Biology subjects

El-Baba, T. J.

Publications and source records attributed to El-Baba, T. J..

6 recordsLinked to original sources

bioRxiv2026

A proteomic feasibility study connecting metabolic and synaptic pathway alterations in serum and extracellular vesicles to characterize treatment-resistant depression

Treatment-resistant depression (TRD) remains a major clinical challenge, yet the biological processes distinguishing TRD from non-treatment-resistant depression (nTRD) are incompletely defined. While circulating serum proteomes reflect broad systemic alterations associated with depression, extracellular vesicles (EVs) could provide a more selective representation of intercellular signaling relevant to treatment resistance. Here, we carried out a pilot study to evaluate the extent that parallel proteomic profiling of serum and serum-derived EVs could distinguish healthy controls (CON), nTRD, and TRD individuals. In this exploratory and hypothesis-generating study, serum proteomes exhibited robust global differences between depression groups and controls, largely reflecting shared systemic biology across nTRD and TRD. In contrast, EV proteomes showed limited global separation but revealed subtype-associated pathway differences. Relative to controls, nTRD EVs were enriched for immune and inflammatory pathways. By contrast, TRD EVs were characterized by enrichment of mitochondrial metabolism, oxidative phosphorylation, translational initiation, and MYC-regulated pathways, together with depletion of synaptic signalling, membrane trafficking, and cytoskeletal pathways. Comparative analysis of pathways significant in both contrasts revealed that these bioenergetic and translational signatures were selectively amplified in TRD relative to nTRD. Our exploratory analyses identified that the circulating EV cargo may reflect a treatment-resistance-specific reorganization of biological pathways not apparent in bulk serum proteomics. This study highlights parallel serum and EV proteomics as a complementary approach for molecular stratification in antidepressant resistance.

neuroscience↗

bioRxiv2026

Defining state-selective lipid binding to brain GPCRs - introducing REVEAL

Membrane lipids are central regulators of G protein-coupled receptor (GPCR) function. Defining receptor-specific lipid interactions in native, fully modified mammalian systems remains challenging. Extensive post-translational modifications generate heterogeneous proteoforms that confound conventional mass spectrometry approaches. Here we introduce REVEAL (REceptor enVironment Elucidation by Activated Lipid-release), an automated native top-down mass spectrometry strategy that discriminates specifically bound lipids from background. Applied here to intact, heterogeneous mammalian membrane protein complexes, incubated with a brain polar lipid extract (>1000 components), we define receptor-specific lipid-binding for two neuronal class C GPCRs. We show that agonism remodels lipid occupancy, selectively enriching a reduced repertoire of bound lipids. Plasmalogen lipids emerge as persistent binders across all conformational states of the metabotropic glycine receptor and are preferentially depleted under oxidative stress, implying a protective role at the receptor surface. These findings position lipids as dynamic regulators of both function and response to the cellular redox environment.

biophysics↗

bioRxiv2026

Molecular dissection of protein complexes isolated from sections of human brain

Molecular studies of brain receptors and transporters have typically relied on recombinant systems, limiting insight into their organization in native tissue. Here, we develop nanobody-based immunoprecipitation coupled with native mass spectrometry to interrogate endogenous protein assemblies from post-mortem mouse and human brain sections. We exemplify our approach by characterizing the synaptic proteins VGluT1 and mGluR2. From a single mouse brain, we discover mGluR2/3 heterodimers, alongside mGluR2 homodimers. Considering regions of human brain heterodimeric mGluR2/3 is highly abundant in the OFC and sgACC ([~]70% and 50%, respectively) and forms regional-specific interactions with additional synaptic proteins. In a modest cohort of biobanked human tissue, associated with depression and suicide, we find increased mGluR2/3 in the OFC. Consistent with this, mice exhibit similar associations between heterodimer levels and stress-susceptibility. Overall, our approach provides a direct means for establishing molecular-behavioural links at the level of receptor organization in brain.

biochemistry↗

bioRxiv2023

Dimerisation of European robin cryptochrome 4a

Homo-dimer formation is important for the function of many proteins. Although dimeric forms of cryptochromes (Cry) have been found by crystallography and were recently observed in vitro for European robin Cry4a, little is known about the dimerisation of avian cryptochromes and the role it could play in the mechanism of magnetic sensing in migratory birds. Here we present a combined experimental and computational investigation of the dimerisation of robin Cry4a resulting from covalent and non-covalent interactions. Experimental studies using native mass spectrometry, mass spectrometric analysis of disulphide bonds, chemical cross-linking and photometric measurements show that disulphide-linked dimers are routinely formed, the most likely cysteines being C317 and C412. Computational modelling and molecular dynamics simulations were used to generate and assess a number of possible dimer structures. The relevance of these findings to the proposed role of Cry4a in avian magnetoreception is discussed.

biochemistry↗

bioRxiv2020

Autoproteolytic Products of the SARS-CoV-2 Nucleocapsid Protein are Primed for Antibody Evasion and Virus Proliferation

The SARS-CoV-2 nucleocapsid (N) protein is the most immunogenic of the structural proteins and plays essential roles in several stages of the virus lifecycle. It is comprised of two major structural domains: the RNA binding domain, which interacts with viral and host RNA, and the oligomerization domain which assembles to form the viral core. Here, we investigate the assembly state and RNA binding properties of the full-length nucleocapsid protein using native mass spectrometry. We find that dimers, and not monomers, of full-length N protein bind RNA, implying that dimers are the functional unit of ribonucleoprotein assembly. In addition, we find that N protein binds RNA with a preference for GGG motifs which are known to form short stem loop structures. Unexpectedly, we found that N undergoes proteolytic processing within the linker region, separating the two major domains. This process results in the formation of at least five proteoforms that we sequenced using electron transfer dissociation, higher-energy collision induced dissociation and corroborated by peptide mapping. The cleavage sites identified are in highly conserved regions leading us to consider the potential roles of the resulting proteoforms. We found that monomers of N-terminal proteoforms bind RNA with the same preference for GGG motifs and that the oligomeric state of a C-terminal proteoform (N156-419) is sensitive to pH. We then tested interactions of the proteoforms with the immunophilin cyclophilin A, a key component in coronavirus replication. We found that N1-209 and N1-273 bind directly to cyclophilin A, an interaction that is abolished by the approved immunosuppressant drug cyclosporin A. In addition, we found the C-terminal proteoform N156-419 generated the highest antibody response in convalescent plasma from patients >6 months from initial COVID-19 diagnosis when compared to the other proteoforms. Overall, the different interactions of N proteoforms with RNA, cyclophilin A, and human antibodies have implications for viral proliferation and vaccine development.

molecular biology↗

bioRxiv2020

Allosteric inhibition of the SARS-CoV-2 main protease - insights from mass spectrometry-based assays

Following translation of the SARS-CoV-2 RNA genome into two viral polypeptides, the main protease Mpro cleaves at eleven sites to release non-structural proteins required for viral replication. MPro is an attractive target for antiviral therapies to combat the coronavirus-2019 disease (COVID-19). Here, we have used native mass spectrometry (MS) to characterize the functional unit of Mpro. Analysis of the monomer-dimer equilibria reveals a dissociation constant of Kd = 0.14 {+/-} 0.03 M, revealing MPro has a strong preference to dimerize in solution. Developing an MS-based kinetic assay we then characterized substrate turnover rates by following temporal changes in the enzyme-substrate complexes, which are effectively "flash-frozen" as they transition from solution to the gas phase. We screened small molecules, that bind distant from the active site, for their ability to modulate activity. These compounds, including one proposed to disrupt the catalytically active dimer, slow the rate of substrate processing by ~35%. This information was readily obtained and, together with analysis of the x-ray crystal structures of these enzyme-small molecule complexes, provides a starting point for the development of more potent molecules that allosterically regulate MPro activity.

biophysics↗