The interaction between LC8 and LCA5 reveals a novel oligomerization function of LC8 in the ciliary-centrosome system

Dynein light chain LC8 is a small dimeric hub protein that recognizes its partners through short linear motifs and is commonly assumed to drive their dimerization. It has more than 100 known binding partners involved in a wide range of cellular processes. Recent large-scale interaction studies suggested that LC8 could also play a role in the ciliary/centrosome system. However, the cellular function of LC8 in this system remains elusive. In this work, we characterized the interaction of LC8 with the centrosomal protein lebercilin (LCA5), which is associated with a specific form of ciliopathy. We showed that LCA5 binds LC8 through two linear motifs. In contrast to the commonly accepted model, LCA5 forms dimers through extensive coiled coil formation in a LC8-independent manner. However, LC8 enhances the oligomerization ability of LCA5 that requires a finely balanced interplay of coiled coil segments and both binding motifs. Based on our results, we propose that LC8 acts as an oligomerization engine that is responsible for the higher order oligomer formation of LCA5. As LCA5 shares several common features with other centrosomal proteins, the presented LC8 driven oligomerization could be widespread among centrosomal proteins, highlighting an important novel cellular function of LC8

Viral Targeting of Host Hubs: Interactions between the Rabies Virus and LC8

As obligate intracellular parasites, viruses rely on host machinery for their own reproduction. Viruses are therefore required to interact with a wide variety of host proteins, despite limitations in viral genome size. The most parsimonious method of success is to hijack central and essential proteins, known as hub proteins. LC8 is a notable example of a hub protein, and has been shown to interact with more than 100 eukaryotic partners in various unrelated pathways. An increasing number of studies have noted interactions between LC8 and viruses, including the Ebola, rabies, and rotaviruses; however, LC8’s role within these systems is unclear. This thesis examines the structural and functional properties of hub proteins, with a particular focus on LC8 and its interactions with the rabies virus. Three chapters of original work include two primary research reports and one review/opinion piece. The first research report, Chapter 2, structurally characterizes the interaction between LC8 and the rabies virus phosphoprotein (RavP). We use insights gleaned from our structural studies to predict and test potential roles for LC8 in the rabies virus infection cycle, and demonstrate that LC8 is important for efficient viral polymerase activity. Chapter 3 is an in-depth study of the LC8 recognition motif, where we examine both the structural and functional plasticity of LC8, and identify and validate many new LC8 interactions. We also develop a tool that can be used to predict LC8 binding motifs in proteins of interest, which will greatly improve the ability of those outside of the LC8 field to recognize, test, and validate partner proteins. In Chapter 4 we present a variety of new ideas about what qualities describe a linear motif-binding hub protein. This work provides ideological and linguistic suggestions for important structural and functional features of hubs that underlie their plasticity. It then further describes how viruses take advantage of these features to more efficiently hijack host pathways. Finally, Chapter 5 discusses the impacts of my thesis work, and outlines some potential future projects. Each individual chapter builds on the previous one to create an expanding view of the importance of linear motif-binding hubs for infectious viruses

Dynein Light Chain 1 Functions as a Cofactor for Post-Transcriptional mRNA Regulation and RNA Granule Assembly

Gene regulation is essential for ensuring maintenance, proliferation, and proper development of a cell. RNA binding proteins (RBPs) regulate gene expression by targeting and binding mRNAs to control their translation and often localize to cytoplasmic assemblies of protein and RNA called RNA granules to facilitate post-transcriptional mRNA regulation. Using C. elegans as a model organism, we report on the function of dynein light chain 1 (DLC-1), a subunit of the dynein motor complex, in post-transcriptional mRNA regulation in the gonad. Previous work suggests that DLC-1 is an RBP cofactor that functions independent of the dynein motor. It is unknown how widespread this regulatory role for DLC-1 may be or what direct interactions between DLC-1 and RBPs make mRNA regulation possible. The work presented in this dissertation suggests that DLC-1 is an important contributor to post-transcriptional mRNA regulation as well as RNA granule assembly. First, we used RNA immunoprecipitation coupled with high throughput sequencing (RIP-seq) to identify the mRNAs associated with DLC-1 through its interaction with RBPs. We found that DLC-1 is involved in post-transcriptional regulation of the oogenic transcriptome and demonstrated that DLC-1-associated transcripts depend on DLC-1 for regulation of their expression in the germline. From this work we identified the RBP OMA-1 as a new interactor of DLC-1 by an in vitro pulldown. Furthermore, we developed a protocol for application of in situ Proximity Ligation Assay (PLA) for use in C. elegans to probe for protein-protein interactions across developmental stages. This allowed us to detect formation of DLC-1/OMA-1 complexes in the gonad. Finally, we used a bioinformatic scan to find additional C. elegans RBPs that might interact with DLC-1. Using in vitro pulldowns, we verified predicted direct interactions between DLC-1 and 4 core RBP components of P granules, which are a subtype of RNA granule. Knockdown or knockout of dlc-1 disrupts embryonic P granule assembly, suggesting that DLC-1 has an important role in this process. As a whole, this work expands upon the alternate and emerging functions of dynein light chains and suggests that cofactors like DLC-1 play critical roles in promoting mRNA regulation

Interactions between two regulatory proteins of microtubule dynamics, HDAC6, TPPP/p25, and the hub protein, DYNLL/LC8

Degradation of unwanted proteins is important in protein quality control cooperating with the dynein/dynactin-mediated trafficking along the acetylated microtubule (MT) network. Proteins associated directly/indirectly with tubulin/MTs play crucial roles in both physiological and pathological processes. Our studies focus on the interrelationship of the tubulin deacetylase HDAC6, the MT-associated TPPP/p25 with its deacetylase inhibitory potency and the hub dynein light chain DYNLL/LC8, constituent of dynein and numerous other protein complexes. In this paper, evidence is provided for the direct interaction of DYNLL/LC8 with TPPP/p25 and HDAC6 and their assembly into binary/ternary complexes with functional potency. The in vitro binding data was obtained with recombinant proteins and used for mathematical modelling. These data and visualization of their localizations by bimolecular fluorescence complementation technology and immunofluorescence microscopy in HeLa cells revealed the promoting effect of TPPP/p25 on the interaction of DYNLL/LC8 with both tubulin and HDAC6.Localization of the LC8-2-TPPP/p25 complex was observed on the MT network in contrast to the LC8-2-HDAC6 complex, which was partly translocated to the nucleus. LC8-2 did not influence directly the acetylation of the MT network. However, the binding of TPPP/p25 to a new binding site of DYNLL/LC8, outside the canonical binding groove, counteracted the TPPP/p25-derived hyperacetylation of the MT network. Our data suggest that multiple associations of the regulatory proteins of the MT network could ensure fine tuning in the regulation of the intracellular trafficking process either by the complexation of DYNLL/LC8 with new partners or indirectly by the modulation of the acetylation level of the MT network

Specificity, Allostery, and Multivalency in Binding to the Hub Protein LC8

Interactions between proteins are essential to life, driving and regulating a majority of processes within all living cells. Study of protein-protein interactions reveals that some proteins act as hubs within networks of interactions, binding to many partner proteins. These hubs therefore are of particular importance to understanding protein function, interwoven as they are with dozens of biological functions. LC8 is one such hub protein, binding to over 100 known clients and playing a role in many unrelated pathways. LC8 binding, mediated by a short linear motif in client proteins, induces a dimeric structure on clients, leading the protein to be referred to as a dimerization engine. This thesis discusses the function of LC8, examining both the general properties of LC8 that facilitate LC8-client binding, and documenting and characterizing new LC8-binding proteins. Each of the three chapters of original work is a report of primary research. Chapter 2 is a detailed investigation of the thermodynamics of LC8 binding, which necessitated the development of a new method of analysis built on principles of Bayesian statistics. This method allowed us to measure detailed thermodynamics of LC8 binding, and demonstrate that LC8 favors a fully bound state, consistent with its function as an engine for dimerization. Chapter 3 is concerned with characterizing the LC8-binding linear motif, and development of a tool for prediction of LC8 binding. We collate a database of LC8-binding proteins and find that residues flanking the core motif sequence play an important role in regulating binding. The predictive tool uses a library of known LC8-binding and non-binding sequences to generate a scoring matrix for potential clients and has already been adopted by researchers studying LC8 interactions. In chapter 4 we present a characterization of a new LC8-binding protein named Kank1. Kank1, a cytoskeletal regulator found at the cell cortex, binds LC8 multivalently, forming a large complex consisting of at least five LC8 dimer units. The complex forms with significant cooperativity, and unlike many multivalent LC8-interacting proteins, forms a homogenous stable oligomer, indicating that the complex may play a structural role, rigidifying the scaffold of Kank1. Lastly, chapter 5 discusses the impact of this work, and highlights of the work presented in each chapter. It additionally presents ongoing and future steps in the study of LC8 interactions. This thesis additionally contains two appendices reporting primary research that is unrelated to LC8. The first is concerned with a protein from the Peroxiredoxin family of redox proteins. Peroxiredoxins unfold during catalysis, and we demonstrated that our model peroxiredoxin unfolds transiently in absence of catalysis, emphasizing that the protein is finely structurally tuned for catalysis. The second appendix discusses the nucleocapsid of the SAR-CoV-2 virus, examining the protein’s interaction with RNA, which is essential to viral replication. We find that the protein can interact both specifically and nonspecifically with RNA, and that nonspecific binding is correlated to liquid-liquid phase separation, which is believed to be essential to some viral functions

IDENTIFYING MOLECULAR FUNCTIONS OF DYNEIN MOTOR PROTEINS USING EXTREME GRADIENT BOOSTING ALGORITHM WITH MACHINE LEARNING

The majority of cytoplasmic proteins and vesicles move actively primarily to dynein motor proteins, which are the cause of muscle contraction. Moreover, identifying how dynein are used in cells will rely on structural knowledge. Cytoskeletal motor proteins have different molecular roles and structures, and they belong to three superfamilies of dynamin, actin and myosin. Loss of function of specific molecular motor proteins can be attributed to a number of human diseases, such as Charcot-Charcot-Dystrophy and kidney disease.  It is crucial to create a precise model to identify dynein motor proteins in order to aid scientists in understanding their molecular role and designing therapeutic targets based on their influence on human disease. Therefore, we develop an accurate and efficient computational methodology is highly desired, especially when using cutting-edge machine learning methods. In this article, we proposed a machine learning-based superfamily of cytoskeletal motor protein locations prediction method called extreme gradient boosting (XGBoost). We get the initial feature set All by extraction the protein features from the sequence and evolutionary data of the amino acid residues named BLOUSM62. Through our successful eXtreme gradient boosting (XGBoost), accuracy score 0.8676%, Precision score 0.8768%, Sensitivity score 0.760%, Specificity score 0.9752% and MCC score 0.7536%.  Our method has demonstrated substantial improvements in the performance of many of the evaluation parameters compared to other state-of-the-art methods. This study offers an effective model for the classification of dynein proteins and lays a foundation for further research to improve the efficiency of protein functional classification

Context matters : assembly and disorder in the regulation of multivalent complexes

Intrinsically disordered proteins (IDPs), protein regions (IDRs), and protein complexes continue to emerge at the forefront of protein science. Proteins and protein regions lacking specific structure are found in all organisms, and often have vital roles in numerous biological processes. Breaking the well-known structure-function paradigm, the understanding of disorder-based functionality is constantly expanding. In addition to their structural plasticity and dynamic conformational flexibility, IDPs/IDRs often interact with their binding partners multivalently to serve regulatory purposes. This thesis reports on two example systems analyzing the impact of disorder and complex assembly on regulation while also showcasing why the context in which we study these systems really matters. Four chapters of original work include one protocol book chapter and three primary research reports. The book chapter, chapter 2, serves as a guide in using NMR to probe interactions of IDPs, using dynein intermediate chain (IC) as a model example. Chapters 3 and 4 report on a domain of the transcription factor, ASCIZ, and interactions with its own product, dynein light chain (LC8). Chapter 3 classifies the domain of study as a predominantly in-register binder of LC8 while chapter 4 further explores how binding motif specificity and the linker length between binding sites impact complex assembly. This work serves as a foundation and model system for more intricate cases, such as the focus of chapter 5, dynein IC subcomplexes. In chapter 5, a regulatory mechanism involving autoinhibition via long range intramolecular interactions that is relieved by multivalent complex assembly is presented. IC is studied and biophysically characterized, for the first time, in context of its entire N-terminal domain, in the reconstitution of subcomplexes, and in the full-length protein. Finally, chapter 6 summarizes the impact and highlights of the reported work and presents suggestions for work moving forward. These studies provide detailed description of two various multivalent and disordered protein assemblies that together serve as a blueprint for studying these systems in context for both the IDP and dynein fields

Removal of antagonistic spindle forces can rescue metaphase spindle length and reduce chromosome segregation defects

Regular Abstracts - Tuesday Poster Presentations: no. 1925Metaphase describes a phase of mitosis where chromosomes are attached and oriented on the bipolar spindle for subsequent segregation at anaphase. In diverse cell types, the metaphase spindle is maintained at a relatively constant length. Metaphase spindle length is proposed to be regulated by a balance of pushing and pulling forces generated by distinct sets of spindle microtubules and their interactions with motors and microtubule-associated proteins (MAPs). Spindle length appears important for chromosome segregation fidelity, as cells with shorter or longer than normal metaphase spindles, generated through deletion or inhibition of individual mitotic motors or MAPs, showed chromosome segregation defects. To test the force balance model of spindle length control and its effect on chromosome segregation, we applied fast microfluidic temperature-control with live-cell imaging to monitor the effect of switching off different combinations of antagonistic forces in the fission yeast metaphase spindle. We show that spindle midzone proteins kinesin-5 cut7p and microtubule bundler ase1p contribute to outward pushing forces, and spindle kinetochore proteins kinesin-8 klp5/6p and dam1p contribute to inward pulling forces. Removing these proteins individually led to aberrant metaphase spindle length and chromosome segregation defects. Removing these proteins in antagonistic combination rescued the defective spindle length and, in some combinations, also partially rescued chromosome segregation defects. Our results stress the importance of proper chromosome-to-microtubule attachment over spindle length regulation for proper chromosome segregation.postprin

Psr1p interacts with SUN/sad1p and EB1/mal3p to establish the bipolar spindle

Regular Abstracts - Sunday Poster Presentations: no. 382During mitosis, interpolar microtubules from two spindle pole bodies (SPBs) interdigitate to create an antiparallel microtubule array for accommodating numerous regulatory proteins. Among these proteins, the kinesin-5 cut7p/Eg5 is the key player responsible for sliding apart antiparallel microtubules and thus helps in establishing the bipolar spindle. At the onset of mitosis, two SPBs are adjacent to one another with most microtubules running nearly parallel toward the nuclear envelope, creating an unfavorable microtubule configuration for the kinesin-5 kinesins. Therefore, how the cell organizes the antiparallel microtubule array in the first place at mitotic onset remains enigmatic. Here, we show that a novel protein psrp1p localizes to the SPB and plays a key role in organizing the antiparallel microtubule array. The absence of psr1+ leads to a transient monopolar spindle and massive chromosome loss. Further functional characterization demonstrates that psr1p is recruited to the SPB through interaction with the conserved SUN protein sad1p and that psr1p physically interacts with the conserved microtubule plus tip protein mal3p/EB1. These results suggest a model that psr1p serves as a linking protein between sad1p/SUN and mal3p/EB1 to allow microtubule plus ends to be coupled to the SPBs for organization of an antiparallel microtubule array. Thus, we conclude that psr1p is involved in organizing the antiparallel microtubule array in the first place at mitosis onset by interaction with SUN/sad1p and EB1/mal3p, thereby establishing the bipolar spindle.postprin

Dichotomic role of NAADP/two-pore channel 2/Ca2+ signaling in regulating neural differentiation of mouse embryonic stem cells

Poster Presentation - Stem Cells and Pluripotency: abstract no. 1866The mobilization of intracellular Ca2+stores is involved in diverse cellular functions, including cell proliferation and differentiation. At least three endogenous Ca2+mobilizing messengers have been identified, including inositol trisphosphate (IP3), cyclic adenosine diphosphoribose (cADPR), and nicotinic adenine acid dinucleotide phosphate (NAADP). Similar to IP3, NAADP can mobilize calcium release in a wide variety of cell types and species, from plants to animals. Moreover, it has been previously shown that NAADP but not IP3-mediated Ca2+increases can potently induce neuronal differentiation in PC12 cells. Recently, two pore channels (TPCs) have been identified as a novel family of NAADP-gated calcium release channels in endolysosome. Therefore, it is of great interest to examine the role of TPC2 in the neural differentiation of mouse ES cells. We found that the expression of TPC2 is markedly decreased during the initial ES cell entry into neural progenitors, and the levels of TPC2 gradually rebound during the late stages of neurogenesis. Correspondingly, perturbing the NAADP signaling by TPC2 knockdown accelerates mouse ES cell differentiation into neural progenitors but inhibits these neural progenitors from committing to the final neural lineage. Interestingly, TPC2 knockdown has no effect on the differentiation of astrocytes and oligodendrocytes of mouse ES cells. Overexpression of TPC2, on the other hand, inhibits mouse ES cell from entering the neural lineage. Taken together, our data indicate that the NAADP/TPC2-mediated Ca2+signaling pathway plays a temporal and dichotomic role in modulating the neural lineage entry of ES cells; in that NAADP signaling antagonizes ES cell entry to early neural progenitors, but promotes late neural differentiation.postprin