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Ufmylation plays a crucial role in various cellular processes including DNA damage response, protein translation, and ER homeostasis. To date, little is known about how the enzymes responsible for ufmylation coordinate their action. Here, we study the details of UFL1 (E3) activity, its binding to UFC1 (E2), and its relation to UBA5 (E1), using a combination of structural modeling, X‐ray crystallography, NMR, and biochemical assays. Guided by Alphafold2 models, we generate an active UFL1 fusion construct that includes its partner DDRGK1 and solve the crystal structure of this critical interaction. This fusion construct also unveiled the importance of the UFL1 N‐terminal helix for binding to UFC1. The binding site suggested by our UFL1‐UFC1 model reveals a conserved interface, and competition between UFL1 and UBA5 for binding to UFC1. This competition changes in the favor of UFL1 following UFM1 charging of UFC1. Altogether, our study reveals a novel, terminal helix‐mediated regulatory mechanism, which coordinates the cascade of E1‐E2‐E3‐mediated transfer of UFM1 to its substrate and provides new leads to target this modification. Synopsis: A combination of AlphaFold2 modeling, X‐ray crystallography, NMR, and biochemical experiments reveals a crucial role for the UFL1 N‐terminal helix in binding to UFC1 and productive ufmylation.Design of a functional UFL1‐DDRGK1 fusion protein using AlphaFold2.The crystal structure of the UFL1‐DDRGK1 fusion protein reveals a shared winged helix domain.The UFL1 N‐terminus competes with UBA5 for binding to UFC1 and is essential for ufmylation activity.The UFM1‐binding site of DDRGK1 contributes to preferential binding to charged UFC1. [ABSTRACT FROM AUTHOR]

Ubiquitination is a critical type of post-translational modification in eukaryotic cells. It is involved in regulating nearly all cellular processes in the cytosol and nucleus. Mitochondria, known as the metabolism heart of the cell, are organelles that evolved from bacteria. Using the subcellular compartment-dependent α-complementation, we detect multiple components of ubiquitination machinery as being eclipsed distributed to yeast mitochondria. Ubiquitin conjugates and mono-ubiquitin can be detected in lysates of isolated mitochondria from cells expressing HA-Ub and treated with trypsin. By expressing MTS (mitochondrial targeting sequence) targeted HA-tagged ubiquitin, we demonstrate that certain ubiquitination events specifically occur in yeast mitochondria and are independent of proteasome activity. Importantly, we show that the E2 Rad6 affects the pattern of protein ubiquitination in mitochondria and provides an in vivo assay for its activity in the matrix of the organelle. This study shows that ubiquitination occurs in the mitochondrial matrix by eclipsed targeted components of the ubiquitin machinery, providing a new perspective on mitochondrial and ubiquitination research. [ABSTRACT FROM AUTHOR]

OTUB1 was previously identified as a K48 linkage-specific deubiquitinating enzyme that contains two distinct ubiquitin-binding sites (Fig. 1a): a distal site and a proximal site that includes the ~45 N-terminal [...], Histones are ubiquitinated in response to DNA double-strand breaks (DSB), promoting recruitment of repair proteins to chromatin (1). UBC13 (also known as UBE2N) is a ubiquitin-conjugating enzyme (E2) that heterodimerizes with UEV1A (2) (also known as UBE2V1) and synthesizes K63-linked polyubiquitin (K63Ub) chains at DSB sites in concert with the ubiquitin ligase (E3), RNF168 (ref. 3). K63Ub synthesis is regulated in a non-canonical manner by the deubiquitinating enzyme, OTUB1 (OTU domain-containing ubiquitin aldehyde-binding protein 1), which binds preferentially to the UBC13~Ub thiolester (4). Residues amino-terminal to the OTU domain, which had been implicated in ubiquitin binding (4), are required for binding to UBC13~Ub and inhibition of K63Ub synthesis (5). Here we describe structural and biochemical studies elucidating how OTUB1 inhibits UBC13 and other E2 enzymes. We unexpectedly find that OTUB1 binding to UBC13~Ub is allosterically regulated by free ubiquitin, which binds to a second site in OTUB1 and increases its affinity for UBC13~Ub, while at the same time disrupting interactions with UEV1A in a manner that depends on the OTUB1 N terminus. Crystal structures of an OTUB1-UBC13 complex and of OTUB1 bound to ubiquitin aldehyde and a chemical UBC13~Ub conjugate show that binding of free ubiquitin to OTUB1 triggers conformational changes in the OTU domain and formation of a ubiquitin-binding helix in the N terminus, thus promoting binding of the conjugated donor ubiquitin in UBC13~Ub to OTUB1. The donor ubiquitin thus cannot interact with the E2 enzyme, which has been shown to be important for ubiquitin transfer (6,7). The N-terminal helix of OTUB1 is positioned to interfere with UEV1A binding to UBC13, as well as with attack on the thiolester by an acceptor ubiquitin, thereby inhibiting K63Ub synthesis. OTUB1 binding also occludes the RING E3 binding site on UBC13, thus providing a further component of inhibition. The general features of the inhibition mechanism explain how OTUB1 inhibits other E2 enzymes (4) in a non-catalytic manner.

Ufmylation is a posttranslational modification in which the modifier UFM1 is attached to target proteins. This conjugation requires the concerted work of three enzymes named UBA5, UFC1, and UFL1. Initially, UBA5 activates UFM1 in a process that ends with UFM1 attached to UBA5's active site Cys. Then, in a trans-thiolation reaction, UFM1 is transferred from UBA5 to UFC1, forming a thioester bond with the latter. Finally, with the help of UFL1, UFM1 is transferred to the final destination—a lysine residue on a target protein. Therefore, not surprisingly, deletion of one of these enzymes abrogates the conjugation process. However, how overexpression of these enzymes affects this process is not yet clear. Here we found, unexpectedly, that overexpression of UBA5, but not UFC1, damages the ability of cells to migrate, in a similar way to cells lacking UBA5 or UFC1. At the mechanistic level, we found that overexpression of UBA5 reverses the trans-thiolation reaction, thereby leading to a back transfer of UFM1 from UFC1 to UBA5. This, as seen in cells lacking UBA5, reduces the level of charged UFC1 and therefore harms the conjugation process. In contrast, co-expression of UBA5 with UFM1 abolishes this effect, suggesting that the reverse transfer of UFM1 from UFC1 to UBA5 depends on the level of free UFM1. Overall, our results propose that the cellular expression level of the UFM1 conjugation enzymes has to be tightly regulated to ensure the proper directionality of UFM1 transfer. [ABSTRACT FROM AUTHOR]

Recent studies on the oral, anaerobic, gram-negative bacterium Fusobacterium nucleatum revealed its presence and involvement in colorectal, esophageal and breast cancer. We previously demonstrated that F. nucleatum binds and activates the human inhibitory receptors TIGIT and CEACAM1 leading to inhibition of T and NK cell anti-tumor immunity. CEACAM1 was found to be bound and activated by the fusobacterial trimeric autotransporter adhesin CbpF. Here we report the generation of a recombinant E. coli expressing full-length CbpF that efficiently binds and activates CEACAM1. [ABSTRACT FROM AUTHOR]

Summary Modification of proteins by ubiquitin or ubiquitin-like proteins (UBLs) is a critical cellular process implicated in a variety of cellular states and outcomes. A prerequisite for target protein modification by a UBL is the activation of the latter by activating enzymes (E1s). Here, we present the crystal structure of the non-canonical homodimeric E1, UBA5, in complex with its cognate UBL, UFM1, and supporting biochemical experiments. We find that UBA5 binds to UFM1 via a trans -binding mechanism in which UFM1 interacts with distinct sites in both subunits of the UBA5 dimer. This binding mechanism requires a region C-terminal to the adenylation domain that brings UFM1 to the active site of the adjacent UBA5 subunit. We also find that transfer of UFM1 from UBA5 to the E2, UFC1, occurs via a trans mechanism, thereby requiring a homodimer of UBA5. These findings explicitly elucidate the role of UBA5 dimerization in UFM1 activation. [ABSTRACT FROM AUTHOR]

OTUB1 is a Lys48-specific deubiquitinating enzyme that forms a complex in vivo with E2 ubiquitin (Ub)-conjugating enzymes including UBC13 and UBCH5. OTUB1 binds E2~Ub thioester intermediates and prevents ubiquitin transfer, thereby noncatalytically inhibiting accumulation of polyubiquitin. We report here that a second role of OTUB1-E2 interactions is to stimulate OTUB1 cleavage of Lys48 polyubiquitin. This stimulation is regulated by the ratio of charged to uncharged E2 and by the concentration of Lys48-linked polyubiquitin and free ubiquitin. Structural and biochemical studies of human and worm OTUB1 and UBCH5B show that the E2 enzyme stimulates binding of the Lys48 polyubiquitin substrate by stabilizing folding of the OTUB1 N-terminal ubiquitin-binding helix. Our results suggest that OTUB1-E2 complexes in the cell are poised to regulate polyubiquitin chain elongation or degradation in response to changing levels of E2 charging and available free ubiquitin. [ABSTRACT FROM AUTHOR]

Voltage-gated K+ channels co-assemble with auxiliary β subunits to form macromolecular complexes. In heart, assembly of Kv7.1 pore-forming subunits with KCNE1 β subunits generates the repolarizing K+ current IKS. However, the detailed nature of their interface remains unknown. Mutations in either Kv7.1 or KCNE1 produce the life-threatening long or short QT syndromes. Here, we studied the interactions and voltage-dependent motions of IKS channel intracellular domains, using fluorescence resonance energy transfer combined with voltage-clamp recording and in vitro binding of purified proteins. The results indicate that the KCNE1 distal C-terminus interacts with the coiled-coil helix C of the Kv7.1 tetramerization domain. This association is important for IKS channel assembly rules as underscored by Kv7.1 current inhibition produced by a dominant-negative C-terminal domain. On channel opening, the C-termini of Kv7.1 and KCNE1 come close together. Co-expression of Kv7.1 with the KCNE1 long QT mutant D76N abolished the K+ currents and gated motions. Thus, during channel gating KCNE1 is not static. Instead, the C-termini of both subunits experience molecular motions, which are disrupted by the D76N causing disease mutation. [ABSTRACT FROM AUTHOR]

It is widely accepted that ubiquitin-conjugating enzymes contain an active site asparagine that serves as an oxyanion hole, thereby stabilizing a negatively charged transition state intermediate and promoting ubiquitin transfer. Using structural and biochemical approaches to study the role of the conserved asparagine to ubiquitin conjugation by Ubc13-Mms2, we conclude that the importance of this residue stems primarily from its structural role in stabilizing an active site loop. [ABSTRACT FROM AUTHOR]

Besides ubiquitin (Ub), humans have a set of ubiquitin-like proteins (UBLs) that can also covalently modify target proteins. To date, less is known about UBLs than Ub and even less is known about the UBL called ubiquitin-fold modifier 1 (UFM1). Currently, our understanding of protein modification by UFM1 (UFMylation) is like a jigsaw puzzle with many missing pieces, and in some cases it is not even clear whether these pieces of data are in the right place. Here we review the current data on UFM1 from structural biology to biochemistry and cell biology. We believe that the physiological significance of protein modification by UFM1 is currently underestimated and there is more to it than meets the eye. [ABSTRACT FROM AUTHOR]

The article discusses a 2022 study on the human ds-prenyltransferase complex (hcis-PT) at the endoplasmic reticulum membrane that has a role in protein glycosylation. Topics covered include the hydrophobic active site core's flanking by regions consistent with separate inlet and outlet orifices, and the product elongation-membrane association correlation. Also noted is the membrane insertion support of the isoprenoid during catalysis, that uncouples active site volume from product length.

Intramuscularly administered vaccines stimulate robust serum neutralizing antibodies, yet they are often less competent in eliciting sustainable "sterilizing immunity" at the mucosal level. Our study uncovers a strong temporary neutralizing mucosal component of immunity, emanating from intramuscular administration of an mRNA vaccine. We show that saliva of BNT162b2 vaccinees contains temporary IgA targeting the receptor-binding domain (RBD) of severe acute respiratory syndrome coronavirus-2 spike protein and demonstrate that these IgAs mediate neutralization. RBD-targeting IgAs were found to associate with the secretory component, indicating their bona fide transcytotic origin and their polymeric multivalent nature. The mechanistic understanding of the high neutralizing activity provided by mucosal IgA, acting at the first line of defense, will advance vaccination design and surveillance principles and may point to novel treatment approaches and new routes of vaccine administration and boosting., Competing Interests: Hebrew University of Jerusalem, Hadassah-Hebrew University Medical Center and Hadassah Academic College has filed a patent application “Compounds and methods for increasing antibody’s neutralization properties and methods for assessing antibody response” on which MSR, SuK, AF, PG, RW, DP, LB, and AR are listed as inventors. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Stolovich-Rain, Kumari, Friedman, Kirillov, Socol, Billan, Pal, Das, Golding, Oiknine-Djian, Sirhan, Sagie, Cohen-Kfir, Gold, Fahoum, Kumar, Elgrably-Weiss, Zhou, Ravins, Gatt, Bhattacharya, Zelig, Wiener, Wolf, Elinav, Strahilevitz, Padawer, Baraz and Rouvinski.)

Mapping co-evolved genes via phylogenetic profiling (PP) is a powerful approach to uncover functional interactions between genes and to associate them with pathways. Despite many successful endeavors, the understanding of co-evolutionary signals in eukaryotes remains partial. Our hypothesis is that 'Clades', branches of the tree of life (e.g. primates and mammals), encompass signals that cannot be detected by PP using all eukaryotes. As such, integrating information from different clades should reveal local co-evolution signals and improve function prediction. Accordingly, we analyzed 1028 genomes in 66 clades and demonstrated that the co-evolutionary signal was scattered across clades. We showed that functionally related genes are frequently co-evolved in only parts of the eukaryotic tree and that clades are complementary in detecting functional interactions within pathways. We examined the non-homologous end joining pathway and the UFM1 ubiquitin-like protein pathway and showed that both demonstrated distinguished co-evolution patterns in specific clades. Our research offers a different way to look at co-evolution across eukaryotes and points to the importance of modular co-evolution analysis. We developed the 'CladeOScope' PP method to integrate information from 16 clades across over 1000 eukaryotic genomes and is accessible via an easy to use web server at http://cladeoscope.cs.huji.ac.il., (© The Author(s) 2021. Published by Oxford University Press on behalf of NAR Genomics and Bioinformatics.)

An acto-myosin contractile ring, which forms after anaphase onset and is highly regulated in time and space, mediates cytokinesis, the final step of mitosis. The chromosomal passenger complex (CPC), composed of Aurora-B kinase, INCENP, borealin and survivin (also known as BIRC5), regulates various processes during mitosis, including cytokinesis. It is not understood, however, how CPC regulates cytokinesis. We show that survivin binds to non-muscle myosin II (NMII), regulating its filament assembly. Survivin and NMII interact mainly in telophase, and Cdk1 regulates their interaction in a mitotic-phase-specific manner, revealing the mechanism for the specific timing of survivin-NMII interaction during mitosis. The survivin-NMII interaction is indispensable for cytokinesis, and its disruption leads to multiple mitotic defects. We further show that only the survivin homodimer binds to NMII, attesting to the biological importance for survivin homodimerization. We suggest a novel function for survivin in regulating the spatio-temporal formation of the acto-NMII contractile ring during cytokinesis and we elucidate the role of Cdk1 in regulating this process.This article has an associated First Person interview with the first author of the paper., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2019. Published by The Company of Biologists Ltd.)

Ubiquitin (Ub)-mediated protein degradation is a key cellular defense mechanism that detects and eliminates defective proteins. A major intracellular site of protein quality control degradation is the endoplasmic reticulum (ER), hence the term ER-associated degradation, or endoplasmic reticulum-associated degradation (ERAD). Yeast ERAD is composed of three Ub-protein conjugation complexes, named according to their E3 Ub-protein ligase components, Hrd1, Doa10, and the Asi complex, which resides at the nuclear envelope (NE). These ER/NE membrane-associated RING-type E3 ligases recognize and ubiquitylate defective proteins in cooperation with the E2 conjugating enzyme Ubc7 and the obligatory Ubc7 cofactor Cue1. Interaction of Ubc7 with the RING domains of its cognate E3 Ub-protein ligases stimulates the formation of isopeptide (amide) Ub-Ub linkages. Each isopeptide bond is formed by transfer of an Ubc7-linked activated Ub to a lysine side chain of an acceptor Ub. Multiple Ub transfer reactions form a poly-Ub chain that targets the conjugated protein for degradation by the proteasome. To study the mechanism of Ub-Ub bond formation, this reaction is reconstituted in a cell-free system consisting of recombinant E1, Ub, Ubc7, its cofactor Cue1, and the RING domain of either Doa10 or Hrd1. Here we provide detailed protocols for the purification of the required recombinant proteins and for the reactions that produce an Ub-Ub bond, specifically, the formation of an Ubc7~Ub thiolester (Ub charging) and subsequent formation of the isopeptide Ub-Ub linkage (Ub transfer). These protocols also provide a useful guideline for similar in vitro ubiquitylation reactions intended to explore the mechanism of other Ub-conjugation systems., (© 2019 Elsevier Inc. All rights reserved.)

The modification of proteins by ubiquitin-fold modifier 1 (UFM1) is implicated in many human diseases. Prior to conjugation, UFM1 undergoes activation by its cognate activating enzyme, UBA5. UBA5 is a non-canonical E1 activating enzyme that possesses an adenylation domain but lacks a distinct cysteine domain. Binding of UBA5 to UFM1 is mediated via an amino acid sequence, known as the UFM1-interacting sequence (UIS), located outside the adenylation domain that is required for UFM1 activation. However, the precise boundaries of the UIS are yet not clear and are still under debate. Here we revisit the interaction of UFM1 with UBA5 by determining the crystal structure of UFM1 fused to 13 amino acids of human UBA5. Using binding and activity assays, we found that His 336 of UBA5, previously not reported to be part of the UIS, occupies a negatively charged pocket on UFM1's surface. This His is involved in UFM1 binding and if mutated perturbs activation of UFM1. Surprisingly, we also found that the interaction between two UFM1 molecules mimics how the UIS binds UFM1. Specifically, UFM1 His 70 resembles UBA5 His336 and enters a negatively charged pocked on the other UFM1 molecule. Our results refine our understanding of UFM1-UBA5 binding.

The slow IKS K+ channel plays a major role in repolarizing the cardiac action potential and consists of the assembly of KCNQ1 and KCNE1 subunits. Mutations in either KCNQ1 or KCNE1 genes produce the long-QT syndrome, a life-threatening ventricular arrhythmia. Here, we show that long-QT mutations located in the KCNQ1 C terminus impair calmodulin (CaM) binding, which affects both channel gating and assembly. The mutations produce a voltage-dependent macroscopic inactivation and dramatically alter channel assembly. KCNE1 forms a ternary complex with wild-type KCNQ1 and Ca(2+)-CaM that prevents inactivation, facilitates channel assembly, and mediates a Ca(2+)-sensitive increase of IKS-current, with a considerable Ca(2+)-dependent left-shift of the voltage-dependence of activation. Coexpression of KCNQ1 or IKS channels with a Ca(2+)-insensitive CaM mutant markedly suppresses the currents and produces a right shift in the voltage-dependence of channel activation. KCNE1 association to KCNQ1 long-QT mutants significantly improves mutant channel expression and prevents macroscopic inactivation. However, the marked right shift in channel activation and the subsequent decrease in current amplitude cannot restore normal levels of IKS channel activity. Our data indicate that in healthy individuals, CaM binding to KCNQ1 is essential for correct channel folding and assembly and for conferring Ca(2+)-sensitive IKS-current stimulation, which increases the cardiac repolarization reserve and hence prevents the risk of ventricular arrhythmias.