Biology subjects

Mehlman, T.

Publications and source records attributed to Mehlman, T..

9 recordsLinked to original sources

bioRxiv2024

Discovery of potent SARS-CoV-2 nsp3 macrodomain inhibitors uncovers lack of translation to cellular antiviral response

A strategy for pandemic preparedness is the development of antivirals against a wide set of viral targets with complementary mechanisms of action. SARS-CoV-2 nsp3-mac1 is a viral macrodomain with ADP-ribosylhydrolase activity, which counteracts host immune response. Targeting the virus immunomodulatory functionality offers a differentiated strategy to inhibit SARS-CoV-2 compared to approved therapeutics, which target viral replication directly. Here we report a fragment-based lead generation campaign guided by computational approaches. We discover tool compounds which inhibit nsp3-mac1 activity at low nanomolar concentrations, and with responsive structure-activity relationships, high selectivity, and drug-like properties. Using our inhibitors, we show that inhibition of nsp3-mac1 increases ADP-ribosylation, but surprisingly does not translate to demonstrable antiviral activity in cell culture and iPSC-derived pneumocyte models. Further, no synergistic activity is observed in combination with interferon gamma, a main protease inhibitor, nor a papain-like protease inhibitor. Our results question the extent to which targeting modulation of innate immunity-driven ADP-ribosylation can influence SARS-CoV-2 replication. Moreover, these findings suggest that nsp3-mac1 might not be a suitable target for antiviral therapeutics development.

microbiology↗

bioRxiv2024

Structures of human PTP1B variants reveal allosteric sites to target for weight loss therapy

Protein Tyrosine Phosphatase 1B (PTP1B) is a negative regulator of leptin signaling whose disruption protects against diet-induced obesity in mice. We investigated whether structural characterization of human PTP1B variant proteins might reveal allosteric sites to target for weight loss therapy. To do so, we selected 12 rare variants for functional characterization from exomes from 997 people with persistent thinness and 200,000 people from UK Biobank. Seven of 12 variants impaired PTP1B function by increasing leptin-stimulated STAT3 phosphorylation in human cells. Focusing on the variants in and near the ordered catalytic domain, we ascribed structural mechanism to their functional effects using in vitro enzyme activity assays, room-temperature X-ray crystallography, and local hydrogen-deuterium exchange mass spectrometry (HDX-MS). By combining these complementary structural biology experiments for multiple variants, we characterize an inherent allosteric network in PTP1B that differs from previously reported allosteric inhibitor-driven mechanisms mediated by catalytic loop motions. The most functionally impactful variant sites map to highly ligandable surface sites, suggesting untapped opportunities for allosteric drug design. Overall, these studies can inform the targeted design of allosteric PTP1B inhibitors for the treatment of obesity. SignificanceObesity is a growing public health concern worldwide. The human enzyme PTP1B is a validated obesity drug target, but to date, effective PTP1B inhibitors have not been developed. Allosteric drugs, which target parts of a protein distant from the active site, offer advantages for specific inhibition -- but finding promising allosteric sites that bind ligands and convey allosteric signals remains challenging. To address this knowledge gap, we used human genetic studies in thin people to identify amino acid variants that diminish PTP1B function. We then used complementary structural biology methods to show that the variants exploit distinct allosteric wiring within PTP1B that includes ligand-enriched binding sites. This work demonstrates how a unique combination of genetics and biophysics can unveil promising allosteric sites in challenging drug target proteins.

biophysics↗

bioRxiv2024

An expanded view of ligandability in the allosteric enzyme PTP1B from computational reanalysis of large-scale crystallographic data

The recent advent of crystallographic small-molecule fragment screening presents the opportunity to obtain unprecedented numbers of ligand-bound protein crystal structures from a single high-throughput experiment, mapping ligandability across protein surfaces and identifying useful chemical footholds for structure-based drug design. However, due to the low binding affinities of most fragments, detecting bound fragments from crystallographic datasets has been a challenge. Here we report a trove of 65 new fragment hits across 59 new liganded crystal structures for PTP1B, an "undruggable" therapeutic target enzyme for diabetes and cancer. These structures were obtained from computational analysis of data from a large crystallographic screen, demonstrating the power of this approach to elucidate many ([~]50% more) "hidden" ligand-bound states of proteins. Our new structures include a fragment hit found in a novel binding site in PTP1B with a unique location relative to the active site, one that validates another new binding site recently identified by simulations, one that links adjacent allosteric sites, and, perhaps most strikingly, a fragment that induces long-range allosteric protein conformational responses via a previously unreported intramolecular conduit. Altogether, our research highlights the utility of computational analysis of crystallographic data, makes publicly available dozens of new ligand-bound structures of a high-value drug target, and identifies novel aspects of ligandability and allostery in PTP1B.

biophysics↗

bioRxiv2023

High-energy demand and nutrient exhaustion in MTCH2 knockout cells

Mitochondrial carrier homolog 2 (MTCH2) is a regulator of apoptosis, mitochondrial dynamics, and metabolism. Loss of MTCH2 results in mitochondrial fragmentation, an increase in whole-body energy utilization, and protection from diet-induced obesity. We now show using temporal metabolomics that MTCH2 deletion results in a high ATP demand, an oxidized environment, a high lipid/amino acid/carbohydrate metabolism, and in the decrease of many metabolites. Lipidomics analyses show a strategic adaptive decrease in membrane lipids and an increase in storage lipids in MTCH2 knockout cells. Importantly, all the metabolic changes in the MTCH2 knockout cells were rescued by MTCH2 re-expression. Interestingly, this imbalance in energy metabolism and reductive potential triggered by MTCH2-deletion inhibits adipocyte differentiation, an energy consuming reductive biosynthetic process. In summary, loss of MTCH2 results in an increase in energy demand that triggers a catabolic and oxidizing environment, which fails to fuel the anabolic processes during adipocyte differentiation.

cell biology↗

bioRxiv2023

High-resolution double vision of the archetypal protein tyrosine phosphatase

Protein tyrosine phosphatase 1B (PTP1B) plays important roles in cellular homeostasis and is a highly validated therapeutic target for multiple human ailments including diabetes, obesity, and breast cancer. However, much remains to be learned about how conformational changes may convey information through the structure of PTP1B to enable allosteric regulation by ligands or functional responses to mutations. High-resolution X-ray crystallography can offer unique windows into protein conformational ensembles, but comparison of even high-resolution structures is often complicated by differences between datasets including non-isomorphism. Here we present the highest-resolution crystal structure of apo wildtype (WT) PTP1B to date, out of [~]350 total PTP1B structures in the PDB. Our structure is in a crystal form that is rare for PTP1B, with two unique copies of the protein that exhibit distinct patterns of conformational heterogeneity, allowing a controlled comparison of local disorder across the two chains within the same asymmetric unit. We interrogate the conformational differences between these chains in our apo structure, and between several recently reported high-resolution ligand-bound structures. We also examine electron density maps in a high-resolution structure of a recently reported activating double mutant, and discover unmodeled alternate conformations in the mutant structure that coincide with regions of enhanced conformational heterogeneity in our new WT structure. Our results validate the notion that these mutations operate by enhancing local dynamics, and suggest a latent susceptibility to such changes in the WT enzyme. Together, our new data and analysis provide a freshly detailed view of the conformational ensemble of PTP1B, and highlight the utility of high-resolution crystallography for elucidating conformational heterogeneity with potential relevance for function.

biophysics↗

bioRxiv2023

The SARM1 TIR domain produces glycocyclic ADPR molecules as minor products

Sterile alpha and TIR motif-containing 1 (SARM1) is a protein involved in programmed death of injured axons. Following axon injury or a drug-induced insult, the TIR domain of SARM1 degrades the essential molecule nicotinamide adenine dinucleotide (NAD+), leading to a form of axonal death called Wallerian degeneration. Degradation of NAD+ by SARM1 is essential for the Wallerian degeneration process, but accumulating evidence suggest that other activities of SARM1, beyond the mere degradation of NAD+, may be necessary for programmed axonal death. In this study we show that the TIR domains of both human and fruit fly SARM1 produce 1''-2' and 1''-3' glycocyclic ADP-ribose (gcADPR) molecules as minor products. As previously reported, we observed that SARM1 TIR domains mostly convert NAD+ to ADPR (for human SARM1) or cADPR (in the case of SARM1 from Drosophila melanogaster). However, we now show that human and Drosophila SARM1 additionally convert [~]0.1-0.5% of NAD+ into gcADPR molecules. We find that SARM1 TIR domains produce gcADPR molecules both when purified in vitro and when expressed in bacterial cells. Given that gcADPR is a second messenger involved in programmed cell death in bacteria and likely in plants, we propose that gcADPR may play a role in SARM1-induced programmed axonal death in animals.

molecular biology↗

bioRxiv2023

A conserved family of immune effectors cleaves cellular ATP upon viral infection

During viral infection, cells can deploy immune strategies that deprive viruses of molecules essential for their replication. In this study, we report a family of immune effectors in bacteria which, in response to phage infection, degrade cellular ATP and dATP by cleaving the N-glycosidic bond between the adenine and sugar moieties. These ATP nucleosidase effectors are widely distributed within multiple bacterial defense systems including CBASS, prokaryotic argonautes and NLR-like proteins, and we show that degradation of (d)ATP during infection halts phage propagation and aborts infection. By analyzing homologs of the immune ATP nucleosidase domain, we discover and characterize Detocs, a new family of bacterial defense systems with a two-component phosphotransfer signaling architecture. The immune ATP nucleosidase domain is also encoded within a diverse set of eukaryotic proteins that have immune-like architectures, and we show biochemically that these eukaryotic homologs preserve the ATP nucleosidase activity. Our findings suggest that ATP and dATP degradation is a cell-autonomous innate immune strategy conserved across the tree of life.

microbiology↗

bioRxiv2022

The differential regulation of placenta trophoblast bisphosphoglycerate mutase in fetal growth restriction: preclinical study in mice and observational histological study of human placenta.

BackgroundFetal growth restriction (FGR) is a pregnancy complication in which a newborn fails to achieve its growth potential, increasing the risk of perinatal morbidity and mortality. Chronic maternal gestational hypoxia, as well as placental insufficiency are associated with increased FGR incidence; however, the molecular mechanisms underlying FGR remain unknown. MethodsIn a case control study of murine and human control and FGR placentae, we implied MR imaging, IHC and metabolomics to assess the levels of BPGM and 2,3 BPG to elucidate the impact of maternal gestational hypoxia, and the molecular mechanisms underlying human FGR. ResultsWe show that murine acute and chronic gestational hypoxia recapitulates FGR phenotype and affects placental structure and morphology. Gestational hypoxia decreased labyrinth area, increased the incidence of red blood cells (RBCs) in the labyrinth while expanding the placental spiral arteries (SpA) diameter. Hypoxic placentae exhibited higher hemoglobin-oxygen affinity compared to the control. Placental abundance of bisphosphoglycerate mutase (BPGM) was upregulated in the syncytiotrophoblast and spiral artery trophoblast cells (SpA TGCs) in the murine gestational hypoxia groups compared to the control. In contrast, human FGR placentae exhibited reduced BPGM levels in the syncytiotrophoblast layer compared to placentae from healthy uncomplicated pregnancies. Levels of 2,3 BPG, the product of BPGM, were lower in cord serum of human FGR placentae compared to control. Polar expression of BPGM, was found in both human and mouse placentae syncytiotrophoblast, with higher expression facing the maternal circulation. Moreover, in the murine SpA TGCs expression of BPGM was concentrated exclusively in the apical cell side, in direct proximity to the maternal circulation. ConclusionsThis study suggests a possible involvement of placental BPGM in maternal-fetal oxygen transfer, and in the pathophysiology of FGR. FundingThis work was supported by the Weizmann - Ichilov (Tel Aviv Sourasky Medical Center) Collaborative Grant in Biomedical Research (to MN) and by the Israel Science Foundation KillCorona grant 3777/19 (to MN, MK). Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=167 HEIGHT=200 SRC="FIGDIR/small/507932v1_ufig1.gif" ALT="Figure 1"> View larger version (37K): org.highwire.dtl.DTLVardef@500160org.highwire.dtl.DTLVardef@8a68a9org.highwire.dtl.DTLVardef@dd60c3org.highwire.dtl.DTLVardef@1fbb209_HPS_FORMAT_FIGEXP M_FIG C_FIG

physiology↗

bioRxiv2020

Bringing BOS to light: Uncovering the key enzyme in the biosynthesis of the neurotoxin β-ODAP in Grass Pea (Lathyrus sativus L.)

Grass pea (Lathyrus sativus L.) is a grain legume commonly grown in parts of Asia and Africa for food and forage. While being a highly nutritious and robust crop, able to survive both drought and floods, it produces a neurotoxic compound, {beta}-N-oxalyl-L-,{beta}-diaminopropionic acid ({beta}-ODAP), which can cause a severe neurological disorder if consumed as a main diet component. So far, the enzyme that catalyzes the formation of {beta}-ODAP has not been identified. By combining protein purification and enzymatic assays with transcriptomic and proteomic analyses, we were able to identify the enzyme {beta}-ODAP synthetase (BOS) from grass pea. We show that BOS is an HXXXD-type acyltransferase of the BAHD superfamily and that its crystal structure is highly similar to that of plant hydroxycinnamoyl transferases. The identification of BOS, more than 50 years after it was proposed, paves the way towards the generation of non-toxic grass pea cultivars safe for human and animal consumption.

plant biology↗