Molecular basis of interchain disulfide-bond formation in BMP-9 and BMP-10.

BMP-9 and BMP-10 are TGF-β family signaling ligands naturally secreted into blood. They act on endothelial cells and are required for proper development and maintenance of the vasculature. In hereditary hemorrhagic telangiectasia, regulation is disrupted due to mutations in the BMP-9/10 pathway, namely in the type I receptor ALK1 or the co-receptor endoglin.

Single particle cryo-electron microscopy with an enhanced 200 kV cryo-TEM configuration achieves near-atomic resolution.

Single particle cryogenic electron microscopy (cryo-EM) as a structural biology methodology has become increasingly attractive and accessible to investigators in both academia and industry as this ever-advancing technology enables successful structural determination of a wide range of protein and nucleic acid targets. Although data for many high resolution cryo-EM structures are still obtained using a 300 kV cryogenic transmission electron microscope (cryo-TEM), a modern 200 kV cryo-TEM equipped with an advanced direct electron detector and energy filter is a cost-effective choice for most single particle applications, routinely achieving sub 3 angstrom (Å) resolution. Here, we systematically evaluate performance of one such high-end configuration - a 200 kV Glacios microscope coupled with a Falcon 4 direct electron detector and Selectris energy filter (Glacios-F4-S).

Development and crystal structures of a potent second-generation dual degrader of BCL-2 and BCL-xL.

Overexpression of BCL-xL and BCL-2 play key roles in tumorigenesis and cancer drug resistance. Advances in PROTAC technology facilitated recent development of the first BCL-xL/BCL-2 dual degrader, 753b, a VHL-based degrader with improved potency and reduced toxicity compared to previous small molecule inhibitors. Here, we determine crystal structures of VHL/753b/BCL-xL and VHL/753b/BCL-2 ternary complexes.

TATDN2 resolution of R-loops is required for survival of BRCA1-mutant cancer cells.

BRCA1-deficient cells have increased IRE1 RNase, which degrades multiple microRNAs. Reconstituting expression of one of these, miR-4638-5p, resulted in synthetic lethality in BRCA1-deficient cancer cells. We found that miR-4638-5p represses expression of TATDN2, a poorly characterized member of the TATD nuclease family.

Cryo-EM structures of Uba7 reveal the molecular basis for ISG15 activation and E1-E2 thioester transfer.

ISG15 plays a crucial role in the innate immune response and has been well-studied due to its antiviral activity and regulation of signal transduction, apoptosis, and autophagy. ISG15 is a ubiquitin-like protein that is activated by an E1 enzyme (Uba7) and transferred to a cognate E2 enzyme (UBE2L6) to form a UBE2L6-ISG15 intermediate that functions with E3 ligases that catalyze conjugation of ISG15 to target proteins. Despite its biological importance, the molecular basis by which Uba7 catalyzes ISG15 activation and transfer to UBE2L6 is unknown as there is no available structure of Uba7.

Structural insights into BCDX2 complex function in homologous recombination.

Homologous recombination (HR) fulfils a pivotal role in the repair of DNA double-strand breaks and collapsed replication forks. HR depends on the products of several paralogues of RAD51, including the tetrameric complex of RAD51B, RAD51C, RAD51D and XRCC2 (BCDX2). BCDX2 functions as a mediator of nucleoprotein filament assembly by RAD51 and single-stranded DNA (ssDNA) during HR, but its mechanism remains undefined.

Ebselen derivatives inhibit SARS-CoV-2 replication by inhibition of its essential proteins: PL and M proteases, and nsp14 guanine N7-methyltransferase.

Proteases encoded by SARS-CoV-2 constitute a promising target for new therapies against COVID-19. SARS-CoV-2 main protease (M, 3CL) and papain-like protease (PL) are responsible for viral polyprotein cleavage-a process crucial for viral survival and replication. Recently it was shown that 2-phenylbenzisoselenazol-3(2H)-one (ebselen), an organoselenium anti-inflammatory small-molecule drug, is a potent, covalent inhibitor of both the proteases and its potency was evaluated in enzymatic and antiviral assays.

Characterization of the Ubiquitin and ISG15 Deconjugase Activity of SARS-CoV-1 and SARS-CoV-2 Papain-Like Protease.

Both severe acute respiratory syndrome coronavirus 1 and 2 (SARS-CoV-1 and SARS-CoV-2) encode a papain-like protease (PLpro), which plays a vital role in viral propagation. PLpro accomplishes this function by processing the viral polyproteins essential for viral replication and removing the small proteins, ubiquitin and ISG15 from the host's key immune signaling proteins, thereby preventing the host's innate immune response. Although PLpro from both SARS-CoV-1 and SARS-CoV-2 are structurally highly similar (83% sequence identity), they exhibit functional variability.

Evaluation of the anti-SARS-CoV-2 properties of essential oils and aromatic extracts.

Essential oils and aromatic extracts (oleoresins, absolutes, concretes, resinoids) are often used as food flavorings and constituents of fragrance compositions. The flavor and fragrance industry observed significant growth in the sales of some natural materials during the COVID-19 outbreak. Some companies worldwide are making false claims regarding the effectiveness of their essential oils or blends (or indirectly point toward this conclusion) against coronaviruses, even though the available data on the activity of plant materials against highly pathogenic human coronaviruses are very scarce.

Crystal structures reveal catalytic and regulatory mechanisms of the dual-specificity ubiquitin/FAT10 E1 enzyme Uba6.

The E1 enzyme Uba6 initiates signal transduction by activating ubiquitin and the ubiquitin-like protein FAT10 in a two-step process involving sequential catalysis of adenylation and thioester bond formation. To gain mechanistic insights into these processes, we determined the crystal structure of a human Uba6/ubiquitin complex. Two distinct architectures of the complex are observed: one in which Uba6 adopts an open conformation with the active site configured for catalysis of adenylation, and a second drastically different closed conformation in which the adenylation active site is disassembled and reconfigured for catalysis of thioester bond formation.

Clearing the air: Uniquely engaging furin as an approach to cystic fibrosis therapy.

In this issue of Cell Chemical Biology, Douglas et al. describe a potent, specific, and cell-permeable furin inhibitor that interacts with a cryptic binding site to rescue hallmarks of cystic fibrosis in human ex vivo models. BOS-318 holds promise for development of therapeutics targeting an array of furin-dependent pathologies.

Targeting SARS-CoV-2 Proteases for COVID-19 Antiviral Development.

The emergence of severe acute respiratory syndrome (SARS-CoV-2) in 2019 marked the third occurrence of a highly pathogenic coronavirus in the human population since 2003. As the death toll surpasses 5 million globally and economic losses continue, designing drugs that could curtail infection and disease progression is critical. In the US, three highly effective Food and Drug Administration (FDA)-authorized vaccines are currently available, and Remdesivir is approved for the treatment of hospitalized patients.

Development of a BCL-xL and BCL-2 dual degrader with improved anti-leukemic activity.

PROteolysis-TArgeting Chimeras (PROTACs) have emerged as an innovative drug development platform. However, most PROTACs have been generated empirically because many determinants of PROTAC specificity and activity remain elusive. Through computational modelling of the entire NEDD8-VHL Cullin RING E3 ubiquitin ligase (CRL)/PROTAC/BCL-xL/UbcH5B(E2)-Ub/RBX1 complex, we find that this complex can only ubiquitinate the lysines in a defined band region on BCL-xL.

A molecular sensor determines the ubiquitin substrate specificity of SARS-CoV-2 papain-like protease.

The SARS-CoV-2 papain-like protease (PLpro) is a target for antiviral drug development. It is essential for processing viral polyproteins for replication and functions in host immune evasion by cleaving ubiquitin (Ub) and ubiquitin-like protein (Ubl) conjugates. While highly conserved, SARS-CoV-2 and SARS-CoV PLpro have contrasting Ub/Ubl substrate preferences.

PRMT5-mediated arginine methylation activates AKT kinase to govern tumorigenesis.

AKT is involved in a number of key cellular processes including cell proliferation, apoptosis and metabolism. Hyperactivation of AKT is associated with many pathological conditions, particularly cancers. Emerging evidence indicates that arginine methylation is involved in modulating AKT signaling pathway.

Crystal structures of an E1-E2-ubiquitin thioester mimetic reveal molecular mechanisms of transthioesterification.

E1 enzymes function as gatekeepers of ubiquitin (Ub) signaling by catalyzing activation and transfer of Ub to tens of cognate E2 conjugating enzymes in a process called E1-E2 transthioesterification. The molecular mechanisms of transthioesterification and the overall architecture of the E1-E2-Ub complex during catalysis are unknown. Here, we determine the structure of a covalently trapped E1-E2-ubiquitin thioester mimetic.

Activity profiling and crystal structures of inhibitor-bound SARS-CoV-2 papain-like protease: A framework for anti-COVID-19 drug design.

Viral papain-like cysteine protease (PLpro, NSP3) is essential for SARS-CoV-2 replication and represents a promising target for the development of antiviral drugs. Here, we used a combinatorial substrate library and performed comprehensive activity profiling of SARS-CoV-2 PLpro. On the scaffold of the best hits from positional scanning, we designed optimal fluorogenic substrates and irreversible inhibitors with a high degree of selectivity for SARS PLpro.

Structural insights into E1 recognition and the ubiquitin-conjugating activity of the E2 enzyme Cdc34.

Ubiquitin (Ub) signaling requires the sequential interactions and activities of three enzymes, E1, E2, and E3. Cdc34 is an E2 that plays a key role in regulating cell cycle progression and requires unique structural elements to function. The molecular basis by which Cdc34 engages its E1 and the structural mechanisms by which its unique C-terminal extension functions in Cdc34 activity are unknown.

Structural basis for adenylation and thioester bond formation in the ubiquitin E1.

The ubiquitin (Ub) and Ub-like (Ubl) protein-conjugation cascade is initiated by E1 enzymes that catalyze Ub/Ubl activation through C-terminal adenylation, thioester bond formation with an E1 catalytic cysteine, and thioester bond transfer to Ub/Ubl E2 conjugating enzymes. Each of these reactions is accompanied by conformational changes of the E1 domain that contains the catalytic cysteine (Cys domain). Open conformations of the Cys domain are associated with adenylation and thioester transfer to E2s, while a closed conformation is associated with pyrophosphate release and thioester bond formation.

TGFβ promotes breast cancer stem cell self-renewal through an ILEI/LIFR signaling axis.

FAM3C/Interleukin-like EMT Inducer (ILEI) is an oncogenic member of the FAM3 cytokine family and serves essential roles in both epithelial-mesenchymal transition (EMT) and breast cancer metastasis. ILEI expression levels are regulated through a non-canonical TGFβ signaling pathway by 3'-UTR-mediated translational silencing at the mRNA level by hnRNP E1. TGFβ stimulation or silencing of hnRNP E1 increases ILEI translation and induces an EMT program that correlates with enhanced invasion and migration.

Molecular mechanism of a covalent allosteric inhibitor of SUMO E1 activating enzyme.

E1 enzymes activate ubiquitin (Ub) and ubiquitin-like modifiers (Ubls) in the first step of Ub/Ubl conjugation cascades and represent potential targets for therapeutic intervention in cancer and other life-threatening diseases. Here, we report the crystal structure of the E1 enzyme for the Ubl SUMO in complex with a recently discovered and highly specific covalent allosteric inhibitor (COH000). The structure reveals that COH000 targets a cryptic pocket distinct from the active site that is completely buried in all previous SUMO E1 structures and that COH000 binding to SUMO E1 is accompanied by a network of structural changes that altogether lock the enzyme in a previously unobserved inactive conformation.

Crystal structure of a human ubiquitin E1-ubiquitin complex reveals conserved functional elements essential for activity.

Ubiquitin (Ub) signaling plays a key regulatory role in nearly every aspect of eukaryotic biology and is initiated by E1 enzymes that activate and transfer Ub to E2 Ub-conjugating enzymes. Despite Ub E1's fundamental importance to the cell and its attractiveness as a target for therapeutic intervention in cancer and other diseases, its only available structural information is derived from yeast orthologs of human ubiquitin-like modifier-activating enzyme 1 (hUBA1). To illuminate structural differences between yeast and hUBA1 structures that might be exploited for the development of small-molecule therapeutics, we determined the first crystal structure of a hUBA1-Ub complex.