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The Janus kinase-signal transducer and activator of transcription (JAK-STAT) signalling pathway is a key player in animal development and physiology. Although it functions in a variety of processes, the net output of JAK-STAT signalling depends on its spatiotemporal activation, as well as extensive crosstalk with other signalling pathways. Drosophila, with its relatively simple signal transduction pathways and plethora of genetic analysis tools, is an ideal system for dissecting JAK-STAT signalling interactions. In this review, we explore studies in Drosophila revealing that JAK-STAT signalling lies at the nexus of a complex network of interlinked pathways, including epidermal growth factor receptor (EGFR), c-Jun N-terminal kinase (JNK), Notch, Insulin, Hippo, bone morphogenetic protein (BMP), Hedgehog (Hh) and Wingless (Wg). These pathways can synergise with or antagonise one another to produce a variety of outcomes. Given the conserved nature of signal transduction pathways, we conclude with our perspective on the implication of JAK-STAT signalling dysregulation in human diseases, and how studies in Drosophila have the potential to inform and influence clinical research.

RETRACTION: L. Zhao, H. Wang, X. Sun, and Y. Ding, “Comparative Proteomic Analysis Identifies Proteins Associated with the Development and Progression of Colorectal Carcinoma,” The FEBS Journal 227, no. 20 (2010): 4195–4204, https://doi.org/10.1111/j.1742-4658.2010.07808.x.

The above article, published online on 31 August 2010 in Wiley Online Library (wileyonlinelibrary.com), has been retracted by agreement between the journal Editor-in-Chief; the Federation of European Biochemical Societies; and John Wiley & Sons Ltd. The retraction has been agreed upon following an investigation into concerns raised by a third party, which revealed the unattributed reproduction of the authors' previously published figure (Fig. 2C GAPDH lane). The investigation uncovered significant primary data redundancy and textual overlap with two previously published articles by the same group of authors elsewhere, which leaves the article with incremental original content of its own.

Pathogenic variants in HMGCR were recently linked to a limb-girdle muscular dystrophy (LGMD) phenotype. The protein product HMG CoA reductase (HMGCR) catalyzes a key component of the cholesterol synthesis pathway. The two other muscle diseases associated with HMGCR, statin-associated myopathy (SAM) and autoimmune anti-HMGCR myopathy, are not inherited in a Mendelian pattern. Statins inhibit HMGCR activity to generate their cholesterol-lowering effects and are known to cause multiple types of adverse effects on skeletal muscle, while the antibodies associated with anti-HMGCR myopathy specifically target this enzyme. The mechanism linking pathogenic variants in HMGCR with skeletal muscle dysfunction is unclear. We knocked down Hmgcr in mouse skeletal myoblasts, knocked down hmgcr in Drosophila, and expressed three pathogenic HMGCR variants (c.1327C>T, p.Arg443Trp; c.1522_1524delTCT, p.Ser508del; and c.1621G>A, p.Ala541Thr) in Hmgcr knockdown mouse myoblasts. Hmgcr deficiency was associated with decreased proliferation, increased apoptosis, and impaired myotube fusion. Transcriptome sequencing of Hmgcr knockdown versus control myoblasts revealed differential expression involving mitochondrial function, with corresponding differences in cellular oxygen consumption rates. Both ubiquitous and muscle-specific knockdown of hmgcr in Drosophila led to lethality. Overexpression of reference HMGCR cDNA rescued myotube fusion in knockdown cells, whereas overexpression of the pathogenic variants of HMGCR cDNA did not. These results suggest that the three HMGCR-related muscle diseases share disease mechanisms related to skeletal muscle development.

Previous studies have identified three families of knotted phytochrome photoreceptors in cyanobacteria. We describe a fourth type: ‘hybrid’ phytochromes with putative bilin-binding cysteine residues in both their N-terminal ‘knot’ extensions and cGMP-phosphodiesterase/adenylate cyclase/FhlA (GAF) domains, which we designate as dual-cysteine bacteriophytochromes (DCBs). Recombinant expression of DCBs in Escherichia coli yields photoactive phycocyanobilin (PCB) adducts with red/far-red photocycles similar to those of the GAF-Cys-containing cyanobacterial phytochromes (Cph1s). Incorporation of the PCB precursor, biliverdin IXα (BV), gave multiple populations, one of which appears similar to those of cyanobacterial bacteriophytochromes (cBphPs). A crystal structure of FiDCB bound to BV exhibits two thioether linkages between the GAF- and ‘PAS-knot’-Cys residues and the C3¹ and C3² atoms of BV. When expressed in Synechocystis sp. PCC 6803, DCBs incorporate PCB rather than BV. DCBs can be converted to photoactive cBphP-, Cph1-, and tandem-cysteine cyanobacterial phytochrome (TCCP) analogs by removal and/or addition of a cysteine residue by site-directed mutagenesis. This structural plasticity contrasts with our inability to generate functional photosensor analogs by analogous site-directed mutagenesis of TCCP and Cph1 representatives. Phylogenetic analysis demonstrates that DCBs do not form a monophyletic clade and also suggest that Cph1 and TCCP families independently emerged from different lineages of cBphPs, possibly via DCB intermediates.

Vesicle trafficking is pivotal in heparan sulfate (HS) biosynthesis, influencing its spatial and temporal regulation within distinct Golgi compartments. This regulation modulates the sulfation pattern of HS, which is crucial for governing various biological processes. Here, we investigate the effects of silencing Rab1A and Rab2A expression on the localisation of 3-O-sulfotransferase-5 (3OST5) within Golgi compartments and subsequent alterations in HS structure and levels. Interestingly, silencing Rab1A led to a shift in 3OST5 localization towards the trans-Golgi, resulting in increased HS levels within 24 and 48 h, while silencing Rab2A caused 3OST5 accumulation in the cis-Golgi, with a delayed rise in HS content observed after 48 h. Furthermore, a compensatory mechanism was evident in Rab2A-silenced cells, where increased Rab1A protein expression was detected. This suggests a dynamic interplay between Rab1A and Rab2A in maintaining the fine balance of vesicle trafficking processes involved in HS biosynthesis. Additionally, we demonstrate that the trafficking of 3OST5 in COPI vesicles is facilitated by GOLPH3 protein. These findings identify novel vesicular transport mechanisms regulating HS biosynthesis and reveal a compensatory relationship between Rab1A and Rab2A in maintaining baseline HS production.

Creatine is essential for ATP regeneration in energy-demanding cells. Creatine deficiency results in severe neurodevelopmental impairments. In the brain, creatine is synthesized locally by oligodendrocytes to supply neighboring neurons. Neuronal uptake is mediated by SLC6A8. However, it is still unknown how creatine is released from the producing cells. Here, we investigated the function of the transporter SLC22A15, which exhibits strikingly high amino acid sequence conservation. The release of substrates from 293 cells via heterologously expressed human and rat SLC22A15 was analyzed by mass spectrometry. A number of zwitterions were identified as substrates, with similar efflux transport efficiencies. However, in absolute numbers, the efflux of creatine far outweighed all other substrates. In contrast to the permanent creatine efflux mediated by SLC16A12 and SLC16A9, SLC22A15 was, by default, completely inactive, thereby preventing continuous creatine loss from producing cells. External substrates such as guanidinoacetic acid, GABA, or MPP⁺ trigger creatine release through a one-to-one exchange. Human and mouse mRNA profiles indicate that SLC22A15 expression is highest in oligodendrocytes and bone marrow. Single-cell RNA sequencing data substantiate the hypothesis that SLC22A15 depends on high intracellular creatine concentrations: high SLC22A15 counts, as in oligodendrocytes and macrophages, correlate with high counts of the creatine synthesis enzymes AGAT and GAMT in both humans and mice, whereas in proximal tubular cells and hepatocytes, AGAT counts are high, but SLC22A15 is absent. Our findings establish SLC22A15 as the pivotal transporter for controlled creatine release from oligodendrocytes, filling a critical gap in understanding creatine metabolism in the brain.

In this study, we explored the intricate relationship between Pannexin 1 (PANX1) and the Hippo signaling pathway effector, Yes-associated protein (YAP). Analysis of The Cancer Genome Atlas (TCGA) data revealed a significant positive correlation between PANX1 mRNA and core Hippo components, Yes-associated protein 1 [YAP], Transcriptional coactivator with PDZ-binding motif [TAZ], and Hippo scaffold, Ras GTPase-activating-like protein IQGAP1 [IQGAP1], in invasive cutaneous melanoma and breast carcinoma. Furthermore, we demonstrated that PANX1 expression is upregulated in invasive melanoma cell lines and is associated with increased YAP protein levels. Notably, our investigations uncovered a previously unrecognized interaction between endogenous PANX1 and the Hippo scaffold protein IQGAP1 in melanoma cells. Moreover, our findings revealed that IQGAP1 exhibits differential expression in melanoma cells and plays a regulatory role in cellular morphology. Functional studies involving PANX1 knockdown provided compelling evidence that PANX1 modulates YAP protein levels and its cotranscriptional activity in melanoma and breast carcinoma cells. Importantly, our study highlights the potential therapeutic significance of targeting PANX1. Pharmacological inhibition of PANX1 using selective FDA-approved inhibitors or PANX1 knockdown reduced YAP levels in melanoma cells. Furthermore, our Clariom™ S analysis unveiled key genes implicated in cell proliferation, such as neuroglin1 (NRG1), β-galactoside binding protein and galectin-3 (LGALS3), that are affected in PANX1-deficient cells. In summary, our investigation delves into the intricate interplay between PANX1 and YAP in the context of invasive melanoma, offering valuable insights into potential therapeutic strategies for effective treatment.

Neutrophil elastase (NE) is released by activated neutrophils during an inflammatory response and exerts proteolytic activity on elastin and other extracellular matrix components. This protease is rapidly inhibited by the plasma serine protease inhibitor alpha-1-antitrypsin (AAT), and the importance of this protective activity on lung tissue is highlighted by the development of early onset emphysema in individuals with AAT deficiency. As a serpin, AAT presents a surface-exposed reactive centre loop (RCL) whose sequence mirrors the target protease specificity. Following binding of NE in a 'Michaelis' encounter complex, cleavage of the RCL results in an irreversible complex between the two molecules. Here, the structure of the AAT-NE encounter complex was studied by molecular dynamics, mutagenesis and enzyme kinetics. Exploration of the geometry of interaction between the two molecules revealed the possibility that the interaction interface extends beyond the RCL; a persistent feature of the simulations was the interaction between a region located upstream of β-strand 4C of AAT, comprising three acidic residues (Asp202, Glu199 and Glu204), and Arg147 of NE. Mutation of the acidic residues to either alanine or serine, or a D202R substitution, resulted in a reduced rate of association between recombinant AAT and NE. Addition of salt to the buffer had little effect for these mutants but substantially reduced the rate of interaction of the wild-type protein. These data are consistent with a role for this acidic region on AAT as an exosite that contributes to an optimal interaction with its physiological protease target.

The FEBS Journal publishes primary papers as well as reviews in the molecular life sciences relating to the molecules and mechanisms underpinning biological processes. Editor-in-Chief Seamus Martin shares some thoughts on the nature of conducting research, some highlights of the past year at the journal, and what is in store for 2025.

Cellulases are an ensemble of enzymes that hydrolyze cellulose chains into fermentable glucose and hence are widely used in bioethanol production. The last enzyme of the cellulose degradation pathway, β-glucosidase, is inhibited by its product, glucose. The product inhibition by glucose hinders cellulose hydrolysis limiting the saccharification during bioethanol production. Thus, engineered β-glucosidases with enhanced glucose tolerance and catalytic efficiency are essential. This study focuses on the rational engineering of β-glucosidase from Acetivibrio thermocellus (WT-AtGH1). Recombinant WT-AtGH1 exhibited activity on cellobiose and p-nitrophenyl-β-d-glucoside as substrates and retained around 80% of its activity over 48 h at 55 °C, pH 5.5. However, WT-AtGH1 showed low glucose tolerance of 380 mm as compared to the required IC₅₀ value of > 800 mm for industrial use. Thus, a rational design approach was utilized to enhance the glucose tolerance of this enzyme. We determined the 3 Å resolution crystal structure of WT-AtGH1. The structure-based engineered G168W-AtGH1 and S242W-AtGH1 mutants exhibited improved glucose tolerance of 840 and 612 mm, respectively. Surprisingly, S242L-AtGH1 mutant showed ~ 2.5-fold increase in the catalytic efficiency as compared to WT-AtGH1. A combinatorial effect of improved glucose tolerance, as well as enhanced catalytic efficiency, was observed for the G168W-S242L-AtGH1 mutant. All the mutants with enhanced properties showed considerable stability at industrial operating conditions of 55 °C and pH 5.5. Thus, we present mutants of WT-AtGH1 with improved glucose tolerance and kinetic properties that have the potential to increase the efficiency of saccharification during biofuel production.