Journal of Biological Chemistry

Fisher, Claire E.;Bak, Daniel W.;Miller, Kennedy E.;Washington-Hughes, Clorissa L.;Dickfoss, Anna M.;Weerapana, Eranthie;Py, Béatrice;Outten, F. Wayne

doi: 10.1016/j.jbc.2024.107506pmid: N/A

<h2>Abstract</h2><p>Iron-sulfur (Fe-S) clusters are required for essential biological pathways, including respiration and isoprenoid biosynthesis. Complex Fe-S cluster biogenesis systems have evolved to maintain an adequate supply of this critical protein cofactor. In <i>Escherichia coli,</i> two Fe-S biosynthetic systems, the "housekeeping" Isc and "stress responsive" Suf pathways, interface with a network of cluster trafficking proteins, such as ErpA, IscA, SufA, and NfuA. GrxD, a Fe-S cluster-binding monothiol glutaredoxin, also participates in Fe-S protein biogenesis in both prokaryotes and eukaryotes. Previous studies in <i>E. coli</i> showed that the Δ<i>grxD</i> mutation causes sensitivity to iron depletion, spotlighting a critical role for GrxD under conditions that disrupt Fe-S homeostasis. Here, we utilized a global chemoproteomic mass spectrometry (MS) approach to analyse the contribution of GrxD to the Fe-S proteome. Our results demonstrate that 1) GrxD is required for biogenesis of a specific subset of Fe-S proteins under iron-depleted conditions, 2) GrxD is required for cluster delivery to ErpA under iron limitation, 3) GrxD is functionally distinct from other Fe-S trafficking proteins and, 4) GrxD Fe-S cluster binding is responsive to iron limitation. All these results lead to the proposal that GrxD is required to maintain Fe-S cluster delivery to the essential trafficking protein ErpA during iron limitation conditions.</p>

Romero, Miguel F.;Krall, Jeffrey B.;Nichols, Parker J.;Vantreeck, Jillian;Henen, Morkos A.;Dejardin, Emmanuel;Schulz, Frederik;Vicens, Quentin;Vögeli, Beat;Diallo, Mamadou Amadou

doi: 10.1016/j.jbc.2024.107504pmid: N/A

<h2>SUMMARY</h2><p>Z-nucleic acid structures play vital roles in cellular processes and have implications in innate immunity due to their recognition by Zα domains containing proteins (Z-DNA/Z-RNA binding proteins, ZBPs). Although Zα domains have been identified in six proteins, including viral E3L, ORF112, and I73R, as well as, cellular ADAR1, ZBP1, and PKZ, their prevalence across living organisms remains largely unexplored. In this study, we introduce a computational approach to predict Zα domains, leading to the revelation of previously unidentified Zα domain-containing proteins in eukaryotic organisms, including non-metazoan species. Our findings encompass the discovery of new ZBPs in previously unexplored giant viruses, members of the <i>Nucleocytoviricota</i> phylum. Through experimental validation, we confirm the Zα functionality of select proteins, establishing their capability to induce the B-to-Z conversion. Additionally, we identify Zα-like domains within bacterial proteins. While these domains share certain features with Zα domains, they lack the ability to bind to Z-nucleic acids or facilitate the B-to-Z DNA conversion. Our findings significantly expand the ZBP family across a wide spectrum of organisms and raise intriguing questions about the evolutionary origins of Zα-containing proteins. Moreover, our study offers fresh perspectives on the functional significance of Zα domains in virus sensing and innate immunity and opens avenues for exploring hitherto undiscovered functions of ZBPs.</p>

Anazia, Kara;Koenekoop, Lucien;Ferré, Guillaume;Petracco, Enzo;Gutiérrez-de-Teran, Hugo;Eddy, Matthew T.

doi: 10.1016/j.jbc.2024.107497pmid: 38925329

<h2>Abstract</h2><p>Activation of G proteins through nucleotide exchange initiates intracellular signaling cascades essential for life processes. Under normal conditions, nucleotide exchange is regulated by the formation of G protein–G protein-coupled receptor (GPCR) complexes. Single point mutations in the Gα subunit of G proteins bypass this interaction, leading to loss-of-function or constitutive gain-of-function, which is closely linked with the onset of multiple diseases. Despite the recognized significance of Gα mutations in disease pathology, structural information for most variants is lacking, potentially due to inherent protein dynamics that pose challenges for crystallography. To address this, we leveraged an integrative spectroscopic and computational approach to structurally characterize seven of the most frequently observed clinically-relevant mutations in the stimulatory Gα subunit, GαS. A previously proposed allosteric model of Gα activation linked structural changes in the nucleotide binding pocket with functionally important changes in interactions between switch regions. We investigated this allosteric connection in GαS by integrating data from variable temperature CD spectroscopy, which measured changes in global protein structure and stability, and molecular dynamics (MD) simulations, which observed changes in interaction networks between GαS switch regions. Further, saturation-transfer difference NMR (STD–NMR) spectroscopy was applied to observe changes in nucleotide interactions with residues within the nucleotide binding site. These data have enabled testing of predictions regarding how mutations in GαS result in loss or gain of function and evaluation of proposed structural mechanisms. The integration of experimental and computational data allowed us to propose a more nuanced classification of mechanisms underlying GαS gain-of-function and loss-of-function mutations.</p>

Dey, Dhritiman;Chakravarti, Rudra;Bhattacharjee, Oindrila;Majumder, Satyabrata;Chaudhuri, Dwaipayan;Ahmed, Kazi Tawsif;Roy, Dipanjan;Bhattacharya, Bireswar;Arya, Mansi;Gautam, Anupam;Singh, Rajveer;Gupta, Rahul;Ravichandiran, Velayutham;Chattopadhyay, Dhrubajyoti;Ghosh, Abhrajyoti;Giri, Kalyan;Roy, Syamal;Ghosh, Dipanjan

Goto, Yota;Miyafusa, Takamitsu;Honda, Shinya

doi: 10.1016/j.jbc.2024.107464pmid: N/A

<h2>SUMMARY</h2><p>IFNγ can initiate immune responses by inducing the expression of major histocompatibility complex molecules, suggesting its potential for cancer immunotherapy. However, it also has an immunosuppressive function that limits its application as a therapeutic agent. IFNγ has a characteristic domain-swapped dimer structure with two of the six α-helices exchanged with each other. As we hypothesized that the contrasting functions of IFNγ could be attributed to its unique domain-swapped structure, we designed monomeric IFNγ by transforming the domain-swapped dimer structure of wild-type IFNγ. We conjectured the evolution of this domain-swapped dimer and hypothesized that the current IFNγ structure emerged through shortening of the loop structure at the base of the swapped domain and the accumulation of hydrophobic amino acids at the newly generated interface during domain-swapping. We then designed and generated a stable monomeric IFNγ by retracing this evolutionary process, complementing the lost loop structure with a linker and replacing the accumulated hydrophobic amino acids with hydrophilic ones. We determined that the designed variant was a monomer based on molecular size and number of epitopes and exhibited activity in cell-based assays. Notably, the monomeric IFNγ showed a qualitatively similar balance between immunostimulatory and immunosuppressive gene expression as wild-type IFNγ. This study demonstrates that the structural format of IFNγ affects the strength of its activity rather than regulating the fate of downstream gene expression.</p>

Wei, Lin;Zou, Ruyi;Du, Min;Zhang, Qingdong;Lu, Danrong;Xu, Yingying;Xu, Xiangyu;Wang, Wenshuang;Zhang, Yu-Zhong;Li, Fuchuan

doi: 10.1016/j.jbc.2024.107466pmid: N/A

<h2>Abstract</h2><p>Glycosaminoglycan (GAG) lyases are often strictly substrate specific, and it is especially difficult to simultaneously degrade GAGs with different types of glycosidic bonds. Herein, we found a new class of GAG lyases (GAGases) from different bacteria. These GAGases belong to polysaccharide lyase 35 family and share quite low homology with the identified GAG lyases. The most surprising thing is that GAGases can not only degrade three types of GAGs: HA, CS and HS, but even one of them can also degrade alginate. Further investigation of structural preferences revealed that GAGases selectively act on GAG domains composed of non/6-<i>O</i>-/<i>N</i>-sulfated hexosamines and d-glucoronic acids, as well as on alginate domains composed of d-mannuronic acids. Additionally, GAG lyases were once speculated to have evolved from alginate lyases, but no transitional enzymes have been found. The discovery of GAGases not only broadens the category of GAG lyases, provides new enzymatic tools for the structural and functional studies of GAGs with specific structures, but also provides candidates for the evolution of GAG lyases.</p>

Kolesiński, Piotr;McGowan, Matthew;Botteaux, Anne;Smeesters, Pierre R.;Ghosh, Partho

doi: 10.1016/j.jbc.2024.107478pmid: N/A

<h2>Abstract</h2><p>Antigenically sequence variable M proteins of the major bacterial pathogen <i>Streptococcus pyogenes</i> (Strep A) are responsible for recruiting human C4b-binding protein (C4BP) to the bacterial surface, which enables Strep A to evade destruction by the immune system. The most sequence divergent portion of M proteins, the hypervariable region (HVR), is responsible for binding C4BP. Structural evidence points to the conservation of two C4BP-binding sequence patterns (M2 and M22) in the HVR of numerous M proteins, with this conservation applicable to vaccine immunogen design. These two patterns, however, only partially explain C4BP-binding by Strep A. Here, we identified several M proteins that lack these patterns but still bind C4BP, and determined the structures of two, M68 and M87 HVRs, in complex with a C4BP fragment. Mutagenesis of these M proteins led to identification of amino acids that are crucial for C4BP-binding, enabling formulation of new C4BP-binding patterns. Mutagenesis was also carried out on M2 and M22 proteins to refine or generate experimentally grounded C4BP-binding patterns. The M22 pattern was the most prevalent among M proteins, followed by the M87 and M2 patterns, while the M68 pattern was rare. These patterns, except for M68, were also evident in numerous M-like Enn proteins. Binding of C4BP via these patterns to Enn proteins was verified. We conclude that C4BP-binding patterns occur frequently in Strep A strains of differing M types, being present in their M or Enn proteins, or frequently both, providing further impetus for their use as vaccine immunogens.</p>

Goodrich, Andrew C.;Leclair, Norbert;Shillova, Nita;Morton, William D.;Wittwer, Arthur J.;Loyet, Kelly M.;Hannoush, Rami N.

doi: 10.1016/j.jbc.2024.107467pmid: N/A

<h2>ABSTRACT</h2><p>The complement system plays a critical role in the innate immune response, acting as a first line of defense against invading pathogens. However, dysregulation of the complement system is implicated in the pathogenesis of numerous diseases, ranging from Alzheimer's to age-related macular degeneration (AMD) and rare blood disorders. As such, complement inhibitors have enormous potential to alleviate disease burden. While a few complement inhibitors are in clinical use, there is still a significant unmet medical need for the discovery and development of novel inhibitors to treat patients suffering from disorders of the complement system. A key hurdle in the development of complement inhibitors has been the determination of their mechanism of action. Progression along the complement cascade involves the formation of numerous multimeric protein complexes, creating the potential for inhibitors to act at multiple nodes in the pathway. This is especially true for molecules that target the central component C3 and its fragment C3b, which serve a dual role as a substrate for the C3 convertases and as a scaffolding protein in both the C3 and C5 convertases. Here, we report a step-by-step <i>in vitro</i> reconstitution of the complement alternative pathway using bio-layer interferometry. By physically uncoupling each step in the pathway, we were able to determine the kinetic signature of inhibitors that act at single steps in the pathway and delineate the full mechanism of action of known and novel C3 inhibitors. The method could have utility in drug discovery and further elucidating the biochemistry of the complement system.</p>

Leimu, Laura;Holm, Patrik;Gąciarz, Anna;Haavisto, Oskar;Prince, Stuart;Pesonen, Ullamari;Huovinen, Tuomas;Lamminmäki, Urpo

doi: 10.1016/j.jbc.2024.107507pmid: N/A

<h2>Abstract</h2><p>Aggregation of aberrant fragment of plasma gelsolin, AGelD187N, is a crucial event underlying the pathophysiology of Finnish gelsolin amyloidosis, an inherited form of systemic amyloidosis. The amyloidogenic gelsolin fragment AGelD187N does not play any physiological role in the body, unlike most aggregating proteins related to other protein misfolding diseases. However, no therapeutic agents that specifically and effectively target and neutralize AGelD187N exist. We employed phage display technology to identify novel single-chain variable fragments (scFvs) that bind to different epitopes in the monomeric AGelD187N that were further maturated by variable domain shuffling and converted to antigen-binding fragment (Fab) antibodies. The generated antibody fragments had nanomolar binding affinity for full-length AGelD187N, as evaluated by biolayer interferometry. Importantly, all four Fabs selected for functional studies efficiently inhibited the amyloid formation of full-length AGelD187N as examined by thioflavin fluorescence assay and transmission electron microscopy. Two Fabs, neither of which bound to the previously proposed fibril-forming region of AGelD187N, completely blocked the amyloid formation of AGelD187N. Moreover, no small soluble aggregates, which are considered pathogenic species in protein misfolding diseases, were formed after successful inhibition of amyloid formation by the most promising aggregation inhibitor, as investigated by size exclusion chromatography combined with multi-angle light scattering. We conclude that all regions of the full-length AGelD187N are important in modulating its assembly into fibrils and that the discovered epitope-specific anti-AGelD187N antibody fragments provide a promising starting point for a disease-modifying therapy for gelsolin amyloidosis, which is currently lacking.</p>

Andersen, Harvey M.;Tai, Hua-Chia;Rubakhin, Stanislav S.;Yau, Peter M.;Sweedler, Jonathan V.

doi: 10.1016/j.jbc.2024.107458pmid: 38857862

<h2>Abstract</h2><p>The function of endogenous cell-cell signaling peptides rely on their interactions with cognate receptors, which in turn are influenced by the peptides' structures, necessitating a comprehensive understanding of the suite of post-translational modifications (PTMs) of the peptide. Herein, we report the initial characterization of putative peptide isomerase enzymes extracted from <i>R. norvegicus</i>, <i>A. californica</i>, and <i>B. taurus</i> tissues. These enzymes are both tissue and substrate-specific across all three organisms. Notably, the lungs of the mammalian species, and the central nervous system of the mollusk displayed the highest isomerase activity among the examined tissues. <i>In-vitro</i> enzymatic conversion was observed for several endogenous peptides, such as the tetrapeptide GFFD in <i>A. californica</i>, and mammalian neuropeptide FF in <i>R. norvegicus</i> and <i>B. taurus</i>. To understand their mode of action, we explored the effects of several inhibitors on these enzymes, which suggests common active site residues. While further characterization of these enzymes is required, the investigations emphasize a widespread and overlooked enzyme activity related to the creation of bioactive peptides.</p>