Hepatic fibrosis is the primary determinant of mortality in patients with metabolic dysfunction-associated steatohepatitis (MASH). Transforming growth factor-β (TGFβ), a master profibrogenic ...cytokine, is a promising therapeutic target that has not yet been translated into an effective therapy in part because of liabilities associated with systemic TGFβ antagonism. We have identified that soluble folate receptor γ (FOLR3), which is expressed in humans but not in rodents, is a secreted protein that is elevated in the livers of patients with MASH but not in those with metabolic dysfunction-associated steatotic liver disease, those with type II diabetes, or healthy individuals. Global proteomics showed that FOLR3 was the most highly significant MASH-specific protein and was positively correlated with increasing fibrosis stage, consistent with stimulation of activated hepatic stellate cells (HSCs), which are the key fibrogenic cells in the liver. Exposure of HSCs to exogenous FOLR3 led to elevated extracellular matrix (ECM) protein production, an effect synergistically potentiated by TGFβ1. We found that FOLR3 interacts with the serine protease HTRA1, a known regulator of TGFBR, and activates TGFβ signaling. Administration of human FOLR3 to mice induced severe bridging fibrosis and an ECM pattern resembling human MASH. Our study thus uncovers a role of FOLR3 in enhancing fibrosis.
The TAM family of receptor tyrosine kinases is implicated in multiple distinct oncogenic signaling pathways. However, to date, there are no FDA-approved small molecule inhibitors for the TAM kinases. ...Inhibitor design and screening rely on tools to study the kinase activity. Our goal was to address this gap by designing a set of synthetic peptide substrates for each of the TAM family members: Tyro3, Axl, and Mer. We used an in vitro phosphoproteomics workflow to determine the substrate profile of each TAM kinase and input the identified substrates into our data processing pipeline, KINATEST-ID, producing a position-specific scoring matrix for each target kinase and generating a list of candidate synthetic peptide substrates. We synthesized and characterized a set of those substrate candidates, systematically measuring their initial phosphorylation rate with each TAM kinase by LC-MS. We also used the multimer modeling function of AlphaFold2 (AF2) to predict peptide–kinase interactions at the active site for each of the novel candidate peptide sequences against each of the TAM family kinases and observed that, remarkably, every sequence for which it predicted a putative catalytically competent interaction was also demonstrated biochemically to be a substrate for one or more of the TAM kinases. This work shows that kinase substrate design can be achieved using a combination of preference motifs and structural modeling, and it provides the first demonstration of peptide–protein interaction modeling with AF2 for predicting the likelihood of constructive catalytic interactions.
Traditional drug targets have historically included signaling proteins that respond to small molecules and enzymes that use small molecules as substrates. Increasing attention is now being directed ...toward other types of protein targets, in particular those that exert their function by interacting with nucleic acids or other proteins rather than small-molecule ligands. Here, we systematically compare existing examples of inhibitors of protein–protein interactions to inhibitors of traditional drug targets. While both sets of inhibitors bind with similar potency, we find that the inhibitors of protein–protein interactions typically bury a smaller fraction of their surface area upon binding to their protein targets. The fact that an average atom is less buried suggests that more atoms are needed to achieve a given potency, explaining the observation that ligand efficiency is typically poor for inhibitors of protein–protein interactions. We then carried out a series of docking experiments and found a further consequence of these relatively exposed binding modes is that structure-based virtual screening may be more difficult: such binding modes do not provide sufficient clues to pick out active compounds from decoy compounds. Collectively, these results suggest that the challenges associated with such non-traditional drug targets may not lie with identifying compounds that potently bind to the target protein surface, but rather with identifying compounds that bind in a sufficiently buried manner to achieve good ligand efficiency and, thus, good oral bioavailability. While the number of available crystal structures of distinct protein interaction sites bound to small-molecule inhibitors is relatively small at present (only 21 such complexes were included in this study), these are sufficient to draw conclusions based on the current state of the field; as additional data accumulate it will be exciting to refine the viewpoint presented here. Even with this limited perspective however, we anticipate that these insights, together with new methods for exploring protein conformational fluctuations, may prove useful for identifying the “low-hanging fruit” among non-traditional targets for therapeutic intervention.
Multidrug-resistant enterococcal strains emerged in the early 1980s and are now among the leading causes of drug-resistant bacterial infection worldwide. We used functional genomics to study an early ...bacterial outbreak in patients in a Wisconsin hospital between 1984 and 1988 that was caused by multidrug-resistant
The goal was to determine how a clonal lineage of
became adapted to growth and survival in the human bloodstream. Genome sequence analysis revealed a progression of increasingly fixed mutations and repeated independent occurrences of mutations in a relatively small set of genes. Repeated independent mutations suggested selection within the host during the course of infection in response to pressures such as host immunity and antibiotic treatment. We observed repeated independent mutations in a small number of loci, including a little studied polysaccharide utilization pathway and the
locus. Functional studies showed that mutating these loci rendered
better able to withstand antibiotic pressure and innate immune defenses in the human bloodstream. We also observed a shift in mutation pattern that corresponded to the introduction of carbapenem antibiotics in 1987. This work identifies pathways that allow enterococci to survive the transition from the human gut into the bloodstream, enabling them to cause severe bacteremia associated with high mortality.
Despite intense interest and considerable effort via high-throughput screening, there are few examples of small molecules that directly inhibit protein-protein interactions. This suggests that many ...protein interaction surfaces may not be intrinsically "druggable" by small molecules, and elevates in importance the few successful examples as model systems for improving our fundamental understanding of druggability. Here we describe an approach for exploring protein fluctuations enriched in conformations containing surface pockets suitable for small molecule binding. Starting from a set of seven unbound protein structures, we find that the presence of low-energy pocket-containing conformations is indeed a signature of druggable protein interaction sites and that analogous surface pockets are not formed elsewhere on the protein. We further find that ensembles of conformations generated with this biased approach structurally resemble known inhibitor-bound structures more closely than equivalent ensembles of unbiased conformations. Collectively these results suggest that "druggability" is a property encoded on a protein surface through its propensity to form pockets, and inspire a model in which the crude features of the predisposed pocket(s) restrict the range of complementary ligands; additional smaller conformational changes then respond to details of a particular ligand. We anticipate that the insights described here will prove useful in selecting protein targets for therapeutic intervention.
Abstract TP53 is the most frequently mutated gene in cancer, and its encoded protein p53 has many tumor-suppressive functions. There are several different classes of mutant p53 acquired in human ...cancer and inherited in cancer-prone families with Li-Fraumeni Syndrome, where individuals have an 80-90% increased risk of cancer. These include structural mutations that are generally (e.g R175H) or locally (e.g Y220C) misfolded, DNA contact mutations (e.g R273H); and oligomerization mutations (A347D). Cooperativity mutations affect binding of the p53 tetramer to DNA via disruption of a salt bridge formed by the negatively charged glutamic acid (E) 180 residue of one p53 monomer and the positively charged arginine (R) 181 residue of another. p53 “cooperativity” mutations at the R181 residue (R181H and R181C) have been increasingly identified in cancer-prone families undergoing genetic testing; however, the mechanism by which these variants disrupt p53 tumor suppression in humans is not understood. We show that the purified DNA binding domains of p53 variants R181H and R181C bind less cooperatively to the p53 binding site in the CDKN1A (p21) promoter, despite retaining wild-type levels of structural stability. RNA-sequencing of CRISPR knock-in colorectal and breast cancer cell lines shows reduced ability of R181H and R181C to transactivate a curated set of ~300 known p53 target genes. Upon treatment with p53 activating molecule Nutlin-3a, the R181-mutant cells fail to cell cycle arrest in the G1 phase and maintain high proliferative rates. Interestingly, we observe some residual apoptotic activity in R181H and R181C mutant cells treated with DNA-damaging agent 5-fluorouracil, despite losing the transactivation of proapoptotic p53 targets; this suggests that these mutants retain the p53 transcription-independent mechanism of apoptosis which is observed in other p53 variants such as P47S and A347D. These studies will define the pathogenicity of R181 variants and guide therapeutic intervention for patients that harbor these p53 mutations. Citation Format: Renyta Moses, Ryan Hausler, Gregory Kelly, Alexandra Indeglia, Sven Miller, John Karanicolas, Maureen Murphy, Kara Maxwell. Germline p53 R181 variants and DNA binding cooperativity in tumorigenesis abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3025.
Nonsynonymous polymorphisms can significantly impact the function of a protein. However, so far, the outcome of such mutations at non‐conserved positions are difficult to predict. Previous work ...showed that some non‐conserved positions have an unexpected outcome when they are mutated: if multiple amino acids are substituted, the results show progressive alterations like a “rheostat”, rather than an “all‐or‐nothing” effect often seen for mutations at conserved positions (“toggle”). To begin to define rules for predicting mutation outcomes at rheostat positions, we used the Na+/Taurocholate Cotransporting Polypeptide (NTCP) as a model transmembrane transporter. NTCP is a sodium dependent bile acid transporter expressed at the basolateral membrane of human hepatocytes. NTCP is important for the enterohepatic circulation of bile acids and also mediates the transport of steroid hormones like estrone‐3‐sulfate (E3S) or drugs like rosuvastatin. We initially focused on the S267F polymorphism that decreased taurocholate transport, stimulated rosuvastatin uptake, and showed no effect on E3S transport. We replaced the serine with all other nineteen amino acids by site directed mutagenesis, transfected the cDNAs into human embryonic kidney cells (HEK293), and determined the uptake of tritiated substrates. In addition, we identified the conserved G102 and the non‐conserved Y146 positions using multiple sequences alignments and homology modeling based on the structures of two bacterial homologues. Both positions fall in mobile regions of NTCP. Interestingly, mutations at S267 resulted in a clear rheostat behavior for taurocholate uptake, with S267F showing only 15% of the function of wild‐type NTCP. Similarly, rheostat behavior was observed for E3S and rosuvastatin, but several mutants transported these substrates 2‐ to 10‐fold higher than wild‐type NTCP, demonstrating that the different amino acid replacements resulted in substrate‐dependent effects. As expected, the non‐conserved Y146 demonstrated rheostat behavior for all three substrates. However, the conserved G102 turned out to be a rheostat position for taurocholate transport but a toggle for the uptake of E3S and rosuvastatin. In summary, we have determined that rheostat positions exist in human NTCP. Furthermore, the functional consequences of single amino acid mutations at the three positions studied so far, varied depending on the substrate tested. Therefore, functional predictions of amino acid mutations seem to be more challenging and will require further experiments at additional positions to potentially define a unifying set of rules.
Support or Funding Information
NIH R01 GM077336, P30 GM118247, W.M. Keck Foundation
This is from the Experimental Biology 2018 Meeting. There is no full text article associated with this published in The FASEB Journal.
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