Glia-derived secretory fatty acid binding protein Obp44a regulates lipid storage and efflux in the developing Drosophila brain.

Glia derived secretory factors play diverse roles in supporting the development, physiology, and stress responses of the central nervous system (CNS). Through transcriptomics and imaging analyses, we have identified Obp44a as one of the most abundantly produced secretory proteins from Drosophila CNS glia. Protein structure homology modeling and Nuclear Magnetic Resonance (NMR) experiments reveal Obp44a as a fatty acid binding protein (FABP) with a high affinity towards long-chain fatty acids in both native and oxidized forms. Further analyses demonstrate that Obp44a effectively infiltrates the neuropil, traffics between neuron and glia, and is secreted into hemolymph, acting as a lipid chaperone and scavenger to regulate lipid and redox homeostasis in the developing brain. In agreement with this essential role, deficiency of Obp44a leads to anatomical and behavioral deficits in adult animals and elevated oxidized lipid levels. Collectively, our findings unveil the crucial involvement of a noncanonical lipid chaperone to shuttle fatty acids within and outside the brain, as needed to maintain a healthy brain lipid environment. These findings could inspire the design of novel approaches to restore lipid homeostasis that is dysregulated in CNS diseases.

Xplor-NIH: Better parameters and protocols for NMR protein structure determination.

The present work describes an update to the protein covalent geometry and atomic radii parameters in the Xplor-NIH biomolecular structure determination package. In combination with an improved treatment of selected non-bonded interactions between atoms three bonds apart, such as those involving methyl hydrogens, and a previously developed term that affects the system's gyration volume, the new parameters are tested using structure calculations on 30 proteins with restraints derived from nuclear magnetic resonance data. Using modern structure validation criteria, including several formally adopted by the Protein Data Bank, and a clear measure of structural accuracy, the results show superior performance relative to previous Xplor-NIH implementations. Additionally, the Xplor-NIH structures compare favorably against originally determined NMR models.

Structural Relationships to Efficacy for Prazole-Derived Antivirals.

Here, an in vitro characterization of a family of prazole derivatives that covalently bind to the C73 site on Tsg101 and assay their ability to inhibit viral particle production is presented. Structurally, increased steric bulk on the 4-pyridyl of the prazole expands the prazole site on the UEV domain toward the ß-hairpin in the Ub-binding site and is coupled to increased inhibition of virus-like particle production in HIV-1. Increased bulk also increased toxicity, which is alleviated by increasing flexibility. Further, the formation of a novel secondary Tsg101 adduct for several of the tested compounds and the commercial drug lansoprazole. The secondary adduct involved the loss of the 4-pyridyl substituent to form an irreversible species, with implications for increasing the half-life of the active species or its specificity toward Tsg101 UEV. It is also determined that sulfide derivatives display effective viral inhibition, presumably through cellular sulfoxidation, allowing for delayed conversion within the cellular environment, and identify SARS-COV-2 as a target of prazole inhibition. These results open multiple avenues for the design of prazole derivatives for antiviral applications.

Identification of Neural (N)-Cadherin as a Novel Endothelial Cell Receptor for Fibrin and Localization of the Complementary Binding Sites.

Based on the high structural homology between vascular endothelial (VE)-cadherin and neural (N)-cadherin, we hypothesized that fibrin, which is known to interact with VE-cadherin and promote angiogenesis through this interaction, may also interact with N-cadherin. To test this hypothesis, we prepared fibrin and its plasmin-produced and recombinant fragments covering practically all parts of the fibrin molecule. We also prepared the soluble extracellular portion of N-cadherin (sN-cadherin), which includes all five extracellular N-cadherin domains, and studied its interaction with fibrinogen, fibrin, and the aforementioned fibrin fragments using two independent methods, ELISA and SPR. The experiments confirmed our hypothesis, revealing that fibrin interacts with sN-cadherin with high affinity. Furthermore, the experiments localized the N-cadherin binding site within the fibrin ßN-domains. Notably, the recombinant dimeric (ß15-66)2 fragment, corresponding to these domains and mimicking their dimeric arrangement in fibrin, preserved the N-cadherin-binding properties of fibrin. To localize the fibrin binding site within N-cadherin, we performed ELISA and SPR experiments with (ß15-66)2 and recombinant N-cadherin fragments representing its individual extracellular domains and combinations thereof. The results obtained indicate that the interaction of fibrin with N-cadherin occurs through the third and fifth extracellular domains of the latter. This is in contrast to our previous study, which revealed that fibrin interacts only with the third extracellular domain of VE-cadherin. In conclusion, our study identified N-cadherin as a novel receptor for fibrin and localized complementary binding sites within both fibrin and N-cadherin. The pathophysiological role of this interaction remains to be established.

Observation of pH-Dependent Residual Structure in the Pmel17 Repeat Domain and the Implication for Its Amyloid Formation.

The varying conformational states of amyloid-forming protein monomers can determine their fibrillation outcome. In this study, we utilize solution NMR and the paramagnetic relaxation enhancement (PRE) effect to observe monomer properties of the repeat domain (RPT) from a human functional amyloid, premelanosomal protein, Pmel17. After excision from the full-length protein, RPT can self-assemble into amyloid fibrils, functioning as a scaffold for melanin deposition. Here, we report possible conformational states of the short RPT (sRPT) isoform, which has been demonstrated to be a fibrillation nucleator. NMR experiments were performed to determine conformational differences in sRPT by comparing aggregation-prone vs nonaggregating solution conditions. We observed significant chemical shift perturbations localized to residues near the C-terminus, demonstrating that the local chemical environment of the amyloid core region is highly sensitive to changes in pH. Next, we introduced cysteine point mutations for the covalent attachment of PRE ligands to sRPT to facilitate the observation of intramolecular interactions. We also utilized solvent PRE molecules with opposing charges to measure changes in the electrostatic potential of sRPT in different pH environments. These observed PRE effects offer insight into initial molecular events that might promote intermolecular interactions, which can trigger fibrillation. Taken together, our results show that sRPT monomers adopt a conformation inconsistent with a fully random coil at neutral pH and undergo conformational changes at lower pH values. These observations highlight regulatory mechanisms via organelle-associated pH conditions that can affect the fibrillation activity of proteins like RPT.

Evidence of natural selection in the mitochondrial-derived peptides humanin and SHLP6.

Mitochondrial-derived peptides are encoded by mitochondrial DNA but have biological activity outside mitochondria. Eight of these are encoded by sequences within the mitochondrial 12S and 16S ribosomal genes: humanin, MOTS-c, and the six SHLP peptides, SHLP1-SHLP6. These peptides have various effects in cell culture and animal models, affecting neuroprotection, insulin sensitivity, and apoptosis, and some are secreted, potentially having extracellular signaling roles. However, except for humanin, their importance in normal cell function is unknown. To gauge their importance, their coding sequences in vertebrates have been analyzed for synonymous codon bias. Because they lie in RNA genes, such bias should only occur if their amino acids have been conserved to maintain biological function. Humanin and SHLP6 show strong synonymous codon bias and sequence conservation. In contrast, SHLP1, SHLP2, SHLP3, and SHLP5 show no significant bias and are poorly conserved. MOTS-c and SHLP4 also lack significant bias, but contain highly conserved N-terminal regions, and their biological importance cannot be ruled out. An additional potential mitochondrial-derived peptide sequence was discovered preceding SHLP2, named SHLP2b, which also contains a highly conserved N-terminal region with synonymous codon bias.

Nix interacts with WIPI2 to induce mitophagy.

Nix is a membrane-anchored outer mitochondrial protein that induces mitophagy. While Nix has an LC3-interacting (LIR) motif that binds to ATG8 proteins, it also contains a minimal essential region (MER) that induces mitophagy through an unknown mechanism. We used chemically induced dimerization (CID) to probe the mechanism of Nix-mediated mitophagy and found that both the LIR and MER are required for robust mitophagy. We find that the Nix MER interacts with the autophagy effector WIPI2 and recruits WIPI2 to mitochondria. The Nix LIR motif is also required for robust mitophagy and converts a homogeneous WIPI2 distribution on the surface of the mitochondria into puncta, even in the absence of ATG8s. Together, this work reveals unanticipated mechanisms in Nix-induced mitophagy and the elusive role of the MER, while also describing an interesting example of autophagy induction that acts downstream of the canonical initiation complexes.

Expression and purification of Drosophila OBP44a with the aids of LC-MS and NMR.

The production of highly purified native soluble proteins in large quantities is crucial for studying protein structure and function. Odorant binding proteins (OBPs) are small, soluble, extracellular proteins with multiple disulfide bonds, whose functions include, but are not limited to, binding hydrophobic molecules and delivering them to their corresponding receptors expressed on insect olfactory receptor neurons. Expression of proteins with multiple disulfide bonds like OBPs usually results in insolubility and low yield, which has been a significant barrier to understanding their biological roles and physiological functions. In the E. coli system, expression of OBPs often results in insoluble inclusion bodies or a limited amount of periplasmic soluble proteins. Although expression of OBPs in eukaryotic systems such as Sf9 insect cells or yeast Pichia pastoris can increase the solubility of the protein, the process remains insufficient. Additionally, monitoring the purity and native apo state of the protein is critical for establishing the correct conformation of the protein. In this study, we employed an E. coli host with an altered intracellular environment to produce cytosolic soluble OBP44a protein, which yielded over 100 mg/L. We monitored the integrity of disulfide bonds throughout the purification process using LC-MS and used NMR to ensure the final product adopted a single conformation. Our study presents an efficient method for obtaining large quantities of soluble proteins in a single conformation, which enables extensive in vitro studies of secreted proteins like OBPs.

Co-crystal structures of the fluorogenic aptamer Beetroot show that close homology may not predict similar RNA architecture.

Beetroot is a homodimeric in vitro selected RNA that binds and activates DFAME, a conditional fluorophore derived from GFP. It is 70% sequence-identical to the previously characterized homodimeric aptamer Corn, which binds one molecule of its cognate fluorophore DFHO at its interprotomer interface. We have now determined the Beetroot-DFAME co-crystal structure at 1.95 Å resolution, discovering that this RNA homodimer binds two molecules of the fluorophore, at sites separated by ~30 Å. In addition to this overall architectural difference, the local structures of the non-canonical, complex quadruplex cores of Beetroot and Corn are distinctly different, underscoring how subtle RNA sequence differences can give rise to unexpected structural divergence. Through structure-guided engineering, we generated a variant that has a 12-fold fluorescence activation selectivity switch toward DFHO. Beetroot and this variant form heterodimers and constitute the starting point for engineered tags whose through-space inter-fluorophore interaction could be used to monitor RNA dimerization.

HECT domain interaction with ubiquitin binding sites on Tsg101-UEV controls HIV-1 egress, maturation, and infectivity.

The HECT domain of HECT E3 ligases consists of flexibly linked N- and C-terminal lobes, with a ubiquitin (Ub) donor site on the C-lobe that is directly involved in substrate modification. HECT ligases also possess a secondary Ub binding site in the N-lobe, which is thought to play a role in processivity, specificity, or regulation. Here, we report the use of paramagnetic solution NMR to characterize a complex formed between the isolated HECT domain of neural precursor cell-expressed developmentally downregulated 4-1 and the ubiquitin E2 variant (UEV) domain of tumor susceptibility gene 101 (Tsg101). Both proteins are involved in endosomal trafficking, a process driven by Ub signaling, and are hijacked by viral pathogens for particle assembly; however, a direct interaction between them has not been described, and the mechanism by which the HECT E3 ligase contributes to pathogen formation has not been elucidated. We provide evidence for their association, consisting of multiple sites on the neural precursor cell-expressed developmentally downregulated 4-1 HECT domain and elements of the Tsg101 UEV domain involved in noncovalent ubiquitin binding. Furthermore, we show using an established reporter assay that HECT residues perturbed by UEV proximity define determinants of viral maturation and infectivity. These results suggest the UEV interaction is a determinant of HECT activity in Ub signaling. As the endosomal trafficking pathway is hijacked by several human pathogens for egress, the HECT-UEV interaction could represent a potential novel target for therapeutic intervention.

Incorporation of residual chemical shift anisotropy into the treatment of 15N pseudocontact shifts for structural refinement.

Paramagnetic NMR experiments, including the pseudocontact shift experiment, have seen increasing use due to recently developed probes and labeling strategies. The pseudocontact shift experiment can provide valuable intra- or inter-molecular distance and orientation information. However, the use of 1H/13C or 1H/15N PCS data in structure calculations is currently complicated by the contribution of residual chemical shift anisotropy to the 13C or 15N datasets. Here, we present a corrected PCS energy term for the software package Xplor-NIH with the appropriate residual chemical shift anisotropy correction and show its suitability for model refinements of ubiquitin labeled at residue 57 with a Tm-M8-SPy tag. For data taken at 800 MHz, the improvement with the corrected energy term is sufficient to make the quality of the fit for the 15N dataset comparable to that of the 1H dataset, for which no correction is needed. The corrected energy term is expected to become more relevant with increased use of higher field instruments and as new paramagnetic probes with larger magnetic susceptibility tensors continue to be developed.

Tsg101/ESCRT-I recruitment regulated by the dual binding modes of K63-linked diubiquitin.

The ESCRT-I protein Tsg101 plays a critical role in viral budding and endocytic sorting. Although Tsg101 is known to recognize monoubiquitin (Ub1), here we show that it can also bind several diubiquitins (K48-Ub2, N-Ub2, and K63-Ub2), with a preference for K63-linked Ub2. The NMR structure of the Tsg101:K63-Ub2 complex showed that while the Ub1-binding site accommodates the distal domain of Ub2, the proximal domain alternatively binds two different sites, the vestigial active site and an N-terminal helix. Mutation of each site results in distinct phenotypes regarding the recruitment of Tsg101 partners. Mutation in the vestigial active site abrogates interaction between Tsg101 and the HIV-1 protein Gag but not Hrs, a cellular protein. Mutation at the N-terminal helix alters Gag but not Hrs-Tsg101 localization. Given the broad involvement of Tsg101 in diverse cellular functions, this discovery advances our understanding of how the ESCRT protein recognizes binding partners and sorts endocytic cargo.

The fluorescent aptamer Squash extensively repurposes the adenine riboswitch fold.

Squash is an RNA aptamer that strongly activates the fluorescence of small-molecule analogs of the fluorophore of green fluorescent protein (GFP). Unlike other fluorogenic aptamers, isolated de novo from random-sequence RNA, Squash was evolved from the bacterial adenine riboswitch to leverage its optimized in vivo folding and stability. We now report the 2.7-Å resolution cocrystal structure of fluorophore-bound Squash, revealing that while the overall fold of the riboswitch is preserved, the architecture of the ligand-binding core is dramatically transformed. Unlike previously characterized aptamers that activate GFP-derived fluorophores, Squash does not harbor a G-quadruplex, sandwiching its fluorophore between a base triple and a noncanonical base quadruple in a largely apolar pocket. The expanded structural core of Squash allows it to recognize unnatural fluorophores that are larger than the simple purine ligand of the parental adenine riboswitch, and suggests that stable RNA scaffolds can tolerate larger variation than has hitherto been appreciated.

Structural Basis for the Interaction of Fibrin with the Very Low-Density Lipoprotein Receptor Revealed by NMR and Site-Directed Mutagenesis.

Interaction of fibrin with the very low-density lipoprotein receptor (VLDLR) promotes transendothelial migration of leukocytes and thereby inflammation. To establish the structural basis for this interaction, we have previously localized the VLDLR-binding site to fibrin ßN-domains including fibrin ß chain sequence 15-64 and determined the NMR solution structure of the VLDLR(2-4) fragment containing fibrin-binding CR domains 2-4 of VLDLR. In this study, we identified amino acid residues in VLDLR and the ßN-domains that are involved in the interaction using NMR and site-directed mutagenesis. The results obtained revealed that Lys47 and Lys53 of the second and third positively charged clusters of the ßN-domain, respectively, interact with Trp20 and Asp25 of the CR2 domain and Trp63 and Glu68 of the CR3 domain, respectively. This finding indicates that Lys residues of the ßN-domain interact with the Lys-binding site of the CR domains in a manner proposed earlier for the interaction of other members of the LDL receptor family with their ligands. In addition, Gly15 of the ßN-domain and its first positively charged cluster contribute to the high-affinity interaction with VLDLR. Molecular modeling based on the results obtained and analysis of the previously published structures of such domains complexed with RAP and HRV2 allowed us to propose a model of interaction of fibrin ßN-domains with the fibrin-binding CR domains of the VLDL receptor.

Novel Tsg101 Binding Partners Regulate Viral L Domain Trafficking.

Two decades ago, Tsg101, a component of the Endosomal Sorting Complexes Required for Transport (ESCRT) complex 1, was identified as a cellular factor recruited by the human immunodeficiency virus type 1 (HIV-1) to facilitate budding of viral particles assembled at the cell periphery. A highly conserved Pro-(Thr/Ser)-Ala-Pro [P(T/S)AP] motif in the HIV-1 structural polyprotein, Gag, engages a P(T/S)AP-binding pocket in the Tsg101 N-terminal domain. Since the same domain in Tsg101 that houses the pocket was found to bind mono-ubiquitin (Ub) non-covalently, Ub binding was speculated to enhance P(T/S)AP interaction. Within the past five years, we found that the Ub-binding site also accommodates di-Ub, with Lys63-linked di-Ub exhibiting the highest affinity. We also identified small molecules capable of disrupting Ub binding and inhibiting budding. The structural similarity of these molecules, prazoles, to nucleosides prompted testing for nucleic acid binding and led to identification of tRNA as a Tsg101 binding partner. Here, we discuss these recently identified interactions and their contribution to the viral assembly process. These new partners may provide additional insight into the control and function of Tsg101 as well as identify opportunities for anti-viral drug design.

Prazoles Targeting Tsg101 Inhibit Release of Epstein-Barr Virus following Reactivation from Latency.

Epstein-Barr virus (EBV) is a ubiquitous herpesvirus responsible for several diseases, including cancers of lymphoid and epithelial cells. EBV cancers typically exhibit viral latency; however, the production and release of EBV through its lytic phase are essential for cancer development. Antiviral agents that specifically target EBV production do not currently exist. Previously, we reported that the proton pump inhibitor tenatoprazole, which blocks the interaction of ubiquitin with the ESCRT-1 factor Tsg101, inhibits production of several enveloped viruses, including EBV. Here, we show that three structurally distinct prazoles impair mature particle formation postreactivation and identify the impact on stages of replication. The prazoles did not impair expression of lytic genes representative of the different kinetic classes but interfered with capsid maturation in the nucleus as well as virion transport from the nucleus. Replacement of endogenous Tsg101 with a mutant Tsg101 refractory to prazole-mediated inhibition rescued EBV release. These findings directly implicate Tsg101 in EBV nuclear egress and identify prazoles as potential therapeutic candidates for conditions that rely on EBV replication, such as chronic active EBV infection and posttransplant lymphoproliferative disorders. IMPORTANCE Production of virions is necessary for the ubiquitous Epstein-Barr virus (EBV) to persist in humans and can set the stage for development of EBV cancers in at-risk individuals. In our attempts to identify inhibitors of the EBV lytic phase, we previously found that a prazole proton pump inhibitor, known to block the interaction of ubiquitin with the ESCRT-1 factor Tsg101, blocks production of EBV. We now find that three structurally distinct prazoles impair maturation of EBV capsids and virion transport from the nucleus and, by interfering with Tsg101, prevent EBV release from lytically active cells. Our findings not only implicate Tsg101 in EBV production but also identify widely used prazoles as candidates to prevent development of posttransplant EBV lymphomas.

Inducible fold-switching as a mechanism to fibrillate pro-apoptotic BCL-2 proteins.

Neurodegenerative diseases often are associated with cellular dysregulation that results in premature cell death or apoptosis. A common example is the accumulation of amyloid plaques that promotes the excessive expression of p38 mitogen-activated protein kinase. The increased abundance of this enzyme leads to mass phosphorylation and activation of a protein from the B-cell lymphoma 2 (BCL-2) family, BAX. BAX is the central regulatory protein for mitochondrial outer membrane permeabilization (MOMP), a poration process that commits cells to apoptosis by releasing death-propagating factors from the mitochondria. Recent reports identify a naturally occurring peptide, Humanin (HN), that could block amyloid-beta-associated neuronal apoptosis by interacting with BCL-2 proteins. We recently showed humanin interaction leads to the amyloid-like fibrillation of BAX and a second BCL-2 family member, BID. We proposed this as a novel anti-apoptotic mechanism that inhibits pro-apoptotic BCL-2 proteins from initiating MOMP by sequestering them into fibrils, a heretofore unprecedented phenomenon that involves refolding globular BCL-2 proteins rapidly into fibrils where they undergo significant alpha-helix to beta-sheet fold-switching. Here we seek to further characterize the fibrillation and fold-switch in conditions that are known to induce amyloid fibrillation.

Simultaneous measurement of 1HC/N-R2's for rapid acquisition of backbone and sidechain paramagnetic relaxation enhancements (PREs) in proteins.

Paramagnetic relaxation enhancements (PREs) are routinely used to provide long-range distance restraints for the determination of protein structures, to resolve protein dynamics, ligand-protein binding sites, and lowly populated species, using Nuclear Magnetic Resonance Spectroscopy (NMR). Here, we propose a simultaneous 1H-15 N, 1H-13C SESAME based pulse scheme for the rapid acquisition of 1HC/N-R2 relaxation rates for the determination of backbone and sidechain PREs of proteins. The 1HN-R2 rates from the traditional and our approach on Ubiquitin (UBQ) are well correlated (R2 = 0.99), revealing their potential to be used quantitatively. Comparison of the S57C UBQ calculated and experimental PREs provided backbone and side chain Q factors of 0.23 and 0.24, respectively, well-fitted to the UBQ NMR structure, showing that our approach can be used to acquire accurate PRE rates from the functionally important sites of proteins but in at least half the time as traditional methods.

Humanin selectively prevents the activation of pro-apoptotic protein BID by sequestering it into fibers.

Members of the B-cell lymphoma (BCL-2) protein family regulate mitochondrial outer membrane permeabilization (MOMP), a phenomenon in which mitochondria become porous and release death-propagating complexes during the early stages of apoptosis. Pro-apoptotic BCL-2 proteins oligomerize at the mitochondrial outer membrane during MOMP, inducing pore formation. Of current interest are endogenous factors that can inhibit pro-apoptotic BCL-2 mitochondrial outer membrane translocation and oligomerization. A mitochondrial-derived peptide, Humanin (HN), was reported being expressed from an alternate ORF in the mitochondrial genome and inhibiting apoptosis through interactions with the pro-apoptotic BCL-2 proteins. Specifically, it is known to complex with BAX and BID. We recently reported the fibrillation of HN and BAX into ß-sheets. Here, we detail the fibrillation between HN and BID. These fibers were characterized using several spectroscopic techniques, protease fragmentation with mass analysis, and EM. Enhanced fibrillation rates were detected with rising temperatures or pH values and the presence of a detergent. BID fibers are similar to those produced using BAX; however, the structures differ in final conformations of the BCL-2 proteins. BID fibers display both types of secondary structure in the fiber, whereas BAX was converted entirely to ß-sheets. The data show that two distinct segments of BID are incorporated into the fiber structure, whereas other portions of BID remain solvent-exposed and retain helical structure. Similar analyses show that anti-apoptotic BCL-xL does not form fibers with humanin. These results support a general mechanism of sequestration of pro-apoptotic BCL-2 proteins into fibers by HN to inhibit MOMP.

Author Correction: Structural basis for polyglutamate chain initiation and elongation by TTLL family enzymes.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.