Caulfield, Thomas R

A rare APOE variant APOE3 -Jac reduces self-aggregation and promotes healthy brain aging.

PLEXIND1 is upregulated in several cancers, including pancreatic ductal adenocarcinoma (PDAC). It is an established mediator of semaphorin signaling, and neuropilins are its known coreceptors. Herein, we report data to support the proposal that PLEXIND1 acts as a transforming growth factor beta (TGFβ) coreceptor, modulating cell growth through SMAD3 signaling. Our findings demonstrate that PLEXIND1 plays a pro-tumorigenic role in PDAC cells with oncogenic KRAS (KRASmut). We show in KRASmut PDAC cell lines (PANC-1, AsPC-1,4535) PLEXIND1 downregulation results in decreased cell viability (in vitro) and reduced tumor growth (in vivo). Conversely, PLEXIND1 acts as a tumor suppressor in the PDAC cell line (BxPC-3) with wild-type KRAS (KRASwt), as its reduced expression results in higher cell viability (in-vitro) and tumor growth (in vivo). Additionally, we demonstrate that PLEXIND1-mediated interactions can be selectively disrupted using a peptide based on its C-terminal sequence (a PDZ domain-binding motif), an outcome that may possess significant therapeutic implications. To our knowledge, this is the first report showing that (1) PLEXIND1 acts as a TGFβ coreceptor and mediates SMAD3 signaling, and (2) differential roles of PLEXIND1 in PDAC cell lines correlate with KRASmut and KRASwt status.

COVID-19 is a devastating respiratory and inflammatory illness caused by a new coronavirus that is rapidly spreading throughout the human population. Over the past 12 months, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus responsible for COVID-19, has already infected over 160 million (>20% located in United States) and killed more than 3.3 million people around the world (>20% deaths in USA). As we face one of the most challenging times in our recent history, there is an urgent need to identify drug candidates that can attack SARS-CoV-2 on multiple fronts. We have therefore initiated a computational dynamics drug pipeline using molecular modeling, structure simulation, docking and machine learning models to predict the inhibitory activity of several million compounds against two essential SARS-CoV-2 viral proteins and their host protein interactors—S/Ace2, Tmprss2, Cathepsins L and K, and Mpro—to prevent binding, membrane fusion and replication of the virus, respectively. All together, we generated an ensemble of structural conformations that increase high-quality docking outcomes to screen over >6 million compounds including all FDA-approved drugs, drugs under clinical trial (>3000) and an additional >30 million selected chemotypes from fragment libraries. Our results yielded an initial set of 350 high-value compounds from both new and FDA-approved compounds that can now be tested experimentally in appropriate biological model systems. We anticipate that our results will initiate screening campaigns and accelerate the discovery of COVID-19 treatments.

Chloroethylagelastatin A (CEAA) is an analogue of agelastatin A (AA), a natural alkaloid derived from a marine sponge. It is under development for therapeutic use against brain tumors as it has excellent central nervous system (CNS) penetration and pre-clinical therapeutic activity against brain tumors. Recently, AA was shown to inhibit protein synthesis by binding to the ribosomal A-site. In this study, we developed a novel virtual screening platform to perform a comprehensive screening of various AA analogues showing that AA analogues with proven therapeutic activity including CEAA have significant ribosomal binding capacity whereas therapeutically inactive analogues show poor ribosomal binding and revealing structural fingerprint features essential for drug-ribosome interactions. In particular, CEAA was found to have greater ribosomal binding capacity than AA. Biological tests showed that CEAA binds the ribosome and contributes to protein synthesis inhibition. Our findings suggest that CEAA may possess ribosomal inhibitor activity and that our virtual screening platform may be a useful tool in discovery and development of novel ribosomal inhibitors.

Single-minded homologue 1 (SIM1) is a transcription factor with numerous different physiological and developmental functions. SIM1 is a member of the class I basic helix-loop-helix-PER-ARNT-SIM (bHLH–PAS) transcription factor family, that includes several other conserved proteins, including the hypoxia-inducible factors, aryl hydrocarbon receptor, neuronal PAS proteins, and the CLOCK circadian regulator. Recent studies of HIF-a-ARNT and CLOCK-BMAL1 protein complexes have revealed the organization of their bHLH, PASA, and PASB domains and provided insight into how these heterodimeric protein complexes form; however, experimental structures for SIM1 have been lacking. Here, we describe the first full-length atomic structural model for human SIM1 with its binding partner ARNT in a heterodimeric complex and analyze several pathogenic variants utilizing state-of-the-art simulations and algorithms. Using local and global positional deviation metrics, deductions to the structural basis for the individual mutants are addressed in terms of the deleterious structural reorganizations that could alter protein function. We propose new experiments to probe these hypotheses and examine an interesting SIM1 dynamic behavior. The conformational dynamics demonstrates conformational changes on local and global regions that represent a mechanism for dysfunction in variants presented. In addition, we used our ab initio hybrid model for further prediction of variant hotspots that can be engineered to test for counter variant (restoration of wild-type function) or basic research probe.

We describe the phenotype of a patient with extensive aortic, carotid, and abdominal dissections. The proband was found to have a heterozygous deletion of exons 21–34 inMYLK, which is a rare finding, as deletions in this gene have been infrequently reported. We describe this finding following detection in a proband with an extensive history of aortic, carotid, and abdominal dissections. Neoteric molecular modeling techniques to help determine the impact of this deletion on protein function indicated loss of function due to lack of any kinase domain. We also provide the electrostatics calculations from the wild type and mutant variant. Through a combined multiomic approach of clinical, functional, and protein informatics, we arrive at a data fusion for determination of pathogenicity embedded within the genetic code for this particular genetic variant, which, as a platform, continues to broaden its scope across the field of variants of uncertain significance classification.

Background. The ATP-binding cassette, subfamily D, member 1 (ABCD1) protein is a peroxisomal half-transporter that allows for very long chain fatty acid (VLCFA) degradation. Pathogenic variants of ABCD1 cause VLCFAs to build up in various tissues and bodily fluids, resulting in a disorder called X-linked adrenoleukodystrophy (X-ALD). This disorder is most commonly marked by adrenocortical insufficiency and high VLCFA concentration, and has varying levels of neurological involvement depending on phenotype. For example, the Addison-only form of X-ALD has no neurological impact, while the cerebral form of X-ALD often causes severe sensory loss, motor function impairment, cognitive decline, and death. Methods. A newly characterized and suspected pathogenic variant in ABCD1 was analyzed using our protein informatics platform (PIP). Personalized protein-level molecular studies were completed on genetic testing data, complementing the analysis and clinical study. Results. A case of adult onset adrenomyeloneuropathy (AMN) and a novel ABCD1 variant are presented. The unique ABCD1 protein is discussed, and the proband’s case is compared to existing reports of AMN. Conclusions. Data fusion from multiple sources was combined in a comprehensive approach yielding an enriched assessment of the patient’s disease and prognosis. Molecular modeling was performed on the variant to better characterize its clinical significance and confirm pathogenicity.

Abstract Context Single-minded homologue 1 (SIM1) is a transcription factor with several physiological and developmental functions. Haploinsufficiency of SIM1 is associated with early-onset obesity with or without Prader-Willi–like (PWL) features and may exhibit incomplete penetrance. Case Description Next-generation sequencing was performed for 2 male patients with obesity, including 1 man presenting with intellectual disability (ID), body mass index (BMI) of 47.4, and impulse-control disorder, and the other man with early obesity (BMI of 36); sequencing revealed a missense variant in SIM1 (c.2144G>T; p.G715V) in both individuals. Previous studies have identified several disease-associated variants that fall near the p.G715V variant within the C-terminal domain of SIM1. We examined p.G715V variant stability and activity in a doxycycline-inducible stable cell line transfected with an artificial reporter construct and either ARNT or ARNT2 as a partner protein. Conclusions Functional testing of the p.G715V variant revealed a significant reduction in SIM1-mediated transcriptional activity. We also generated the first ab initio hybrid protein model for full-length SIM1 to show the predicted spatial relationship between p.G715V and other previously described variants in this region and identified a putative mutation hotspot within the C-terminus. Significant clinical heterogeneity has been observed in patients with SIM1 variants, particularly with regards to the PWL phenotype. In the patient with ID, a second variant of uncertain significance in CHD2 was identified that may contribute to his ID and behavioral disturbances, emphasizing the role of additional genetic modifiers.

In-frame decoding in the ribosome occurs through canonical or wobble Watson–Crick pairing of three mRNA codon bases (a triplet) with a triplet of anticodon bases in tRNA. Departures from the triplet–triplet interaction can result in frameshifting, meaning downstream mRNA codons are then read in a different register. There are many mechanisms to induce frameshifting, and most are insufficiently understood. One previously proposed mechanism is doublet decoding, in which only codon bases 1 and 2 are read by anticodon bases 34 and 35, which would lead to −1 frameshifting. In E. coli, tRNASer3GCU can induce −1 frameshifting at alanine (GCA) codons. The logic of the doublet decoding model is that the Ala codon’s GC could pair with the tRNASer3′s GC, leaving the third anticodon residue U36 making no interactions with mRNA. Under that model, a U36C mutation would still induce −1 frameshifting, but experiments refute this. We perform all-atom simulations of wild-type tRNASer3, as well as a U36C mutant. Our simulations revealed a hydrogen bond between U36 of the anticodon and G1 of the codon. The U36C mutant cannot make this interaction, as it lacks the hydrogen-bond-donating H3. The simulation thus suggests a novel, non-doublet decoding mechanism for −1 frameshifting by tRNASer3 at Ala codons.