Liquid-Liquid Phase Separation to Study the Association of Proteins in Solution.

Liquid-liquid phase separation (LLPS) is a reversible biological process that contributes to the formation of critical concentration of proteins, forming membraneless compartments that are physiologically and pathologically relevant. Several proteins have been shown to demix into liquid droplets under in vitro crowding conditions. These studies are mainly conducted in isolation using purified recombinant proteins. Recently, we used LLPS to study the association between two proteins that are co-aggregated in Alzheimer's disease brain, tau, and EFhd2. Here, we describe how we used LLPS to determine the molecular components that contribute to the transition of these two proteins from liquid droplets to solid-like structures.

Large-Scale SARS-CoV-2 Testing Utilizing Saliva and Transposition Sample Pooling.

Identification and isolation of contagious individuals along with quarantine of close contacts, is critical for slowing the spread of COVID-19. Large-scale testing in a surveillance or screening capacity for asymptomatic carriers of COVID-19 provides both data on viral spread and the follow-up ability to rapidly test individuals during suspected outbreaks. The COVID-19 early detection program at Michigan State University has been utilizing large-scale testing in a surveillance or screening capacity since fall of 2020. The methods adapted here take advantage of the reliability, large sample volume, and self-collection benefits of saliva, paired with a cost-effective, reagent conserving two-dimensional pooling scheme. The process was designed to be adaptable to supply shortages, with many components of the kits and the assay easily substituted. The processes outlined for collecting and processing SARS-CoV-2 samples can be adapted to test for future viral pathogens reliably expressed in saliva. By providing this blueprint for universities or other organizations, preparedness plans for future viral outbreaks can be developed.

Beta2-adrenoreceptor agonist clenbuterol produces transient decreases in alpha-synuclein mRNA but no long-term reduction in protein.

ß2-adrenoreceptor (ß2AR) agonists have been associated with a decreased risk of developing Parkinson's disease (PD) and are hypothesized to decrease expression of both alpha-synuclein mRNA (Snca) and protein (α-syn). Effects of ß2AR agonist clenbuterol on the levels of Snca mRNA and α-syn protein were evaluated in vivo (rats and mice) and in rat primary cortical neurons by two independent laboratories. A modest decrease in Snca mRNA in the substantia nigra was observed after a single acute dose of clenbuterol in rats, however, this decrease was not maintained after multiple doses. In contrast, α-syn protein levels remained unchanged in both single and multiple dosing paradigms. Furthermore, clenbuterol did not decrease Snca in cultured rat primary cortical neurons, or decrease Snca or α-syn in mice. Additionally, compared to the single-dose paradigm, repeat dosing resulted in substantially lower levels of clenbuterol in plasma and brain tissue in rodents. Based on our observations of a transient decrease in Snca and no effect on α-syn protein in this preclinical study, these data support the conclusion that clenbuterol is not likely a viable disease-modifying strategy for PD.

EFhd2 brain interactome reveals its association with different cellular and molecular processes.

EFhd2 is a conserved calcium-binding protein that is highly expressed in the central nervous system. We have shown that EFhd2 interacts with tau protein, a key pathological hallmark in Alzheimer's disease and related dementias. However, EFhd2's physiological and pathological functions in the brain are still poorly understood. To gain insights into its physiological function, we identified proteins that co-immunoprecipitated with EFhd2 from mouse forebrain and hindbrain, using tandem mass spectrometry (MS). In addition, quantitative mass spectrometry was used to detect protein abundance changes due to the deletion of the Efhd2 gene in mouse forebrain and hindbrain regions. Our data show that mouse EFhd2 is associated with cytoskeleton components, vesicle trafficking modulators, cellular stress response-regulating proteins, and metabolic proteins. Moreover, proteins associated with the cytoskeleton, vesicular transport, calcium signaling, stress response, and metabolic pathways showed differential abundance in Efhd2(-/-) mice. This study presents, for the first time, an EFhd2 brain interactome that it is associated with different cellular and molecular processes. These findings will help prioritize further studies to investigate the mechanisms by which EFhd2 modulates these processes in physiological and pathological conditions of the nervous system.

Validation of recombinant human protein purified from bacteria: An important step to increase scientific rigor.

E. coli is a common host for generating human recombinant proteins in in vitro studies that seek to understand the biochemical and structural properties of proteins and in drug discovery. Validation of this biological resource is crucial to avoid misinterpretations and assay interference. Here, we demonstrate the use of tandem mass spectrometry to detect inadvertent post-translational modifications on human recombinant proteins produced in E. coli. Additionally, we identified co-purified E. coli proteins orthologous to known human interacting proteins. The results confirmed the importance of mass spectrometry in validating bacterial purified recombinant proteins as part of authenticating this key biological resource.

EFhd2 Affects Tau Liquid-Liquid Phase Separation.

The transition of tau proteins from its soluble physiological conformation to the pathological aggregate forms found in Alzheimer's disease and related dementias, is poorly understood. Therefore, understanding the process that modulates the formation of toxic tau oligomers and their conversion to putative neuroprotective neurofibrillary tangles will lead to better therapeutic strategies. We previously identified that EFhd2 is associated with aggregated tau species in AD brains and the coiled-coil domain in EFhd2 mediates the interaction with tau. To further characterize the association between EFhd2 and tau, we examined whether EFhd2 could affect the liquid-liquid phase separation properties of tau under molecular crowding conditions. We demonstrate that EFhd2 alters tau liquid phase behavior in a calcium and coiled-coil domain dependent manner. Co-incubation of EFhd2 and tau in the absence of calcium leads to the formation of solid-like structures containing both proteins, while in the presence of calcium these two proteins phase separate together into liquid droplets. EFhd2's coiled-coil domain is necessary to alter tau's liquid phase separation, indicating that protein-protein interaction is required. The results demonstrate that EFhd2 affects the liquid-liquid phase separation of tau proteins in vitro, suggesting that EFhd2 modulates the structural dynamics of tau proteins.

Tau13 Antibody Preferentially Immunoprecipitates High Molecular Weight Tau Proteins.

The accumulation of tau protein aggregates is a pathological hallmark in Alzheimer's disease (AD) and other neurodegenerative diseases. However, the identity of the toxic tau conformation that propagates and induces neurodegeneration is still unknown. Anti-tau antibodies are a common tool used to differentiate between normal and pathological-associated tau forms or as passive immunotherapy in the quest to interfere with tau-mediated neurodegeneration. Here, we show that Tau13, a tau N-terminal antibody, preferentially enriches high molecular weight tau species produced in a tauopathy mouse model and AD. The data suggest that Tau13 has higher affinity to specific tau conformation presence in higher molecular weight tau species.

A functional proteomics platform to reveal the sequence determinants of lysine methyltransferase substrate selectivity.

Lysine methylation is a key regulator of histone protein function. Beyond histones, few connections have been made to the enzymes responsible for the deposition of these posttranslational modifications. Here, we debut a high-throughput functional proteomics platform that maps the sequence determinants of lysine methyltransferase (KMT) substrate selectivity without a priori knowledge of a substrate or target proteome. We demonstrate the predictive power of this approach for identifying KMT substrates, generating scaffolds for inhibitor design, and predicting the impact of missense mutations on lysine methylation signaling. By comparing KMT selectivity profiles to available lysine methylome datasets, we reveal a disconnect between preferred KMT substrates and the ability to detect these motifs using standard mass spectrometry pipelines. Collectively, our studies validate the use of this platform for guiding the study of lysine methylation signaling and suggest that substantial gaps exist in proteome-wide curation of lysine methylomes.

Tau's Three-Repeat Domain and EFhd2 Co-incubation Leads to Increased Thioflavin Signal.

Aggregation of the protein tau is a pathological hallmark of Alzheimer's disease (AD) and related disorders. However, the molecular mechanisms that lead to tau protein aggregation are still unclear. Previously, we showed that EFhd2 protein is associated with pathological aggregated forms of tau in AD brain. Further, immuno-gold analyses of purified tau aggregates showed that EFhd2 co-localized with filamentous tau structures. We demonstrated that EFhd2's coiled-coil domain is required for its association with tau proteins. However, it is unknown the role that EFhd2 plays in tau aggregation. Here, we show that incubation of K19-tau with substoichiometric amount of EFhd2 promote the formation of amyloid structures in vitro. The result suggests that EFhd2 may play a role in the biogenesis of aggregated tau.

Ezrin Expression is Increased During Disease Progression in a Tauopathy Mouse Model and Alzheimer's Disease.

BACKGROUND: The lack of diagnostic tools and disease-modifying treatments against Alzheimer's disease (AD) and related disorders, collectively known as tauopathies, has led to a socioeconomic burden of epidemic proportion. Proteomics approaches can be used to identify novel proteome changes that could help us understand the pathogenesis of tau-related pathological hallmarks and/or cellular stress responses associated with tauopathy. These studies, however, need to be conducted taking into consideration brain region specificity and stage of neurodegeneration in order to provide insights about the pathological role of the identified proteins. METHODS: We used a tauopathy mouse model (JNPL3) that expresses human tau bearing a P301L mutation and develops motor impairment, the severity of which correlates with the increased accumulation of pathological tau. Tissue was dissected from asymptomatic and severely motor impaired JNPL3 mice as well as non-transgenic littermate controls and subjected to two-dimensional gel electrophoresis. Differentially abundant protein spots were identified by tandem mass spectrometry. Postmortem mild cognitive impairment (MCI), AD and normal aging controls were used to validate the pathological significance of the identified protein. RESULTS: Ezrin was identified as a protein that is upregulated in tau-mediated neurodegeneration. We demonstrate that Ezrin protein abundance increased in JNPL3 mice preceded motor impairment and was sustained in severely motor impaired mice. Ezrin expression was also increased in the temporal cortex of MCI and AD patients. CONCLUSION: The results demonstrate that increased Ezrin protein abundance changes are associated with the early stages of neurodegeneration in tauopathy models and human disease. Understanding the role of Ezrin in tauopathies such as AD may provide new insights for targeting tau-mediated neurodegeneration.