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Dematin (band 4.9) is an F-actin binding and bundling protein best known for its role within red blood cells, where it both stabilizes as well as attaches the spectrin/actin cytoskeleton to the erythrocytic membrane. Here, we investigate the structural consequences of phosphorylating serine 381, a covalent modification that turns off F-actin bundling activity. In contrast to the canonical doctrine, in which phosphorylation of an intrinsically disordered region/protein confers affinity for another domain/ protein, we found the converse to be true of dematin: phosphorylation of the well folded C-terminal villin-type headpiece confers affinity for its intrinsically disordered N-terminal core domain. We employed analytical ultracentrifugation to demonstrate that dematin is monomeric, in contrast to the prevailing view that it is trimeric. Next, using a series of truncation mutants, we verified that dematin has two F-actin binding sites, one in the core domain and the other in the headpiece domain. Although the phosphorylation-mimicking mutant, S381E, was incapable of bundling microfilaments, it retains the ability to bind F-actin. We found that a phosphorylation-mimicking mutant, S381E, eliminated the ability to bundle, but not bind F-actin filaments. Lastly, we show that the S381E point mutant caused the headpiece domain to associate with the core domain, leading us to the mechanism for cAMP-dependent kinase control of dematin's F-actin bundling activity: when unphosphorylated, dematin's two F-actin binding domains move independent of one another permitting them to bind different F-actin filaments. Phosphorylation causes these two domains to associate, forming a compact structure, and sterically eliminating one of these F-actin binding sites. [ABSTRACT FROM AUTHOR]

Viruses form extensive interfaces with host proteins to modulate the biology of the infected cell, frequently via multifunctional viral proteins. These proteins are conventionally considered as assemblies of independent functional modules, where the presence or absence of modules determines the overall composite phenotype. However, this model cannot account for functions observed in specific viral proteins. For example, rabies virus (RABV) P3 protein is a truncated form of the pathogenicity factor P protein, but displays a unique phenotype with functions not seen in longer isoforms, indicating that changes beyond the simple complement of functional modules define the functions of P3. Here, we report structural and cellular analyses of P3 derived from the pathogenic RABV strain Nishigahara (Nish) and an attenuated derivative strain (Ni-CE). We identify a network of intraprotomer interactions involving the globular C-terminal domain and intrinsically disordered regions (IDRs) of the N-terminal region that characterize the fully functional Nish P3 to fluctuate between open and closed states, whereas the defective Ni-CE P3 is predominantly open. This conformational difference appears to be due to the single mutation N226H in Ni-CE P3. We find that Nish P3, but not Ni-CE or N226H P3, undergoes liquid-liquid phase separation and this property correlates with the capacity of P3 to interact with different cellular membrane-less organelles, including those associated with immune evasion and pathogenesis. Our analyses propose that discrete functions of a critical multifunctional viral protein depend on the conformational arrangements of distant individual domains and IDRs, in addition to their independent functions.

Proteome solubility contains latent information on the nature of protein interaction networks in cells and changes in solubility can provide information on rewiring of networks. Here, we report a simple one-step ultracentrifugation method to separate the soluble and insoluble fraction of the proteome. The method involves quantitative proteomics and a bioinformatics strategy to analyze the changes that arise. Because protein solubility changes are also associated with protein misfolding and aggregation in neurodegenerative disease, we also include a protocol for isolating disease-associated protein aggregates with pulse shape analysis (PulSA) by flow cytometry as a complementary approach that can be used alongside the more general measure of solubility or as a stand-alone approach., (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Pulse shape analysis (PulSA) is a flow cytometry-based method that involves the measurement of the pulse width and height of a fluorescently labeled molecule simultaneously, enabling a multidimensional analysis of protein localization in a cell at high speed and throughput. We have used the method to detect morphological changes in organelles such as Golgi fragmentation, track protein trafficking from the cell surface, and also discriminate cells with different target protein localizations such as the Golgi, lyso-endosomal network, and the plasma membrane. Here, we describe the basic experimental setup and analytical methods for performing PulSA to examine membrane trafficking processes. We illustrate in particular the application of PulSA for monitoring the trafficking of the membrane-bound enzyme furin and morphological changes to the Golgi caused by Brefeldin A.

Defining the aggregation process of proteins formed by poly-amino acid repeats in cells remains a challenging task due to a lack of robust techniques for their isolation and quantitation. Sedimentation velocity methodology using fluorescence detected analytical ultracentrifugation is one approach that can offer significant insight into aggregation formation and kinetics. While this technique has traditionally been used with purified proteins, it is now possible for substantial information to be collected with studies using cell lysates expressing a GFP-tagged protein of interest. In this chapter, we describe protocols for sample preparation and setting up the fluorescence detection system in an analytical ultracentrifuge to perform sedimentation velocity experiments on cell lysates containing aggregates formed by poly-amino acid repeat proteins.

Pulse shape analysis (PulSA) is a flow cytometry-based method that can be used to study protein localization patterns in cells. Examples for its use include tracking the formation of inclusion bodies of polyglutamine-expanded proteins and other aggregating proteins. The method can also be used for phenomena relating to protein movements in cells such as translocation from the cytoplasm to the nucleus, trafficking from the plasma membrane to the Golgi, and stress granule formation. An attractive feature is its capacity to quantify these parameters in whole-cell populations very quickly and in high throughput. We describe the basic experimental details for performing PulSA using expression of GFP-tagged proteins, endogenous proteins labelled immunofluorescently, and organelle dyes.

Huntington's disease arises from CAG codon-repeat expansions in the Htt gene, which leads to a Htt gene product with an expanded polyglutamine (polyQ) sequence. The length of the polyQ expansion correlates with an increased tendency to form aggregates and clustering into micrometer-plus sized inclusion bodies in neurons and other cell types. Yet after nearly 20 years since the genetic basis for HD was identified, our knowledge of how polyQ-expanded Htt fragment aggregation relates to disease mechanisms remains fragmentary and controversial. Challenges remain in defining the aggregation process at the molecular level and how this process is influenced by, or influences cellular activities. Insight is further confounded by the term "aggregation" being used to describe a composite of distinct processes that may have opposing consequences to cell health and survival. This review discusses these issues in light of a historic summary of Htt aggregation in the cellular milieu and the intrinsic attributes of polyQ-expanded Htt that lead to aggregation. Finally, discussion centers on strategies forward to improve our knowledge for how aggregation relates to cellular dysfunction.

Twenty-five proteins are known to form amyloid fibrils in vivo in association with disease (Westermark et al., Amyloid 12:1-4, 2005). However, the fundamental ability of a protein to form amyloid-like fibrils is far more widespread than in just the proteins associated with disease, and indeed this property can provide insight into the basic thermodynamics of folding and misfolding pathways. But how does one determine whether a protein has formed amyloid-like fibrils? In this chapter, we cover the basic steps toward defining the amyloid-like properties of a protein and how to measure the kinetics of fibrillization. We describe several basic tests for aggregation and the binding to two classic amyloid-reactive dyes, Congo Red, and thioflavin T, which are key indicators to the presence of fibrils.