. Talin is a large 235-kDa protein composed of an N-terminal 45-kDa FERM (4.1, ezrin-, radixin-, and moesin-related protein) domain, also known as the talin head domain, and a series of helical bundles known as the rod domain. The talin head domain consists of four distinct lobes designated as F0 -F3. Integrin binding and activation are mediated through the F3 region, a critically regulated domain in talin. Regulation of the F3 lobe is accomplished through autoinhibition via anti-parallel dimerization. In the anti-parallel dimerization model, the rod domain region of one talin molecule binds to the F3 lobe on an adjacent talin molecule, thus achieving the state of autoinhibition. Platelet functionality requires integrin activation for adherence and thrombus formation, and thus regulation of talin presents a critical node where pharmacological intervention is possible. A major mechanism of integrin activation in platelets is through heterotrimeric G protein signaling regulating hemostasis and thrombosis. Here, we provide evidence that switch region 2 (SR2) of the ubiquitously expressed G protein (G␣13) directly interacts with talin, relieves its state of autoinhibition, and triggers integrin activation. Biochemical analysis of G␣ 13 shows SR2 binds directly to the F3 lobe of talin's head domain and competes with the rod domain for binding. Intramolecular FRET analysis shows G␣ 13 can relieve autoinhibition in a cellular milieu. Finally, a myristoylated SR2 peptide shows demonstrable decrease in thrombosis in vivo. Altogether, we present a mechanistic basis for the regulation of talin through G␣ 13 .Integrins are transmembrane receptors that regulate dynamic cell/cell and cell/matrix interactions (1, 2). Environmental cues regulate a multitude of cellular processes through integrins, such as cell proliferation, shape, adhesion, and migration. Because of the well established importance of integrin signaling in hemostasis and thrombosis, the mechanisms of integrin regulation remain an area of intense investigation. Upon activation, platelet integrins switch from a low affinity to a high affinity state, thus permitting the binding to multivalent ligands such as von Willebrand factor and fibrinogen to enable clotting (3-7). The most abundant platelet integrin complex, ␣IIb␤3, is activated through the cytoskeletal protein talin (1, 8 -12). Talin is a large 235-kDa protein composed of an N-terminal 45-kDa FERM 2 domain, also known as the talin head domain (THD). THD is connected to a large rod domain composed of a series of ␣-helical bundles. The talin head domain consists of four distinct lobes or regions designated as F0 -F3. Integrin binding and activation are mediated through the F3 region, a critically regulated domain in talin. Regul...