Non-canonical nucleotides, generated as oxidative metabolic by-products, significantly threaten the genome integrity of Plasmodium falciparum and thereby, their survival, owing to their mutagenic effects. PfHAM1, an evolutionarily conserved inosine/xanthosine triphosphate pyrophosphohydrolase, maintains nucleotide homeostasis in the malaria parasite by removing non-canonical nucleotides, although structure-function intricacies are hitherto poorly reported. Here, we report the X-ray crystal structure of PfHAM1, which revealed a homodimeric structure, additionally validated by size-exclusion chromatography-multi-angle light scattering analysis. The two monomeric units in the dimer were aligned in a parallel fashion, and critical residues associated with substrate and metal binding were identified, wherein a notable structural difference was observed in the β-sheet main frame compared to human inosine triphosphate pyrophosphatase. PfHAM1 exhibited Mg++-dependent pyrophosphohydrolase activity and the highest binding affinity to dITP compared to other non-canonical nucleotides as measured by isothermal titration calorimetry. Modifying the pfham1 genomic locus followed by live-cell imaging of expressed mNeonGreen-tagged PfHAM1 demonstrated its ubiquitous presence in the cytoplasm across erythrocytic stages with greater expression in trophozoites and schizonts. Interestingly, CRISPR-Cas9/DiCre recombinase-guided pfham1-null P. falciparum survived in culture under standard growth conditions, indicating its assistive role in non-canonical nucleotide clearance during intra-erythrocytic stages. This is the first comprehensive structural and functional report of PfHAM1, an atypical nucleotide-cleansing enzyme in P. falciparum.
{"title":"Structure-function analysis of nucleotide housekeeping protein HAM1 from human malaria parasite Plasmodium falciparum.","authors":"Debanjan Saha, Atanu Pramanik, Aline Freville, Asim Azhar Siddiqui, Uttam Pal, Chinmoy Banerjee, Shiladitya Nag, Subhashis Debsharma, Saikat Pramanik, Somnath Mazumder, Nakul C Maiti, Saumen Datta, Christiaan van Ooij, Uday Bandyopadhyay","doi":"10.1111/febs.17216","DOIUrl":"https://doi.org/10.1111/febs.17216","url":null,"abstract":"<p><p>Non-canonical nucleotides, generated as oxidative metabolic by-products, significantly threaten the genome integrity of Plasmodium falciparum and thereby, their survival, owing to their mutagenic effects. PfHAM1, an evolutionarily conserved inosine/xanthosine triphosphate pyrophosphohydrolase, maintains nucleotide homeostasis in the malaria parasite by removing non-canonical nucleotides, although structure-function intricacies are hitherto poorly reported. Here, we report the X-ray crystal structure of PfHAM1, which revealed a homodimeric structure, additionally validated by size-exclusion chromatography-multi-angle light scattering analysis. The two monomeric units in the dimer were aligned in a parallel fashion, and critical residues associated with substrate and metal binding were identified, wherein a notable structural difference was observed in the β-sheet main frame compared to human inosine triphosphate pyrophosphatase. PfHAM1 exhibited Mg<sup>++</sup>-dependent pyrophosphohydrolase activity and the highest binding affinity to dITP compared to other non-canonical nucleotides as measured by isothermal titration calorimetry. Modifying the pfham1 genomic locus followed by live-cell imaging of expressed mNeonGreen-tagged PfHAM1 demonstrated its ubiquitous presence in the cytoplasm across erythrocytic stages with greater expression in trophozoites and schizonts. Interestingly, CRISPR-Cas9/DiCre recombinase-guided pfham1-null P. falciparum survived in culture under standard growth conditions, indicating its assistive role in non-canonical nucleotide clearance during intra-erythrocytic stages. This is the first comprehensive structural and functional report of PfHAM1, an atypical nucleotide-cleansing enzyme in P. falciparum.</p>","PeriodicalId":94226,"journal":{"name":"The FEBS journal","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-07-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141604721","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Tumor necrosis factor (TNF) is a pro-inflammatory cytokine and its functional homotrimeric form interacts with the TNF receptor (TNFR) to activate downstream apoptotic, necroptotic, and inflammatory signaling pathways. Excessive activation of these pathways leads to various inflammatory diseases, which makes TNF a promising therapeutic target. Here, 12-mer peptides were selected from the interface of TNF-TNFR based upon their relative binding energies and were named 'TNF-inhibiting decoys' (TIDs). These decoy peptides inhibited TNF-mediated secretion of cytokines and cell death, as well as activation of downstream signaling effectors. Effective TIDs inhibited TNF signaling by disrupting the formation of TNF's functional homotrimeric form. Among derivatives of TIDs, TID3c showed slightly better efficacy in cell-based assays by disrupting TNF trimer formation. Moreover, TID3c oligomerized TNF to a high molecular weight configuration. In silico modeling and simulations revealed that TID3c and its parent peptide, TID3, form a stable complex with TNF through hydrogen bonds and electrostatic interactions, which makes them the promising lead to develop peptide-based anti-TNF therapeutics.
{"title":"Decoy peptides that inhibit TNF signaling by disrupting the TNF homotrimeric oligomer.","authors":"Nasir Javaid, Bilal Ahmad, Mahesh Chandra Patra, Sangdun Choi","doi":"10.1111/febs.17220","DOIUrl":"https://doi.org/10.1111/febs.17220","url":null,"abstract":"<p><p>Tumor necrosis factor (TNF) is a pro-inflammatory cytokine and its functional homotrimeric form interacts with the TNF receptor (TNFR) to activate downstream apoptotic, necroptotic, and inflammatory signaling pathways. Excessive activation of these pathways leads to various inflammatory diseases, which makes TNF a promising therapeutic target. Here, 12-mer peptides were selected from the interface of TNF-TNFR based upon their relative binding energies and were named 'TNF-inhibiting decoys' (TIDs). These decoy peptides inhibited TNF-mediated secretion of cytokines and cell death, as well as activation of downstream signaling effectors. Effective TIDs inhibited TNF signaling by disrupting the formation of TNF's functional homotrimeric form. Among derivatives of TIDs, TID3c showed slightly better efficacy in cell-based assays by disrupting TNF trimer formation. Moreover, TID3c oligomerized TNF to a high molecular weight configuration. In silico modeling and simulations revealed that TID3c and its parent peptide, TID3, form a stable complex with TNF through hydrogen bonds and electrostatic interactions, which makes them the promising lead to develop peptide-based anti-TNF therapeutics.</p>","PeriodicalId":94226,"journal":{"name":"The FEBS journal","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-07-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141604720","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
The metabolic networks of microorganisms are remarkably robust to genetic and environmental perturbations. This robustness stems from redundancies such as gene duplications, isoenzymes, alternative metabolic pathways, and also from non-enzymatic reactions. In the oxidative branch of the pentose phosphate pathway (oxPPP), 6-phosphogluconolactone hydrolysis into 6-phosphogluconate is catalysed by 6-phosphogluconolactonase (Pgl) but in the absence of the latter, the oxPPP flux is thought to be maintained by spontaneous hydrolysis. However, in Δpgl Escherichia coli, an extracellular pathway can also contribute to pentose phosphate synthesis. This raises question as to whether the intracellular non-enzymatic reaction can compensate for the absence of 6-phosphogluconolactonase and, ultimately, on the role of 6-phosphogluconolactonase in central metabolism. Our results validate that the bypass pathway is active in the absence of Pgl, specifically involving the extracellular spontaneous hydrolysis of gluconolactones to gluconate. Under these conditions, metabolic flux analysis reveals that this bypass pathway accounts for the entire flux into the oxPPP. This alternative metabolic route-partially extracellular-sustains the flux through the oxPPP necessary for cell growth, albeit at a reduced rate in the absence of Pgl. Importantly, these findings imply that intracellular non-enzymatic hydrolysis of 6-phosphogluconolactone does not compensate for the absence of Pgl. This underscores the crucial role of Pgl in ensuring the efficient functioning of the oxPPP.
{"title":"6-Phosphogluconolactonase is critical for the efficient functioning of the pentose phosphate pathway.","authors":"Léa Phégnon, Julien Pérochon, Sandrine Uttenweiler-Joseph, Edern Cahoreau, Pierre Millard, Fabien Létisse","doi":"10.1111/febs.17221","DOIUrl":"https://doi.org/10.1111/febs.17221","url":null,"abstract":"<p><p>The metabolic networks of microorganisms are remarkably robust to genetic and environmental perturbations. This robustness stems from redundancies such as gene duplications, isoenzymes, alternative metabolic pathways, and also from non-enzymatic reactions. In the oxidative branch of the pentose phosphate pathway (oxPPP), 6-phosphogluconolactone hydrolysis into 6-phosphogluconate is catalysed by 6-phosphogluconolactonase (Pgl) but in the absence of the latter, the oxPPP flux is thought to be maintained by spontaneous hydrolysis. However, in Δpgl Escherichia coli, an extracellular pathway can also contribute to pentose phosphate synthesis. This raises question as to whether the intracellular non-enzymatic reaction can compensate for the absence of 6-phosphogluconolactonase and, ultimately, on the role of 6-phosphogluconolactonase in central metabolism. Our results validate that the bypass pathway is active in the absence of Pgl, specifically involving the extracellular spontaneous hydrolysis of gluconolactones to gluconate. Under these conditions, metabolic flux analysis reveals that this bypass pathway accounts for the entire flux into the oxPPP. This alternative metabolic route-partially extracellular-sustains the flux through the oxPPP necessary for cell growth, albeit at a reduced rate in the absence of Pgl. Importantly, these findings imply that intracellular non-enzymatic hydrolysis of 6-phosphogluconolactone does not compensate for the absence of Pgl. This underscores the crucial role of Pgl in ensuring the efficient functioning of the oxPPP.</p>","PeriodicalId":94226,"journal":{"name":"The FEBS journal","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-07-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141565492","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Alana Maerivoet, Rebecca Price, Cécile Galmiche, Anthony Scott-Tucker, Jeff Kennedy, Tom Crabbe, Svetlana Antonyuk, Jillian Madine
Light chain amyloidosis (AL), is classified as a plasma cell dyscrasia, whereby a mutant plasma cell multiplies uncontrollably and secretes enormous amounts of immunoglobulin-free light chain (FLC) fragments. These FLCs undergo a process of misfolding and aggregation into amyloid fibrils, that can cause irreversible system-wide damage. Current treatments that focus on depleting the underlying plasma cell clone are often poorly tolerated, particularly in patients with severe cardiac involvement, meaning patient prognosis is poor. An alternative treatment approach currently being explored is the inhibition of FLC aggregation by stabilisation of the native conformer. Here, we aimed to identify and characterise antibody fragments that target FLC domains and promote their stabilisation. Using phage-display screening methods, we identified a variable heavy (VH) domain, termed VH1, targeted towards the FLC. Using differential scanning fluorimetry and surface plasmon resonance, VH1 was characterised to bind and kinetically stabilise an amyloidogenic FLC, whereby a > 5.5 °C increase in thermal stability was noted. This improved stability corresponded to the inhibition of fibril formation, where 10 : 1 LC : VH1 concentration reduced aggregation to baseline levels. X-ray crystallographic structures of the LC : VH1 complex at atomic resolution revealed binding in a 1 : 1 ratio, mimicking the dimeric antigen binding sites of the native immunoglobulin molecule and the native LC homodimer.
{"title":"Enhanced stabilisation and reduced fibril forming potential of an amyloidogenic light chain using a variable heavy domain to mimic the homodimer complex.","authors":"Alana Maerivoet, Rebecca Price, Cécile Galmiche, Anthony Scott-Tucker, Jeff Kennedy, Tom Crabbe, Svetlana Antonyuk, Jillian Madine","doi":"10.1111/febs.17223","DOIUrl":"https://doi.org/10.1111/febs.17223","url":null,"abstract":"<p><p>Light chain amyloidosis (AL), is classified as a plasma cell dyscrasia, whereby a mutant plasma cell multiplies uncontrollably and secretes enormous amounts of immunoglobulin-free light chain (FLC) fragments. These FLCs undergo a process of misfolding and aggregation into amyloid fibrils, that can cause irreversible system-wide damage. Current treatments that focus on depleting the underlying plasma cell clone are often poorly tolerated, particularly in patients with severe cardiac involvement, meaning patient prognosis is poor. An alternative treatment approach currently being explored is the inhibition of FLC aggregation by stabilisation of the native conformer. Here, we aimed to identify and characterise antibody fragments that target FLC domains and promote their stabilisation. Using phage-display screening methods, we identified a variable heavy (VH) domain, termed VH1, targeted towards the FLC. Using differential scanning fluorimetry and surface plasmon resonance, VH1 was characterised to bind and kinetically stabilise an amyloidogenic FLC, whereby a > 5.5 °C increase in thermal stability was noted. This improved stability corresponded to the inhibition of fibril formation, where 10 : 1 LC : VH1 concentration reduced aggregation to baseline levels. X-ray crystallographic structures of the LC : VH1 complex at atomic resolution revealed binding in a 1 : 1 ratio, mimicking the dimeric antigen binding sites of the native immunoglobulin molecule and the native LC homodimer.</p>","PeriodicalId":94226,"journal":{"name":"The FEBS journal","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-07-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141565493","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Sourobh Maji, Mohd Waseem, Manish Kumar Sharma, Maninder Singh, Anamika Singh, Nidhi Dwivedi, Pallabi Thakur, David G. Cooper, Naveen C. Bisht, Jan S. Fassler, Naidu Subbarao, Jitendra P. Khurana, Neel Sarovar Bhavesh, Jitendra Kumar Thakur
The protein–protein interaction (PPI) network of the Mediator complex is very tightly regulated and depends on different developmental and environmental cues. Here, we present an interactive platform for comparative analysis of the Mediator subunits from humans, baker's yeast Saccharomyces cerevisiae, and model plant Arabidopsis thaliana in a user-friendly web-interface database called MediatorWeb. MediatorWeb provides an interface to visualize and analyze the PPI network of Mediator subunits. The database facilitates downloading the untargeted and unweighted network of Mediator complex, its submodules, and individual Mediator subunits to better visualize the importance of individual Mediator subunits or their submodules. Further, MediatorWeb offers network visualization of the Mediator complex and interacting proteins that are functionally annotated. This feature provides clues to understand functions of Mediator subunits in different processes. In an additional tab, MediatorWeb provides quick access to secondary and tertiary structures, as well as residue–level contact information for Mediator subunits in each of the three model organisms. Another useful feature of MediatorWeb is detection of interologs based on orthologous analyses, which can provide clues to understand the functions of Mediator complex in less explored kingdoms. Thus, MediatorWeb and its features can help the user to understand the role of Mediator complex and its subunits in the transcription regulation of gene expression.
{"title":"MediatorWeb: a protein–protein interaction network database for the RNA polymerase II Mediator complex","authors":"Sourobh Maji, Mohd Waseem, Manish Kumar Sharma, Maninder Singh, Anamika Singh, Nidhi Dwivedi, Pallabi Thakur, David G. Cooper, Naveen C. Bisht, Jan S. Fassler, Naidu Subbarao, Jitendra P. Khurana, Neel Sarovar Bhavesh, Jitendra Kumar Thakur","doi":"10.1111/febs.17225","DOIUrl":"10.1111/febs.17225","url":null,"abstract":"<p>The protein–protein interaction (PPI) network of the Mediator complex is very tightly regulated and depends on different developmental and environmental cues. Here, we present an interactive platform for comparative analysis of the Mediator subunits from humans, baker's yeast <i>Saccharomyces cerevisiae</i>, and model plant <i>Arabidopsis thaliana</i> in a user-friendly web-interface database called MediatorWeb. MediatorWeb provides an interface to visualize and analyze the PPI network of Mediator subunits. The database facilitates downloading the untargeted and unweighted network of Mediator complex, its submodules, and individual Mediator subunits to better visualize the importance of individual Mediator subunits or their submodules. Further, MediatorWeb offers network visualization of the Mediator complex and interacting proteins that are functionally annotated. This feature provides clues to understand functions of Mediator subunits in different processes. In an additional tab, MediatorWeb provides quick access to secondary and tertiary structures, as well as residue–level contact information for Mediator subunits in each of the three model organisms. Another useful feature of MediatorWeb is detection of interologs based on orthologous analyses, which can provide clues to understand the functions of Mediator complex in less explored kingdoms. Thus, MediatorWeb and its features can help the user to understand the role of Mediator complex and its subunits in the transcription regulation of gene expression.</p>","PeriodicalId":94226,"journal":{"name":"The FEBS journal","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-07-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141556243","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Matteo Costacurta, Jarrod J Sandow, Belinda Maher, Olivia Susanto, Stephin J Vervoort, Jennifer R Devlin, Daniel Garama, Mark R Condina, Joel R Steele, Hossein V Kahrood, Daniel Gough, Ricky W Johnstone, Jake Shortt
Immunomodulatory imide drugs (IMiDs) are central components of therapy for multiple myeloma (MM). IMiDs bind cereblon (CRBN), an adaptor for the CUL4-DDB1-RBX1 E3 ligase to change its substrate specificity and induce degradation of 'neosubstrate' transcription factors that are essential to MM cells. Mechanistic studies to date have largely focussed on mediators of therapeutic activity and insight into clinical IMiD toxicities is less developed. We adopted BioID2-dependent proximity labelling (BioID2-CRBN) to characterise the CRBN interactome in the presence and absence of various IMiDs and the proteasome inhibitor, bortezomib. We aimed to leverage this technology to further map CRBN interactions beyond what has been achieved by conventional proteomic techniques. In support of this approach, analysis of cells expressing BioID2-CRBN following IMiD treatment displayed biotinylation of known CRBN interactors and neosubstrates. We observed that bortezomib alone significantly modifies the CRBN interactome. Proximity labelling also suggested that IMiDs augment the interaction between CRBN and proteins that are not degraded, thus designating 'neointeractors' distinct from previously disclosed 'neosubstrates'. Here we identify Non-Muscle Myosin Heavy Chain IIA (MYH9) as a putative CRBN neointeractor that may contribute to the haematological toxicity of IMiDs. These studies provide proof of concept for proximity labelling technologies in the mechanistic profiling of IMiDs and related E3-ligase-modulating drugs.
{"title":"Mapping the IMiD-dependent cereblon interactome using BioID-proximity labelling.","authors":"Matteo Costacurta, Jarrod J Sandow, Belinda Maher, Olivia Susanto, Stephin J Vervoort, Jennifer R Devlin, Daniel Garama, Mark R Condina, Joel R Steele, Hossein V Kahrood, Daniel Gough, Ricky W Johnstone, Jake Shortt","doi":"10.1111/febs.17196","DOIUrl":"https://doi.org/10.1111/febs.17196","url":null,"abstract":"<p><p>Immunomodulatory imide drugs (IMiDs) are central components of therapy for multiple myeloma (MM). IMiDs bind cereblon (CRBN), an adaptor for the CUL4-DDB1-RBX1 E3 ligase to change its substrate specificity and induce degradation of 'neosubstrate' transcription factors that are essential to MM cells. Mechanistic studies to date have largely focussed on mediators of therapeutic activity and insight into clinical IMiD toxicities is less developed. We adopted BioID2-dependent proximity labelling (BioID2-CRBN) to characterise the CRBN interactome in the presence and absence of various IMiDs and the proteasome inhibitor, bortezomib. We aimed to leverage this technology to further map CRBN interactions beyond what has been achieved by conventional proteomic techniques. In support of this approach, analysis of cells expressing BioID2-CRBN following IMiD treatment displayed biotinylation of known CRBN interactors and neosubstrates. We observed that bortezomib alone significantly modifies the CRBN interactome. Proximity labelling also suggested that IMiDs augment the interaction between CRBN and proteins that are not degraded, thus designating 'neointeractors' distinct from previously disclosed 'neosubstrates'. Here we identify Non-Muscle Myosin Heavy Chain IIA (MYH9) as a putative CRBN neointeractor that may contribute to the haematological toxicity of IMiDs. These studies provide proof of concept for proximity labelling technologies in the mechanistic profiling of IMiDs and related E3-ligase-modulating drugs.</p>","PeriodicalId":94226,"journal":{"name":"The FEBS journal","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-07-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141556242","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Cullin-based RING ligases (CRLs) comprise the largest family of ubiquitin E3 ligases. CRL activity is tightly regulated by cullin neddylation, which has been associated with various diseases. Although inhibitors of CRLs neddylation have been reported, there is a lack of small molecules that can selectively target individual cullins. Here, we identified a natural product, liquidambaric acid (LDA), with relatively selective inhibition properties against cullin (Cul) 2 neddylation, and found that its target, Tumor Necrosis Factor receptor-associated factor 2 (TRAF2) was required for the activity. TRAF2 associates with the Cul2 neddylation complex and regulates the machinery assembly, especially that of E2 (UBC12) and E3 (RBX1) enzymes. In addition, we demonstrated that by intervention of the associations between TRAF2 and the neddylation machinery, LDA disturbed NEDD8 transfer from E1 to E2, therefore blocking Cul2 neddylation. Taken together, we show that TRAF2 plays a positive role in neddylation cascades, and we have identified a small molecule capable of selective modulation of cullin neddylation.
{"title":"TRAF2 associates with cullin neddylation complex assembly.","authors":"Tiantian Wang, Qi Zhang, Yu Xu, Rong Yan, Yuting Pan, Ying Xuan, Mengzhen Shen, Xianzhi Chen, Hongyan Zhu, Xisong Ke, Yi Qu, Xue Zhang","doi":"10.1111/febs.17222","DOIUrl":"https://doi.org/10.1111/febs.17222","url":null,"abstract":"<p><p>Cullin-based RING ligases (CRLs) comprise the largest family of ubiquitin E3 ligases. CRL activity is tightly regulated by cullin neddylation, which has been associated with various diseases. Although inhibitors of CRLs neddylation have been reported, there is a lack of small molecules that can selectively target individual cullins. Here, we identified a natural product, liquidambaric acid (LDA), with relatively selective inhibition properties against cullin (Cul) 2 neddylation, and found that its target, Tumor Necrosis Factor receptor-associated factor 2 (TRAF2) was required for the activity. TRAF2 associates with the Cul2 neddylation complex and regulates the machinery assembly, especially that of E2 (UBC12) and E3 (RBX1) enzymes. In addition, we demonstrated that by intervention of the associations between TRAF2 and the neddylation machinery, LDA disturbed NEDD8 transfer from E1 to E2, therefore blocking Cul2 neddylation. Taken together, we show that TRAF2 plays a positive role in neddylation cascades, and we have identified a small molecule capable of selective modulation of cullin neddylation.</p>","PeriodicalId":94226,"journal":{"name":"The FEBS journal","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-07-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141560668","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Ana O. Tiroli-Cepeda, Leonardo A. Linhares, Annelize Z. B. Aragão, Jemmyson R. de Jesus, Ana P. Wasilewska-Sampaio, Fernanda G. De Felice, Sérgio T. Ferreira, Júlio C. Borges, Douglas M. Cyr, Carlos H. I. Ramos
A rise in temperature triggers a structural change in the human Type I 40 kDa heat shock protein (Hsp40/DnaJ), known as DNAJA1. This change leads to a less compact structure, characterized by an increased presence of solvent-exposed hydrophobic patches and β-sheet-rich regions. This transformation is validated by circular dichroism, thioflavin T binding, and Bis-ANS assays. The formation of this β-sheet-rich conformation, which is amplified in the absence of zinc, leads to protein aggregation. This aggregation is induced not only by high temperatures but also by low ionic strength and high protein concentration. The aggregated conformation exhibits characteristics of an amyloidogenic structure, including a distinctive X-ray diffraction pattern, seeding competence (which stimulates the formation of amyloid-like aggregates), cytotoxicity, resistance to SDS, and fibril formation. Interestingly, the yeast Type I Ydj1 also tends to adopt a similar β-sheet-rich structure under comparable conditions, whereas Type II Hsp40s, whether human or from yeast, do not. Moreover, Ydj1 aggregates were found to be cytotoxic. Studies using DNAJA1- and Ydj1-deleted mutants suggest that the zinc-finger region plays a crucial role in amyloid formation. Our discovery of amyloid aggregation in a C-terminal deletion mutant of DNAJA1, which resembles a spliced homolog expressed in the testis, implies that Type I Hsp40 co-chaperones may generate amyloidogenic species in vivo.
温度升高会引发人类 I 型 40 kDa 热休克蛋白(Hsp40/DnaJ)(即 DNAJA1)的结构变化。这种变化导致结构变得不那么紧凑,其特点是溶剂暴露的疏水斑块和富含β片的区域增多。圆二色性、硫黄素 T 结合和 Bis-ANS 试验验证了这种转变。这种富含β片的构象的形成在缺锌的情况下会放大,导致蛋白质聚集。这种聚集不仅受高温诱导,而且受低离子强度和高蛋白质浓度诱导。这种聚集构象具有淀粉样结构的特征,包括独特的 X 射线衍射图样、播种能力(可刺激淀粉样聚集体的形成)、细胞毒性、对 SDS 的抗性以及纤维的形成。有趣的是,在类似条件下,酵母 I 型 Ydj1 也倾向于采用类似的富含 β 片层的结构,而 II 型 Hsp40(无论是人类还是酵母)则不然。此外,研究还发现 Ydj1 的聚集体具有细胞毒性。利用DNAJA1-和Ydj1-缺失突变体进行的研究表明,锌指区域在淀粉样蛋白形成过程中起着至关重要的作用。我们发现 DNAJA1 的 C 端缺失突变体(类似于在睾丸中表达的剪接同源物)中存在淀粉样蛋白聚集,这意味着 I 型 Hsp40 协同伴侣可能在体内产生淀粉样蛋白。
{"title":"Type I Hsp40s/DnaJs aggregates exhibit features reminiscent of amyloidogenic structures","authors":"Ana O. Tiroli-Cepeda, Leonardo A. Linhares, Annelize Z. B. Aragão, Jemmyson R. de Jesus, Ana P. Wasilewska-Sampaio, Fernanda G. De Felice, Sérgio T. Ferreira, Júlio C. Borges, Douglas M. Cyr, Carlos H. I. Ramos","doi":"10.1111/febs.17215","DOIUrl":"10.1111/febs.17215","url":null,"abstract":"<p>A rise in temperature triggers a structural change in the human Type I 40 kDa heat shock protein (Hsp40/DnaJ), known as DNAJA1. This change leads to a less compact structure, characterized by an increased presence of solvent-exposed hydrophobic patches and β-sheet-rich regions. This transformation is validated by circular dichroism, thioflavin T binding, and Bis-ANS assays. The formation of this β-sheet-rich conformation, which is amplified in the absence of zinc, leads to protein aggregation. This aggregation is induced not only by high temperatures but also by low ionic strength and high protein concentration. The aggregated conformation exhibits characteristics of an amyloidogenic structure, including a distinctive X-ray diffraction pattern, seeding competence (which stimulates the formation of amyloid-like aggregates), cytotoxicity, resistance to SDS, and fibril formation. Interestingly, the yeast Type I Ydj1 also tends to adopt a similar β-sheet-rich structure under comparable conditions, whereas Type II Hsp40s, whether human or from yeast, do not. Moreover, Ydj1 aggregates were found to be cytotoxic. Studies using DNAJA1- and Ydj1-deleted mutants suggest that the zinc-finger region plays a crucial role in amyloid formation. Our discovery of amyloid aggregation in a C-terminal deletion mutant of DNAJA1, which resembles a spliced homolog expressed in the testis, implies that Type I Hsp40 co-chaperones may generate amyloidogenic species <i>in vivo</i>.</p>","PeriodicalId":94226,"journal":{"name":"The FEBS journal","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-07-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141556245","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Cachexia is a wasting syndrome that manifests in more than half of all cancer patients. Cancer-associated cachexia negatively influences the survival of patients and their quality of life. It is characterized by a rapid loss of adipose and skeletal muscle tissues, which is partly mediated by inflammatory cytokines. Here, we explored the crucial roles of interleukin-6 (IL-6) family cytokines, including IL-6, leukemia inhibitory factor, and oncostatin M, in the development of cancer cachexia. These cytokines have been shown to exacerbate cachexia by promoting the wasting of adipose and muscle tissues, activating mechanisms that enhance lipolysis and proteolysis. Overlapping effects of the IL-6 family cytokines depend on janus kinase/signal transducer and activator of transcription 3 signaling. We argue that the blockade of these cytokine pathways individually may fail due to redundancy and future therapeutic approaches should target common downstream elements to yield effective clinical outcomes.
{"title":"The role of interleukin-6 family cytokines in cancer cachexia","authors":"Samet Agca, Serkan Kir","doi":"10.1111/febs.17224","DOIUrl":"10.1111/febs.17224","url":null,"abstract":"<p>Cachexia is a wasting syndrome that manifests in more than half of all cancer patients. Cancer-associated cachexia negatively influences the survival of patients and their quality of life. It is characterized by a rapid loss of adipose and skeletal muscle tissues, which is partly mediated by inflammatory cytokines. Here, we explored the crucial roles of interleukin-6 (IL-6) family cytokines, including IL-6, leukemia inhibitory factor, and oncostatin M, in the development of cancer cachexia. These cytokines have been shown to exacerbate cachexia by promoting the wasting of adipose and muscle tissues, activating mechanisms that enhance lipolysis and proteolysis. Overlapping effects of the IL-6 family cytokines depend on janus kinase/signal transducer and activator of transcription 3 signaling. We argue that the blockade of these cytokine pathways individually may fail due to redundancy and future therapeutic approaches should target common downstream elements to yield effective clinical outcomes.</p>","PeriodicalId":94226,"journal":{"name":"The FEBS journal","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-07-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1111/febs.17224","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141556244","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Accumulating evidence shows that inflammation is essential for embryo implantation and decidualization. Histamine, a proinflammatory factor that is present in almost all mammalian tissues, is synthesized through decarboxylating histidine by histidine decarboxylase (HDC). Although histamine is known to be essential for decidualization, the underlying mechanism remains undefined. In the present study, histamine had no obvious direct effects on in vitro decidualization in mice. However, the obvious differences in HDC protein levels between day 4 of pregnancy and day 4 of pseudopregnancy, as well as between delayed and activated implantation, suggested that the blastocyst may be involved in regulating HDC expression. Furthermore, blastocyst-derived tumor necrosis factor α (TNFα) significantly increased HDC levels in the luminal epithelium. Histamine increased the levels of amphiregulin (AREG) and disintegrin and metalloproteinase domain-containing protein 17 (ADAM17) proteins, which was abrogated by treatment with famotidine, a specific histamine type 2 receptor (H2R) inhibitor, or by TPAI-1 (a specific inhibitor of ADAM17). Intraluminal injection of urocanic acid (HDC inhibitor) on day 4 of pregnancy significantly reduced the number of implantation sites on day 5 of pregnancy. TNFα-stimulated increases in HDC, AREG and ADAM17 protein levels was abrogated by urocanic acid, a specific inhibitor of HDC. Additionally, AREG treatment significantly promoted in vitro decidualization. Collectively, our data suggests that blastocyst-derived TNFα induces luminal epithelial histamine secretion, and histamine increases mouse decidualization through ADAM17-mediated AREG release.
{"title":"Histamine promotes mouse decidualization through stimulating epithelial amphiregulin release","authors":"Cheng-Kan Liu, Yu-Ying He, Si-Ting Chen, Wen-Wen Shi, Ying Wang, Hui-Na Luo, Zeng-Ming Yang","doi":"10.1111/febs.17219","DOIUrl":"10.1111/febs.17219","url":null,"abstract":"<p>Accumulating evidence shows that inflammation is essential for embryo implantation and decidualization. Histamine, a proinflammatory factor that is present in almost all mammalian tissues, is synthesized through decarboxylating histidine by histidine decarboxylase (HDC). Although histamine is known to be essential for decidualization, the underlying mechanism remains undefined. In the present study, histamine had no obvious direct effects on <i>in vitro</i> decidualization in mice. However, the obvious differences in HDC protein levels between day 4 of pregnancy and day 4 of pseudopregnancy, as well as between delayed and activated implantation, suggested that the blastocyst may be involved in regulating HDC expression. Furthermore, blastocyst-derived tumor necrosis factor α (TNFα) significantly increased HDC levels in the luminal epithelium. Histamine increased the levels of amphiregulin (AREG) and disintegrin and metalloproteinase domain-containing protein 17 (ADAM17) proteins, which was abrogated by treatment with famotidine, a specific histamine type 2 receptor (H2R) inhibitor, or by TPAI-1 (a specific inhibitor of ADAM17). Intraluminal injection of urocanic acid (HDC inhibitor) on day 4 of pregnancy significantly reduced the number of implantation sites on day 5 of pregnancy. TNFα-stimulated increases in HDC, AREG and ADAM17 protein levels was abrogated by urocanic acid, a specific inhibitor of HDC. Additionally, AREG treatment significantly promoted <i>in vitro</i> decidualization. Collectively, our data suggests that blastocyst-derived TNFα induces luminal epithelial histamine secretion, and histamine increases mouse decidualization through ADAM17-mediated AREG release.</p>","PeriodicalId":94226,"journal":{"name":"The FEBS journal","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-07-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141556241","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}