Category Archives: FEBS Letters

Structural and functional insights of itaconyl‐CoA hydratase from Pseudomonas aeruginosa highlight a novel N‐terminal hotdog fold

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Structural and functional insights of itaconyl-CoA hydratase from Pseudomonas aeruginosa highlight a novel N-terminal hotdog fold

Here, we report the first crystal structure of PaIch at 1.98 Å resolution. A unique N-terminal hotdog fold containing a short helical segment α3-, named an “eaten sausage”, slipped away from the conserved β-sheet scaffold, whereas central helices of other N- as well as C-terminal hotdog fold containing hydratases are properly wrapped up by their respective β-sheet scaffold.


Itaconyl-CoA hydratase in Pseudomonas aeruginosa (PaIch) converts itaconyl-CoA to (S)-citramalyl-CoA upon addition of a water molecule, a part of an itaconate catabolic pathway in virulent organisms required for their survival in humans host cells. Crystal structure analysis of PaIch showed that a unique N-terminal hotdog fold containing a 4-residue short helical segment α3-, named as an “eaten sausage”, followed by a flexible loop region slipped away from the conserved β-sheet scaffold, whereas the C-terminal hotdog fold is similar to all MaoC. A conserved hydratase motif with catalytic residues provides mechanistic insights into catalysis, and existence of a longer substrate binding tunnel may suggest the binding of longer CoA derivatives.

Skraban‐Deardorff intellectual disability syndrome‐associated mutations in WDR26 impair CTLH E3 complex assembly

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Skraban-Deardorff intellectual disability syndrome-associated mutations in WDR26 impair CTLH E3 complex assembly

The Skraban-Deardorff intellectual disability syndrome (SKDEAS) is associated with diverse mutations in WDR26, encoding a subunit required to assemble a giant oval-shaped supramolecular CTLH E3 ubiquitin ligase complex and substrate recruitment. Structural modeling of SKDEAS-associated mutations combined with functional assays revealed impaired CTLH E3 complex assembly and interactions, thus providing first mechanistic insights into SKDEAS pathology.


Patients with Skraban-Deardorff syndrome (SKDEAS), a neurodevelopmental syndrome associated with a spectrum of developmental and intellectual delays and disabilities, harbor diverse mutations in WDR26, encoding a subunit of the multiprotein CTLH E3 ubiquitin ligase complex. Structural studies revealed that homodimers of WDR26 bridge two core-CTLH E3 complexes to generate giant, hollow oval-shaped supramolecular CTLH E3 assemblies. Additionally, WDR26 mediates CTLH E3 complex binding to subunit YPEL5 and functions as substrate receptor for the transcriptional repressor HBP1. Here, we mapped SKDEAS-associated mutations on a WDR26 structural model and tested their functionality in complementation studies using genetically engineered human cells lacking CTLH E3 supramolecular assemblies. Despite the diversity of mutations, 15 of 16 tested mutants impaired at least one CTLH E3 complex function contributing to complex assembly and interactions, thus providing first mechanistic insights into SKDEAS pathology.

The scorpion toxin BeKm‐1 blocks hERG cardiac potassium channels using an indispensable arginine residue

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The scorpion toxin BeKm-1 blocks hERG cardiac potassium channels using an indispensable arginine residue

Using computational methods, we constructed a model of the cardiac ion channel hERG in complex with BeKm-1, a scorpion toxin. We identified the crucial role of the toxin residue Arg20 and validated it by in silico and in vitro mutagenesis. The BeKm-1R20K mutant showed dramatically reduced activity, suggesting the significance of Arg20 for channel binding. Our model aids future drug design attempts.


BeKm-1 is a peptide toxin from scorpion venom that blocks the pore of the potassium channel hERG (Kv11.1) in the human heart. Although individual protein structures have been resolved, the structure of the complex between hERG and BeKm-1 is unknown. Here, we used molecular dynamics and ensemble docking, guided by previous double-mutant cycle analysis data, to obtain an in silico model of the hERG–BeKm-1 complex. Adding to the previous mutagenesis study of BeKm-1, our model uncovers the key role of residue Arg20, which forms three interactions (a salt bridge and hydrogen bonds) with the channel vestibule simultaneously. Replacement of this residue even by lysine weakens the interactions significantly. In accordance, the recombinantly produced BeKm-1R20K mutant exhibited dramatically decreased activity on hERG. Our model may be useful for future drug design attempts.

Caspase‐3 targets pro‐interleukin‐1β (IL‐1β) to restrict inflammation

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Caspase-3 targets pro-interleukin-1β (IL-1β) to restrict inflammation

The present study suggests pro-IL-1β as a novel substrate of caspase-3. The activation of apoptotic signaling induces caspase-3 to cleave pro-IL-1β at the Asp26 site, and the generation of the Asp26 site restricts the inflammasome-mediated cleavage at the Asp117 site. Thus, caspase-3 prevents the release of mature IL-1β into the extracellular environment.


The interleukin (IL)-1 family of cytokines plays a pivotal role in immune responses. Among the members of IL-1 family, IL-1β is synthesized as an inactive precursor (pro-IL-1β) and becomes active upon cleavage, which is typically facilitated by inflammasomes through caspase-1. In our research, we explored the potential role of caspase-3 in the cleavage of pro-IL-1β and found that caspase-3 cleaves pro-IL-1β, specifically at Asp26. Moreover, we found that in the absence of caspase-3 cleavage, the release of active IL-1β via the inflammasome is increased. Our study introduces pro-IL-1β as a new substrate for caspase-3 and suggests that caspase-3-mediated cleavage has the potential to suppress IL-1β-mediated inflammatory responses.

Cryo‐EM structure of the Slo1 potassium channel with the auxiliary γ1 subunit suggests a mechanism for depolarization‐independent activation

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Cryo-EM structure of the Slo1 potassium channel with the auxiliary γ1 subunit suggests a mechanism for depolarization-independent activation

γ subunits allow Slo potassium channels to open without an action potential. By cryo-EM structure determination, we show how γ1 binds the voltage-sensor domain (VSD) of Slo1. The kinked transmembrane helix and an extracellular hook of γ1 stabilize the VSD in its active conformation, while an intracellular polybasic stretch locally decreases the resting potential.


Mammalian Ca2+-dependent Slo K+ channels can stably associate with auxiliary γ subunits which fundamentally alter their behavior. By a so far unknown mechanism, the four γ subunits reduce the need for voltage-dependent activation and, thereby, allow Slo to open independently of an action potential. Here, using cryo-EM, we reveal how the transmembrane helix of γ1/LRRC26 binds and presumably stabilizes the activated voltage-sensor domain of Slo1. The activation is further enhanced by an intracellular polybasic stretch which locally changes the charge gradient across the membrane. Our data provide a possible explanation for Slo1 regulation by the four γ subunits and also their different activation efficiencies. This suggests a novel activation mechanism of voltage-gated ion channels by auxiliary subunits.

Gulp1 regulates chondrocyte growth arrest and differentiation via the TGF‐β/SMAD2/3 pathway

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Gulp1 regulates chondrocyte growth arrest and differentiation via the TGF-β/SMAD2/3 pathway

We report a novel function of Gulp1 in chondrocyte differentiation. Gulp1 knockdown in chondrogenic ATDC5 cells reduces the expression of chondrogenic marker genes, impairs cell growth arrest, and decreases p21 levels during differentiation. This knockdown also desrupts the TGF-β/SMAD2/3 pathway activation linked to p21 expression, highlighting Gulp1's involvment in regulating chondrocyte differentiation and growth arrest via the TGF-β/SMAD2/3 pathway.


Chondrocyte differentiation is crucial for cartilage formation. However, the complex processes and mechanisms coordinating chondrocyte proliferation and differentiation remain incompletely understood. Here, we report a novel function of the adaptor protein Gulp1 in chondrocyte differentiation. Gulp1 expression is upregulated during chondrogenic differentiation. Gulp1 knockdown in chondrogenic ATDC5 cells reduces the expression of chondrogenic and hypertrophic marker genes during differentiation. Furthermore, Gulp1 knockdown impairs cell growth arrest during chondrocyte differentiation and reduces the expression of the cyclin-dependent kinase inhibitor p21. The activation of the TGF-β/SMAD2/3 pathway, which is associated with p21 expression in chondrocytes, is impaired in Gulp1 knockdown cells. Collectively, these results demonstrate that Gulp1 contributes to cell growth arrest and chondrocyte differentiation by modulating the TGF-β/SMAD2/3 pathway.

Generation of novel anti‐apoE monoclonal antibodies that selectively recognize apoE isoforms

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Generation of novel anti-apoE monoclonal antibodies that selectively recognize apoE isoforms

Apolipoprotein E (apoE) is a regulator of lipid metabolism, cholesterol transport, and the clearance and aggregation of amyloid β in the brain. Human apoE4 isoform is a risk factor for apoE-related diseases, although only one or two residues are different in other isoforms. Here, we generated novel anti-apoE monoclonal antibodies and constructed a sandwich ELISA system to selectively detect the apoE4 isoform.


Apolipoprotein E (apoE) is a regulator of lipid metabolism, cholesterol transport, and the clearance and aggregation of amyloid β in the brain. The three human apoE isoforms apoE2, apoE3, and apoE4 only differ in one or two residues. Nevertheless, the functions highly depend on the isoform types and lipidated states. Here, we generated novel anti-apoE monoclonal antibodies (mAbs) and obtained an apoE4-selective mAb whose epitope is within residues 110–117. ELISA and bio-layer interferometry measurements demonstrated that the dissociation constants of mAbs are within the nanomolar range. Using the generated antibodies, we successfully constructed sandwich ELISA systems, which can detect all apoE isoforms or selectively detect apoE4. These results suggest the usability of the generated anti-apoE mAbs for selective detection of apoE isoforms.

Comparative structural and functional analysis of the glycine‐rich regions of Class A and B J‐domain protein cochaperones of Hsp70

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Comparative structural and functional analysis of the glycine-rich regions of Class A and B J-domain protein cochaperones of Hsp70

This study provides new perspectives on the functions of the glycine-rich region adjacent to the J-domain of Classes A and B J-domain proteins. Within the glycine-rich region of both Classes A and B, a role for helical segments that are similar in chemical character and conserved across phylogeny in mediating functionally important interactions is indicated—in addition to that already established for Class B.


J-domain proteins are critical Hsp70 co-chaperones. A and B types have a poorly understood glycine-rich region (Grich) adjacent to their N-terminal J-domain (Jdom). We analyzed the ability of Jdom/Grich segments of yeast Class B Sis1 and a suppressor variant of Class A, Ydj1, to rescue the inviability of sis1-∆. In each, we identified a cluster of Grich residues required for rescue. Both contain conserved hydrophobic and acidic residues and are predicted to form helices. While, as expected, the Sis1 segment docks on its J-domain, that of Ydj1 does not. However, data suggest both interact with Hsp70. We speculate that the Grich–Hsp70 interaction of Classes A and B J-domain proteins can fine tune the activity of Hsp70, thus being particularly important for the function of Class B.

Do oral and gut microbiota communicate through redox pathways? A novel asset of the nitrate‐nitrite‐NO pathway

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Do oral and gut microbiota communicate through redox pathways? A novel asset of the nitrate-nitrite-NO pathway

The oral and gut microbiotas are distinct microbial communities with unique compositions and functions but they can communicate and influence each other. Antibiotics disrupt the oral and gut ecosystems, leading to dysbiosis and increased gut epithelial permeability. Nitrate may promote inter-kingdom crosstalk during dysbiosis by either generating redox signalling species or serving as a substrate for bacteria in both ecosystems.


Nitrate may act as a regulator of •NO bioavailability via sequential reduction along the nitrate-nitrite-NO pathway with widespread health benefits, including a eubiotic effect on the oral and gut microbiota. Here, we discuss the molecular mechanisms of microbiota-host communication through redox pathways, via the production of •NO and oxidants by the family of NADPH oxidases, namely hydrogen peroxide (via Duox2), superoxide radical (via Nox1 and Nox2) and peroxynitrite, which leads to downstream activation of stress responses (Nrf2 and NFkB pathways) in the host mucosa. The activation of Nox2 by microbial metabolites is also discussed. Finally, we propose a new perspective in which both oral and gut microbiota communicate through redox pathways, with nitrate as the pivot linking both ecosystems.

ICLAMP: a novel technique to explore adenosine deamination via inosine chemical labeling and affinity molecular purification

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ICLAMP: a novel technique to explore adenosine deamination via inosine chemical labeling and affinity molecular purification

Maleimide-based chemical labeling targets inosine in RNA and DNA to identify A-to-I editing sites. Fluorescein conjugation enables visualization via PAGE, and biotin conjugation facilitates streptavidin-mediated enrichment, enhancing the detection and analysis of nucleic acid modifications.


Recent developments in sequencing and bioinformatics have advanced our understanding of adenosine-to-inosine (A-to-I) RNA editing. Surprisingly, recent analyses have revealed the capability of adenosine deaminase acting on RNA (ADAR) to edit DNA:RNA hybrid strands. However, edited inosines in DNA remain largely unexplored. A precise biochemical method could help uncover these potentially rare DNA editing sites. We explore maleimide as a scaffold for inosine labeling. With fluorophore-conjugated maleimide, we were able to label inosine in RNA or DNA. Moreover, with biotin-conjugated maleimide, we purified RNA and DNA containing inosine. Our novel technique of inosine chemical labeling and affinity molecular purification offers substantial advantages and provides a versatile platform for further discovery of A-to-I editing sites in RNA and DNA.