Modeling the CRL4A ligase complex to predict target protein ubiquitination induced by cereblon-recruiting PROTACs

PROteolysis TArgeting Chimeras (PROTACs) are hetero-bifunctional small molecules that can simultaneously recruit target proteins and E3 ligases to form a ternary complex, promoting target protein ubiquitination and degradation via the Ubiquitin-Proteasome System (UPS). PROTACs have gained increasing attention in recent years due to certain advantages over traditional therapeutic modalities and enabling targeting of previously “undruggable” proteins. To better understand the mechanism of PROTAC-induced Target Protein Degradation (TPD), several computational approaches have recently been developed to study and predict ternary complex formation. However, mounting evidence suggests that ubiquitination can also be a rate-limiting step in PROTAC-induced TPD. Here, we propose a structure-based computational approach to predict target protein ubiquitination induced by cereblon (CRBN)-based PROTACs by leveraging available structural information of the CRL4A ligase complex (CRBN/DDB1/CUL4A/Rbx1/NEDD8/E2/Ub). We generated ternary complex ensembles with Rosetta, modeled multiple CRL4A ligase complex conformations, and predicted ubiquitination efficiency by separating the ternary ensemble into productive and unproductive complexes based on the proximity of the ubiquitin to accessible lysines on the target protein. We validated our CRL4A ligase complex models with published ternary complex structures and additionally employed our modeling workflow to predict ubiquitination efficiencies and sites of a series of cyclin-dependent kinases (CDKs) after treatment with TL12–186, a pan-kinase PROTAC. Our predictions are consistent with CDK ubiquitination and site-directed mutagenesis of specific CDK lysine residues as measured using a NanoBRET ubiquitination assay in HEK293 cells. This work structurally links PROTAC-induced ternary formation and ubiquitination, representing an important step toward prediction of target “degradability.”     

Phyllachora species infecting maize and other grass species in the Americas represents a complex of closely related species

The genus Phyllachora contains numerous obligate fungal parasites that produce raised, melanized structures called stromata on their plant hosts referred to as tar spot. Members of this genus are known to infect many grass species but generally do not cause significant damage or defoliation, with the exception of P. maydis which has emerged as an important pathogen of maize throughout the Americas, but the origin of this pathogen remains unknown. To date, species designations for Phyllachora have been based on host associations and morphology, and most species are assumed to be host specific. We assessed the sequence diversity of 186 single stroma isolates collected from 16 hosts representing 15 countries. Samples included both herbarium and contemporary strains that covered a temporal range from 1905 to 2019. These 186 isolates were grouped into five distinct species with strong bootstrap support. We found three closely related, but genetically distinct groups of Phyllachora are capable of infecting maize in the United States, we refer to these as the P. maydis species complex. Based on herbarium specimens, we hypothesize that these three groups in the P. maydis species complex originated from Central America, Mexico, and the Caribbean. Although two of these groups were only found on maize, the third and largest group contained contemporary strains found on maize and other grass hosts, as well as herbarium specimens from maize and other grasses that include 10 species of Phyllachora. The herbarium specimens were previously identified based on morphology and host association. This work represents the first attempt at molecular characterization of Phyllachora species infecting grass hosts and indicates some Phyllachora species can infect a broad range of host species and there may be significant synonymy in the Phyllachora genus.     

TNFα activation of PKCδ, mediated by NFκB and ER stress,cross-talks with the insulin signaling cascade

TNFα plays key roles in the regulation of inflammation, cell death, and proliferation and its signaling cascade cross-talks with the insulin signaling cascade. PKCδ, a novel PKC isoform, is known to participate in proximal TNFα signaling events. However, it has remained unclear whether PKCδ plays a role in distal TNFα signaling events. Here we demonstrate that PKCδ is activated by TNFα in a delayed fashion that is temporally associated with JNK activation. To investigate the signaling pathways activating PKCδ and JNK, we used pharmacological and genetic inhibitors of NFκB. We found that inhibition of NFκB attenuated PKCδ and JNK activations. Further analysis revealed that ER stress contributes to TNFα-stimulated PKCδ and JNK activations. To investigate the role of PKCδ in TNFα action, we used 29-mer shRNAs to silence PKCδ expression. A reduction of ~90% in PKCδ protein levels reduced TNFα-stimulated stress kinase activation, including JNK. Further, PKCδ was necessary for thapsigargin-stimulated JNK activation. Because thapsigargin is a potent inducer of ER stress, we determined whether PKCδ was necessary for induction of the UPR. Indeed, a reduction in PKCδ protein levels reduced thapsigargin-stimulated CHOP induction, a hallmark of the UPR, but not BiP/GRP78 induction, suggesting that PKCδ does not globally regulate the UPR. Next, the role of PKCδ in TNFα mediated cross-talk with the insulin signaling pathway was investigated in cells expressing human IRS-1 and a 29-mer shRNA to silence PKCδ expression. We found that a reduction in PKCδ protein levels reversed the TNFα-mediated reduction in insulin-stimulated IRS-1 Tyr phosphorylation, Akt activation, and glycogen synthesis. In addition, TNFα-stimulated IRS protein Ser/Thr phosphorylation and degradation were blocked. Our results indicate that: 1) NFκB and ER stress contribute in part to PKCδ activation; 2) PKCδ plays a key role in the propagation of the TNFα signal; and 3) PKCδ contributes to TNFα-induced inhibition of insulin signaling events.     

Extension of Family Planning Service in General Practice

All general practitioners on the list of a single executive council were contacted and 91% were interviewed. They were asked about the type of family planning service currently provided and their willingness to extend the service. Only 3% said that they were not providing any service at the time of interview, but only 4% stated that they provided a complete range of techniques to all patients; almost two thirds of practitioners only provided advice and the “pill”. Lack of training in family planning techniques emerged as the most important factor in determining the type of service provided. At the time of interview 64% of doctors stated that they would like further training, and 35% of all doctors asked for a full course of clinical training. Many general practitioners (81%) were willing to extend their family planning services but 65% wanted financial reimbursement and 50% needed additional administrative support as prerequisites.     

An fMRI Study Exploring the Overlap and Differences between Neural Representations of Physical and Recalled Pain

Implementing a recall paradigm without hypnosis, we use functional MRI (fMRI) to explore and compare nociceptive and centrally-driven pain experiences. We posit that a trace of a recent nociceptive event can be used to create sensory-re-experiencing of pain that can be qualified in terms of intensity and vividness. Fifteen healthy volunteers received three levels of thermal stimuli (warm, low pain and high pain) and subsequently were asked to recall and then rate this experience. Neuroimaging results reveal that recalling a previous sensory experience activates an extensive network of classical pain processing structures except the contralateral posterior insular cortex. Nociceptive-specific activation of this structure and the rated intensity difference between physical and recalled pain events allow us to investigate the link between the quality of the original nociceptive stimulus and the mental trace, as well as the differences between the accompanying neural responses. Additionally, by incorporating the behavioural ratings, we explored which brain regions were separately responsible for generating either an accurate or vivid recall of the physical experience. Together, these observations further our understanding of centrally-mediated pain experiences and pain memory as well as the potential relevance of these factors in the maintenance of chronic pain.     

Ultrastructure of spermiogenesis in the American alligator,Alligator mississippiensis (Reptilia,Crocodylia, Alligatoridae)

Testicular samples were collected to describe the ultrastructure of spermiogenisis in Alligator mississipiensis (American Alligator). Spermiogenesis commences with an acrosome vesicle forming from Golgi transport vesicles. An acrosome granule forms during vesicle contact with the nucleus, and remains posterior until mid to late elongation when it diffuses uniformly throughout the acrosomal lumen. The nucleus has uniform diffuse chromatin with small indices of heterochromatin, and the condensation of DNA is granular. The subacrosome space develops early, enlarges during elongation, and accumulates a thick layer of dark staining granules. Once the acrosome has completed its development, the nucleus of the early elongating spermatid becomes associated with the cell membrane flattening the acrosome vesicle on the apical surface of the nucleus, which aids in the migration of the acrosomal shoulders laterally. One endonuclear canal is present where the perforatorium resides. A prominent longitudinal manchette is associated with the nuclei of late elongating spermatids, and less numerous circular microtubules are observed close to the acrosome complex. The microtubule doublets of the midpiece axoneme are surrounded by a layer of dense staining granular material. The mitochondria of the midpiece abut the proximal centriole resulting in a very short neck region, and possess tubular cristae internally and concentric layers of cristae superficially. A fibrous sheath surrounds only the axoneme of the principal piece. Characters not previously described during spermiogenesis in any other amniote are observed and include (1) an endoplasmic reticulum cap during early acrosome development, (2) a concentric ring of endoplasmic reticulum around the nucleus of early to middle elongating spermatids, (3) a band of endoplasmic reticulum around the acrosome complex of late developing elongate spermatids, and (4) midpiece mitochondria that have both tubular and concentric layers of cristae. J. Morphol., 2010. © 2010 Wiley‐Liss, Inc.     

An integrative, in situ approach to examining K+ flux in resting skeletal muscle

The contributions of Na+/K+-ATPase, K+ channels, and the NaK2Cl cotransporter (NKCC) to total and unidirectional K+ flux were determined in mammalian skeletal muscle at rest. Rat hindlimbs were perfused in situ via the femoral artery with a bovine erythrocyte perfusion medium that contained either 86Rb or 42K, or both simultaneously, to determine differences in ability to trace unidirectional K+ flux in the absence and presence of K+-flux inhibitors. In most experiments, the unidirectional flux of K+ into skeletal muscle (J(in)K) measured using 86Rb was 8-10% lower than J(in)K measured using 42K. Ouabain (5 mM) was used to inhibit Na+/K+-ATPase activity, 0.06 mM bumetanide to inhibit NKCC activity, 1 mM tetracaine or 0.5 mM barium to block K+ channels, and 0.05 mM glybenclamide (GLY) to block ATP-sensitive K+ (K(ATP)) channels. In controls, J(in)K remained unchanged at 0.31 +/- 0.03 micromol x g(-1) x min(-1) during 55 min of perfusion. The ouabain-sensitive Na+/K+-ATPase contributed to 50 +/- 2% of basal J(in)K, K+ channels to 47 +/- 2%, and the NKCC to 12 +/- 1%. GLY had minimal effect on J(in)K, and both GLY and barium inhibited unidirectional efflux of K+ (J(out)K) from the cell through K+ channels. Combined ouabain and tetracaine reduced J(in)K by 55 +/- 2%, while the combination of ouabain, tetracaine, and bumetanide reduced J(in)K by 67 +/- 2%, suggesting that other K+-flux pathways may be recruited because the combined drug effects on inhibiting J(in)K were not additive. The main conclusions are that the NKCC accounted for about 12% of J(in)K, and that K(ATP) channels accounted for nearly all of the J(out)K, in resting skeletal muscle in situ.     

Mechanisms of the renal vasodilation caused by insulin in anesthetized pigs

The present study was planned to determine the mechanisms involved in the renal vasodilation caused by insulin. Changes in flow caused by the intravenous infusion of 0.004 IU/kg/min of insulin at constant heart rate, aortic blood pressure, left ventricular contractility and blood levels of glucose and potassium in the left renal artery were assessed using an electromagnetic flowmeter. In ten pigs, infusion of insulin caused an increase in renal blood flow which averaged 12.8% of the control values. After hemodynamic variables had returned to control values, insulin infusion was repeated in five pigs following blockade of alpha-adrenergic receptors with injection of phentolamine into the renal artery and in the other five pigs following blockade of nitric oxide formation with injection in the same artery of Nomega-nitro-L-arginine methyl ester (L-NAME). After blockade of alpha-adrenergic receptors, insulin infusion caused an increase in renal blood flow which averaged 18.1% of the control values, being significantly enhanced with respect to the increase previously obtained in the same pigs. On the contrary, after blockade of nitric oxide formation insulin infusion caused a decrease in renal blood flow which averaged 6.5% of the control values. These responses were respectively abolished by the subsequent injection into the renal artery of L-NAME and phentolamine. The present study showed that the renal vasodilation caused by insulin in the anesthetized pig was the result of two opposite effects which involved a predominant vasodilation mediated by the release of nitric oxide from the endothelium and a sympathetic vasoconstrictor mechanism mediated by alpha-adrenergic receptors.