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.

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.

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.

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 (G_rich) adjacent to their N-terminal J-domain (J_dom). We analyzed the ability of J_dom/G_rich 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 G_rich 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 G_rich–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.

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.

Discovering new proteins through modular assembly, inspired by both nature's evolution and protein engineering, offers exciting possibilities. By leveraging information on subdomain-sized fragments from the database Fuzzle, we seamlessly integrated a flavodoxin-like fragment into a periplasmic binding protein. The resulting chimera exhibits remarkable folding and stable interfaces, showcasing the adaptability of α/β-proteins. Our work pioneers novel avenues in protein engineering and sheds light on the evolutionary origins of periplasmic binding proteins.

Modular assembly is a compelling pathway to create new proteins, a concept supported by protein engineering and millennia of evolution. Natural evolution provided a repository of building blocks, known as domains, which trace back to even shorter segments that underwent numerous ‘copy-paste’ processes culminating in the scaffolds we see today. Utilizing the subdomain-database Fuzzle, we constructed a fold-chimera by integrating a flavodoxin-like fragment into a periplasmic binding protein. This chimera is well-folded and a crystal structure reveals stable interfaces between the fragments. These findings demonstrate the adaptability of α/β-proteins and offer a stepping stone for optimization. By emphasizing the practicality of fragment databases, our work pioneers new pathways in protein engineering. Ultimately, the results substantiate the conjecture that periplasmic binding proteins originated from a flavodoxin-like ancestor.

In this work, we study the effect of macromolecular crowding on transcription initiation and report a surprising result: while the first step of promoter opening slows down, the subsequent step of RNA synthesis and promoter escape becomes faster in the presence of crowders. This suggests that the crowded cellular environment significantly impacts gene function, with the effect varying between promoters.

Transcription initiation, the first step in gene expression, has been studied extensively in dilute buffer, a condition which fails to consider the crowded environment in live cells. Recent reports indicate the kinetics of promoter escape is altered in crowded conditions for a consensus bacterial promoter. Here, we use a real-time fluorescence enhancement assay to study the kinetics of unwound bubble formation and promoter escape for three separate promoters. We find that the effect of crowding on transcription initiation is complex, with lower rates of unwound bubble formation, higher rates of promoter escape, and large variations depending on promoter identity. Based on our results, we suggest that altered conditions of crowding inside a live cell can trigger global changes.

TG-interacting factor 1 (TGIF1) contributes to the differentiation of white preadipocytes; however, its regulation is not well elucidated. We highlight that the insulin-induced ERK activation phosphorylates the T235 or T239 residue of TGIF1, which is crucial for the promotion of mitotic clonal expansion and adipocyte differentiation.

TG-interacting factor 1 (TGIF1) contributes to the differentiation of murine white preadipocyte and human adipose tissue-derived stem cells; however, its regulation is not well elucidated. Insulin is a component of the adipogenic cocktail that induces ERK signaling. TGIF1 phosphorylation and sustained stability in response to insulin were reduced through the use of specific MEK inhibitor U0126. Mutagenesis at T235 or T239 residue of TGIF1 in preadipocytes led to dephosphorylation of TGIF1. The reduced TGIF1 stability resulted in an increase in p27 ^kip1 expression, a decrease in phosphorylated Rb expression and cellular proliferation, and a reduced accumulation of lipids compared to the TGIF1-overexpressed cells. These findings highlight that insulin/ERK-driven phosphorylation of the T235 or T239 residue at TGIF1 is crucial for adipocyte differentiation.

Using ligand binding and molecular docking, we found that trans-resveratrol, its glucuronide and sulfate conjugates, and dihydro-resveratrol bind with high affinities to polyphenol- and glycosaminoglycan-binding motifs, which are present within the peptide G region of the 67-kDa laminin receptor (67LR). Neuronal cells were protected from death via 67LR/cAMP-mediated signaling pathways by preconditioning with low nanomolar concentrations of resveratrol-glucuronide.

Resveratrol prevents various neurodegenerative diseases in animal models despite reaching only low nanomolar concentrations in the brain after oral administration. In this study, based on the quenching of intrinsic tryptophan fluorescence and molecular docking, we found that trans-resveratrol, its conjugates (glucuronide and sulfate), and dihydro-resveratrol (intestinal microbial metabolite) bind with high affinities (K _d, 0.2–2 nm) to the peptide G palindromic sequence (near glycosaminoglycan-binding motif) of the 67-kDa laminin receptor (67LR). Preconditioning with low concentrations (0.01–10 nm) of these polyphenols, especially resveratrol-glucuronide, protected neuronal cells from death induced by serum withdrawal via activation of cAMP-mediated signaling pathways. This protection was prevented by a 67LR-blocking antibody, suggesting a role for this cell-surface receptor in neuroprotection by resveratrol metabolites.

Vint proteins in unicellular metazoans are novel hedgehog-related proteins that feature von Willebrand factor type A domains and the Hedgehog/INTein (HINT). This study unveiled the HINT structure, including an adduct recognition region, explored interactions with heparin, and suggested a regulatory role in auto-processing. These findings enhance our understanding of HINT fold evolution and its potential biotechnological uses.

Vint proteins have been identified in unicellular metazoans as a novel hedgehog-related gene family, merging the von Willebrand factor type A domain and the Hedgehog/INTein (HINT) domains. We present the first three-dimensional structure of the Vint domain from Tetrahymena thermophila corresponding to the auto-processing domain of hedgehog proteins, shedding light on the unique features, including an adduct recognition region (ARR). Our results suggest a potential binding between the ARR and sulfated glycosaminoglycans like heparin sulfate. Moreover, we uncover a possible regulatory role of the ARR in the auto-processing by Vint domains, expanding our understanding of the HINT domain evolution and their use in biotechnological applications. Vint domains might have played a crucial role in the transition from unicellular to multicellular organisms.