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Microtubules are regulated by posttranslational modifications (PTMs) of tubulin. The ligation and cleavage of the C-terminal tyrosine of α tubulin impact microtubule functions during mitosis, cardiomyocyte contraction, and neuronal processes. Tubulin tyrosination and detyrosination are mediated by tubulin tyrosine ligase (TTL) and the recently discovered tubulin detyrosinases, vasohibin 1 and 2 (VASH1 and VASH2) bound to the small vasohibin-binding protein (SVBP). Here, we report the crystal structures of human VASH1–SVBP alone, in complex with a tyrosine-derived covalent inhibitor, and bound to the natural product parthenolide. The structures and subsequent mutagenesis analyses explain the requirement for SVBP during tubulin detyrosination, and reveal the basis for the recognition of the C-terminal tyrosine and the acidic α tubulin tail by VASH1. The VASH1–SVBP–parthenolide structure provides a framework for designing more effective chemical inhibitors of vasohibins, which can be valuable for dissecting their biological functions and may have therapeutic potential.

OBJECTIVE: To evaluate the effectiveness of the nose ring drain (NRD) in treatment of severe diabetic foot infection. METHODS: The clinical data of 35 patients with severe diabetic foot infection who were treated with NRD between June 2017 and June 2019 were analyzed retrospectively. There were 24 males and 11 females with an average age of 54.5 years (range, 28-82 years). All of them were type 2 diabetic patients. The diabetes duration was 3-20 years, with an average of 9.4 years. The diabetic foot duration was 4 months to 2 years, with an average of 1.16 years. There were 16 cases of left foot and 19 cases of right foot. According to Wagner’s grading, there were 11 cases of grade 2, 20 cases of grade 3, and 4 cases of grade 4, all of which were moderate and severe infection of diabetic foot wound. Postoperative wounds were treated with “nibble-like” debridement until the patient’s epidermis regenerated and healed. During the treatment process, the indexes of bacterial culture type of wound secretions, duration of antibiotic therapy, wound healing method, healing time, amputation rate, and other indicators were analyzed and summarized. RESULTS: All 35 patients were followed up 3-6 months, with an average of 4.5 months. Postoperative bacterial culture of wounds showed that 5 cases of Staphylococcus aureus, 4 cases of Pseudomonas aeruginosa, 5 cases of Escherichia coli, 3 cases of Enterobacter cloacae, 3 cases of coagulase-negative Staphylococcus, and 15 cases of other types were detected. The duration of antibiotic therapy ranged from 3 to 15 days, with an average of 9.1 days. The wound was autolytically healed without skin grafting, and the healing time was 62-82 days, with an average of 72.3 days. During the follow-up, 3 cases (8.6%) had amputation due to the patient’s poor blood glucose control, which led to a large spread of infection. In addition, among the other patients with wound healing, there was no recurrence of wound infection or new ulcer on the original surface. CONCLUSION: The NRD is a simple operation for treatment of severe diabetic foot infection, which can effectively control wound infections and promote wound healing and regeneration without skin grafting.

The dynamic tyrosination-detyrosination cycle of α-tubulin regulates microtubule functions. Perturbation of this cycle impairs mitosis, neural physiology, and cardiomyocyte contraction. The carboxypeptidases vasohibins 1 and 2 (VASH1 and VASH2), in complex with the small vasohibin-binding protein (SVBP), mediate α-tubulin detyrosination. These enzymes detyrosinate microtubules more efficiently than soluble αβ-tubulin heterodimers. The structural basis for this substrate preference is not understood. Using cryo-electron microscopy (cryo-EM), we have determined the structure of human VASH1-SVBP bound to microtubules. The acidic C-terminal tail of α-tubulin binds to a positively charged groove near the active site of VASH1. VASH1 forms multiple additional contacts with the globular domain of α-tubulin, including contacts with a second α-tubulin in an adjacent protofilament. Simultaneous engagement of two protofilaments by VASH1 can only occur within the microtubule lattice, but not with free αβ heterodimers. These lattice-specific interactions enable preferential detyrosination of microtubules by VASH1.

The spindle checkpoint maintains genomic stability and prevents aneuploidy. Unattached kinetochores convert the latent open conformer of the checkpoint protein Mad2 (O-Mad2) to the active closed conformer (C-Mad2), bound to Cdc20. C-Mad2–Cdc20 is incorporated into the mitotic checkpoint complex (MCC), which inhibits the anaphase-promoting complex/cyclosome (APC/C). The C-Mad2-binding protein p31comet and the ATPase TRIP13 promote MCC disassembly and checkpoint silencing. Here, using nuclear magnetic resonance (NMR) spectroscopy, we show that TRIP13 and p31comet catalyze the conversion of C-Mad2 to O-Mad2, without disrupting its stably folded core. We determine the crystal structure of human TRIP13, and identify functional TRIP13 residues that mediate p31comet–Mad2 binding and couple ATP hydrolysis to local unfolding of Mad2. TRIP13 and p31comet prevent APC/C inhibition by MCC components, but cannot reactivate APC/C already bound to MCC. Therefore, TRIP13–p31comet intercepts and disassembles free MCC not bound to APC/C through mediating the local unfolding of the Mad2 C-terminal region., The spindle checkpoint ensures the fidelity of chromosome segregation during mitosis and meiosis. Here the authors use a combination of biochemical and structural biology approaches to show how the TRIP13 ATPase and its adaptor, p31comet, catalyze the conversion of the checkpoint protein Mad2 between latent and active forms

Optineurin is an important autophagy receptor involved in several selective autophagy processes, during which its function is regulated by TBK1. Mutations of optineurin and TBK1 are both associated with neurodegenerative diseases. However, the mechanistic basis underlying the specific interaction between optineurin and TBK1 is still elusive. Here we determine the crystal structures of optineurin/TBK1 complex and the related NAP1/TBK1 complex, uncovering the detailed molecular mechanism governing the optineurin and TBK1 interaction, and revealing a general binding mode between TBK1 and its associated adaptor proteins. In addition, we demonstrate that the glaucoma-associated optineurin E50K mutation not only enhances the interaction between optineurin and TBK1 but also alters the oligomeric state of optineurin, and the ALS-related TBK1 E696K mutation specifically disrupts the optineurin/TBK1 complex formation but has little effect on the NAP1/TBK1 complex. Thus, our study provides mechanistic insights into those currently known disease-causing optineurin and TBK1 mutations found in patients., Mutations in optineurin that cause defects in the interaction with TBK1 are associated with neurodegenerative diseases. Here, the authors report the structure of this complex, and outline a general binding mode for these proteins.

OPTN (optineurin), a ubiquitin-binding scaffold protein, functions as an important macroautophagy/autophagy receptor in selective autophagy processes. Mutations in OPTN have been linked with human neurodegenerative diseases including ALS and glaucoma. However, the mechanistic basis underlying the recognition of ubiquitin by OPTN and its regulation by TBK1-mediated phosphorylation are still elusive. Here, we demonstrate that the UBAN domain of OPTN preferentially recognizes linear ubiquitin chain and forms an asymmetric 2:1 stoichiometry complex with the linear diubiquitin. In addition, our results provide new mechanistic insights into how phosphorylation of UBAN would regulate the ubiquitin-binding ability of OPTN and how disease-associated mutations in the OPTN UBAN domain disrupt its interaction with ubiquitin. Finally, we show that defects in ubiquitin-binding may affect the recruitment of OPTN to linear ubiquitin-decorated mutant Huntington protein aggregates. Taken together, our findings clarify the interaction mode between UBAN and linear ubiquitin chain in general, and expand our knowledge of the molecular mechanism of ubiquitin-decorated substrates recognition by OPTN as well as the pathogenesis of neurodegenerative diseases caused by OPTN mutations.

Summary: The DNA-dependent metalloprotease Spartan (SPRTN) cleaves DNA-protein crosslinks (DPCs) and protects cells from DPC-induced genome instability. Germline mutations of SPRTN are linked to human Ruijs-Aalfs syndrome (RJALS) characterized by progeria and early-onset hepatocellular carcinoma. The mechanism of DNA-mediated activation of SPRTN is not understood. Here, we report the crystal structure of the human SPRTN SprT domain bound to single-stranded DNA (ssDNA). Our structure reveals a Zn2+-binding sub-domain (ZBD) in SprT that shields its active site located in the metalloprotease sub-domain (MPD). The narrow catalytic groove between MPD and ZBD only permits cleavage of flexible substrates. The ZBD contains an ssDNA-binding site, with a DNA-base-binding pocket formed by aromatic residues. Mutations of ssDNA-binding residues diminish the protease activity of SPRTN. We propose that the ZBD contributes to the ssDNA specificity of SPRTN, restricts the access of globular substrates, and positions DPCs, which may need to be partially unfolded, for optimal cleavage. : Li et al. determine the crystal structure of human Spartan bound to DNA. Spartan is a DNA-activated metalloprotease that cleaves DNA-protein crosslinks to preserve genome stability. Spartan mutations cause progeria and liver cancer in humans. The structure provides important insights into how DNA activates the protease activity of Spartan. Keywords: DNA repair, genome instability, DNA-protein crosslinks, metalloprotease, Spartan, Zn2+ binding, RJALS

FYCO1 (FYVE and coiled-coil domain containing 1) functions as an autophagy adaptor in directly linking autophagosomes with the microtubule-based kinesin motor, and plays an essential role in the microtubule plus end-directed transport of autophagic vesicles. The specific association of FYCO1 with autophagosomes is mediated by its interaction with Atg8-family proteins decorated on the outer surface of autophagosome. However, the mechanistic basis governing the interaction between FYCO1 and Atg8-family proteins is largely unknown. Here, using biochemical and structural analyses, we demonstrated that FYCO1 contains a unique LC3-interacting region (LIR), which discriminately binds to mammalian Atg8 orthologs and preferentially binds to the MAP1LC3A and MAP1LC3B. In addition to uncovering the detailed molecular mechanism underlying the FYCO1 LIR and MAP1LC3A interaction, the determined FYCO1-LIR-MAP1LC3A complex structure also reveals a unique LIR binding mode for Atg8-family proteins, and demonstrates, first, the functional relevance of adjacent sequences C-terminal to the LIR core motif for binding to Atg8-family proteins. Taken together, our findings not only provide new mechanistic insight into FYCO1-mediated transport of autophagosomes, but also expand our understanding of the interaction modes between LIR motifs and Atg8-family proteins in general.

The autophagy receptor CALCOCO2/NDP52 functions as a bridging adaptor and plays an essential role in the selective autophagic degradation of invading pathogens by specifically recognizing ubiquitin-coated intracellular pathogens and subsequently targeting them to the autophagic machinery; thereby it is required for innate immune defense against a range of infectious pathogens in mammals. However, the mechanistic basis underlying CALCOCO2-mediated specific recognition of ubiqutinated pathogens is still unknown. Here, using biochemical and structural analyses, we demonstrated that the cargo-binding region of CALCOCO2 contains a dynamic unconventional zinc finger as well as a C2H2-type zinc-finger, and only the C2H2-type zinc finger specifically recognizes mono-ubiquitin or poly-ubiquitin chains. In addition to elucidating the specific ubiquitin recognition mechanism of CALCOCO2, the structure of the CALCOCO2 C2H2-type zinc finger in complex with mono-ubiquitin also uncovers a unique zinc finger-binding mode for ubiquitin. Our findings provide mechanistic insight into how CALCOCO2 targets ubiquitin-decorated pathogens for autophagic degradations.