Nico Tjandra

Glia-derived secretory factors are essential for brain development, physiology, and homeostasis, with their dysfunction linked to a variety of neurological disorders. Through genetic and biochemical approaches, we identified odorant binding protein 44a (Obp44a), a noncanonical α-helical fatty acid binding protein (FABP) highly expressed in Drosophila central nervous system glia. Obp44a binds long-chain fatty acids and shuttles between glia and neurons, acting as a secretory lipid chaperone and scavenger to support lipid storage, efflux, and redox homeostasis. Notably, Obp44a is recruited to apoptotic cells and injured axons, especially when glial engulfment is impaired, demonstrating its role in lipid waste management and clearance of cellular debris during development and in pathological states. Our findings highlight FABPs’ importance in regulating brain lipid dynamics and neuronal response to stress and injury. By visualizing FABP function in vivo, this study provides insights into how defective lipid regulation may contribute to neuronal stress and disease progression.

UEV domains are catalytically dead variants of the E2 enzymes which play an intermediate role in ubiquitin signaling. UEV domain containing proteins, like the ESCRT-I factor Tsg101 often play critical roles in trafficking of ubiquitylated cargos or in modulating ubiquitin processivity, or in determining the type of signal that is transferred to a target protein. Ubiquitin-conjugating enzyme E2 variant (UEV) and lactate/malate dehydrogenase (UEVLD), also known as UEV3, is a human paralogue of Tsg101 with apparent associations to cancer, innate immunity, NF-κB signaling, and autophagy. It contains an N-terminal UEV domain with 56% identity to that of Tsg101 and a C-terminal lactate dehydrogenase domain. Here, we show the backbone assignments of the UEV domain from UEVLD and find that its Cα shifts are consistent with a UEV domain composition. Further experiments suggest that it may have regions corresponding to the known binding pockets of Tsg101, but further structural and functional work will be required to uncover critical determinants of UEV domain function, and the role of these domains in Ubiquitin signaling as a whole.

Paramagnetic relaxation enhancement (PRE) is widely used in biomolecular NMR spectroscopy to obtain long-range distance and orientational information for intra- or intermolecular interactions. In contrast to conventional PRE measurements, which require tethering small molecules containing either a radical or paramagnetic ion to specific sites on the target protein, solvent PRE (sPRE) experiments utilize paramagnetic cosolutes to induce a delocalized PRE effect. Compounds developed as contrast agents in magnetic resonance imaging (MRI) applications typically consist of Gd chelated by a small molecule. Coordinating these Gd-containing small molecules to larger and inert scaffolds has been shown to increase the PRE-effect and produce more effective contrast agents in MRI. Inspired by their use as MRI contrast agent, in this work we evaluate the effectiveness of using a functionalized polyamidoamine (PAMAM) dendrimer for sPRE measurements. Using ubiquitin as a model system, we measured the sPRE effect from a generation 5 PAMAM dendrimer (G5-Gd) as a function of temperature and pH and compared to conventional relaxation agents. We also demonstrated the utility of G5-Gd in sPRE studies to monitor changes in the structures of two proteins as they bind their ligands. These studies highlight the attractive properties of these macromolecular relaxation agents in biomolecular sPRE.

Tsg101 is a highly conserved protein best known as an early-functioning component of cellular ESCRT machinery participating in recognition, sorting, and trafficking of cellular cargo to various intracellular destinations. It shares sequence and structural homology to canonical ubiquitin-conjugating (E2) enzymes and is linked to diverse events regulated by Ub signaling. How it might fulfill these roles is unclear. Here, we show that Tsg101 E2 mimicry permits interactions with diverse ubiquitin ligating (E3) enzymes and underlies its multifunctional capabilities. Coexpression of Tsg101 with the E3 ligase NNedd4-2s protected the enzyme from degradation and, remarkably, other widely divergent ligases as well. Structural alignment with UbcH5, a canonical E2 enzyme, revealed that recognition at the E2-E3 interface, a region broadly conserved despite sequence and structural differences in both E2 and E3 enzymes, was critical for protection. Nevertheless, UbcH5 failed to protect NNedd4-2s, indicating that the UEV chaperone function is unique to the variant. Studies using Cy5-Ub-VME showed that Tsg101-mediated protection reduced accessibility to Cys residues in the ligase. Access to Tsg101 Ub-binding sites was critical: Rabeprazole, which interferes with Tsg101 Ub-binding, diminished E3 ligase protection. Thus, E2 mimicry permitting control of E3 ligase ubiquitin signaling underlies Tsg101’s broad ability to participate in multiple cellular functions. The study provides mechanistic insight into how Tsg101, by partnering with diverse E3 ligases, can contribute to a broad range of cellular activities.

We recently identified N-cadherin as a novel receptor for fibrin and localized complementary binding sites within the fibrin βN-domains and the third and fifth extracellular domains (EC3 and EC5) of N-cadherin. We also hypothesized that the His16 and Arg17 residues of the βN-domains and the (Asp/Glu)-X-(Asp/Glu) motifs present in the EC3 and EC5 domains may play roles in the interaction between fibrin and N-cadherin. The primary objectives of this study were to test these hypotheses and to further clarify the structural basis for this interaction. To test our hypotheses, we first mutated His16 and Arg17 in the recombinant (β15-66)₂ fragment, which mimics the dimeric arrangement of the βN-domains in fibrin, using site-directed mutagenesis. The results revealed that the mutations of both His16 and Arg17 are critical for the interaction. Next, we mutated Asp/Glu residues in the three (Asp/Glu)-X-(Asp/Glu) motifs, M1 (Asp-Phe-Glu), M2 (Glu-Ala-Glu), and M3 (Asp-Tyr-Asp), of the fibrin-binding N-cad(3-5) fragment of N-cadherin. The results showed that Asp292 and Glu294 of M1, and Asp468 and Asp470 of M3, are critical for the interaction. Our molecular modeling of the 3D structure of the EC3-EC4-EC5 domains revealed that these residues are located at the interfaces of EC3-EC4 and EC4-EC5 and that some may also be involved in calcium binding. In conclusion, our study identified amino acid residues in the fibrin βN-domains and the EC3 and EC5 domains of N-cadherin that are critical for the interaction of fibrin with N-cadherin and localized the fibrin-binding residues in the 3D structure of N-cadherin.

Tsg101, a component of the endosomal sorting complex required for transport (ESCRT), is responsible for recognition of events requiring the machinery, as signaled by cargo tagging with ubiquitin (Ub), and for recruitment of downstream acting subunits to the site. Although much is known about the latter function, little is known about its role in the earlier event. The N-terminal domain of Tsg101 is a structural homologue of Ub conjugases (E2 enzymes) and the protein associates with Ub ligases (E3 enzymes) that regulate several cellular processes including virus budding. A pocket in the domain recognizes a motif, PT/SAP, that permits its recruitment. PT/SAP disruption makes budding dependent on Nedd4L E3 ligases. Using HIV-1 encoding a PT/SAP mutation that makes budding Nedd4L-dependent, we identified as critical for rescue the residues in the catalytic (HECT) domain of the E3 enzyme that lie in proximity to sites in Tsg101 that bind Ub non-covalently. Mutation of these residues impaired rescue by Nedd4L but the same mutations had no apparent effect in the context of a Nedd4 isomer, Nedd4-2s, whose N-terminal (C2) domain is naturally truncated, precluding C2-HECT auto-inhibition. Surprisingly, like small molecules that disrupt Tsg101 Ub-binding, small molecules that interfered with Nedd4 substrate recognition arrested budding at an early stage, supporting the conclusion that Tsg101–Ub–Nedd4 interaction promotes enzyme activation and regulates Nedd4 signaling for viral egress. Tsg101 regulation of E3 ligases may underlie its broad ability to function as an effector in various cellular activities, including viral particle assembly and budding.

The odorant binding protein, OBP44a is one of the most abundant proteins expressed in the brain of the developing fruit fly Drosophila melanogaster. Its cellular function has not yet been determined. The OBP family of proteins is well established to recognize hydrophobic molecules. In this study, NMR is employed to structurally characterize OBP44a. NMR chemical shift perturbation measurements confirm that OBP44a binds to fatty acids. Complete assignments of the backbone chemical shifts and secondary chemical shift analysis demonstrate that the apo state of OBP44a is comprised of six α-helices. Upon binding 8(Z)-eicosenoic acid (8(Z)-C20:1), the OBP44a C-terminal region undergoes a conformational change, from unstructured to α-helical. In addition to C-terminal restructuring upon ligand binding, some hydrophobic residues show dramatic chemical shift changes. Surprisingly, several charged residues are also strongly affected by lipid binding. Some of these residues could represent key structural features that OBP44a relies on to perform its cellular function. The NMR chemical shift assignment is the first step towards characterizing the structure of OBP44a and how specific residues might play a role in lipid binding and release. This information will be important in deciphering the biological function of OBP44a during fly brain development.