<p>Protein biosynthesis is a core process in all living organisms. Assembly of the protein chain from aminoacids is catalysed by the ribosome, ancient and extremely complex macromolecular machine. Several different classes of accessory molecules are involved in translation, and one set of them, called translational GTPases (trGTPases), was in the focus of this work. </p><p>In this thesis properties of two trGTPases– EF-G and eRF3 - were studied by means of direct biochemical experiments. EF-G is a bacterial trGTPase involved in two steps of translation: translocation and ribosomal recycling. Translocation is a process of the ribosomal movement along the mRNA, and recycling as the step when upon completion of the protein ribosome is released from the mRNA via splitting in two ribosomal subunits. We found that off the ribosome EF-G has similar affinities to GDP and GTP, and thus given the predominance of the latter in the cell, EF-G should be present mostly in the complex with GTP. However, binding to the ribosome increases factors affinity to GTP drastically, ensuring that it is in the GTP-bound state. GDP can not promote neither translocation, not recycling, and GDPNP can not promote recycling. It can, however, promote translocation, but in so doing it results in an intermediate ribosomal state and translocation process can be reversed by addition of GDP, which is not the case for the EF-G•GTP-catalyzed reaction.</p><p>The second trGTPase we investigated is eukaryotic termination factor eRF3. This protein together with another factor, eRF1, is involved translation termination, which is release of the synthesized protein from the ribosome. We demonstrateed, that eRF3 alone has basically no propensity to bind GTP and thus resides in the GDP-bound state. Complex formation between eRF1 and eRF3 promotes GTP binding by the latter, resulting in the formation of the ternary complex eRF1•eRF3•GTP, which in turn is catalyzing the termination event.</p><p>Experimental investigations of trGTPases where rationalized within a generalized thermodynamical framework, accommoding the existent experimental observations, both structural and biochemical. </p>

Rad/Rem/Rem2/Gem/Kir (RGK) proteins are Ras-like GTPases with diverse (and expanding) functions including: regulating cytoskeleton dynamics, cell proliferation, synaptogenesis, and inhibition of high voltage-dependent calcium (CaV) channels. Furthermore, they have tissue-specific distribution with Rem and Rad most highly expressed in the heart. Indeed, the importance of Rem and Rad in the cardiovascular system is underscored by a number of studies linking them to disease states including cardiac hypertrophy, cardiac fibrosis, and inflammation. A hallmark feature of RGK proteins is their ability to inhibit current through CaV channels (ICa) and in fact, they are recognized as the most potent endogenous inhibitors of ICa. However, how RGK proteins are regulated and what their physiological role is are unknown. Understanding these points is critical for defining the patho-physiological roles of RGK proteins. My thesis work contributes towards the RGK field on two fronts: First, we demonstrate that RGK proteins are non-canonical G-proteins in the context of their ability to undergo nucleotide regulation and second, we reveal a novel paradigm of RGK-mediated inhibition on CaV channels. In Chapters 2 and 3, we show that Rem and Rad are are non-canonical G-proteins with respect to the regulatory role of their guanine nucleotide binding pocket (GNBP). Canonical Raslike G-proteins contain a conserved G-domain that encompass a GNBP and is important for guanine nucleotide binding and hydrolysis. Since RGK proteins also possess a G-domain and GNBP as well as demonstrate bona fide nucleotide binding, it was initially thought that they were regulated in a manner similar to other Ras proteins. However, subsequent studies suggested that RGK proteins may not obey such a classical model and as a result, the regulatory role of their GNBP in the G-domain was unclear. By using a wide range of functional measurements (CaV1.2 currents, Ca2+ transients, β-subunit binding), we demonstrate that RGK proteins Rem and Rad are non-canonical G-proteins. Utilizing point mutants that abolish GTPbinding and prevent GTPase activity (RemT94N and RadS105N), we show that only some cellular functions are dependent on an operational nucleotide binding pocket while others are unperturbed. Specifically, Rem- and Rad-mediated inhibition of ICa is independent of guanine nucleotide regulation whereas protein interactions with the b-subunit of CaV channels (CaVβ) and protein stability are sensitive to nucleotide regulation. We also discover skeletal and cardiac actin to be novel binding partners of Rem. And lastly, we observe differences between the effects of Rem and Rad on their degree of ICa inhibition in cardiac myocytes. Thus, Rem and Rad are non-canonical G-proteins with respect to the regulatory role of their GNBP. In collaboration with a close colleague, Akil Puckerin, Chapter 4 reveals a novel mechanism behind RGK-mediated inhibition of ICa. Together, we show RGK proteins display different modes of inhibition against specific CaV channels and that we can utilize this property to design calcium channel blockers which inhibit CaV channels in an isoform specific manner. We demonstrate this by designing Rem and Rad mutants which have diminished CaVβ capacity, termed Rem-βNULL and Rad-βNULL, respectively. Characterization of these mutants using wholecell patch clamp experiments revealed that Rem-βNULL inhibits only CaV1.2 whereas Rad-βNULL inhibits only CaV1.2 and CaV2.2. Thus, our results describe the first genetically encoded calcium channel blocker that can selectively distinguish amongst L-type channels. Altogether, this thesis work contributes towards our understanding of RGK protein regulation function and the underlying mechanisms by which they inhibit ICa. These findings advance the field both from a mechanistic and physiological standpoint, and will be of great importance towards investigating the patho-physiological role of RGK proteins.

The Chromosomal Passenger Complex (CPC) is a major regulator of mitosis composed of the catalytic subunit Aurora B, the inner centromere protein INCENP, Survivin and Borealin/Dasra B. The CPC controls aspects of mitosis, ranging from chromosome and spindle structure to the correction of kinetochore-microtubule attachment errors, regulation of mitotic progression and completion of cytokinesis (Carmena et al., 2012). Knocking down any one CPC component induces delocalization of the others and disrupts mitotic progression (Adams et al., 2001 ; Carvalho et al., 2003; Lens et al., 2003 ; Gassmann et al., 2004; Vader et al., 2006). Telophase Disc (TD-60), also known as RCC2, is a putative Guanine Exchange Factor (GEF) that has been suggested to be involved in completion of cytokinesis through GTPase activation (Mollinari et al., 2003). However, its mechanism of action is still unclear. Interestingly, TD-60 has a typical Chromosomal Passenger Complex (CPC) localization (Andreassen et al., 1991) and its down-regulation alters CPC localisation during early mitosis. However, it is not a member of the CPC immunoprecipitated from mitotic cells (Gassmann et al., 2004). Here, I improved human TD-60 recombinant protein production by expressing a synthetic cDNA in the baculovirus expression system. This allowed me to characterize TD-60-associated GEF activity in vitro and study its possible influence on core CPC activity in vivo. I tested purified human TD-60 against a broad selection of GTPase targets, representing each GTPase family, in an established GEF assay. My data demonstrated that TD-60 has consistent high GEF activity in vitro towards the Ras-like protein A, RalA. To understand if TD-60 links RalA GTPase function to the CPC in vivo, I performed TD-60 and RalA RNAi experiments in HeLa cells. Interestingly, both TD-60 and RalA-depleted cells exhibit destabilized kinetochore fibers, a similar defective prometaphase-like bipolar spindle structure, and an abnormal centromeric accumulation of the CPC in early mitosis. In order to confirm that phenotypes seen after TD-60 depletion were due to lack of RalA activation in vivo, I generated a constitutively active RalA mutant that I transfected into TD-60- deficient cells. Strikingly, the RalA Q72L active mutant (mimicking the GTP-bound form) rescued the abnormal bipolar spindle structure, corrected the defective kinetochore-microtubules attachments, and rescued the atypical CPC distributions observed at centromeres after TD-60 depletion. These results suggest that TD-60-associated RalA GEF activity stabilizes kinetochore-microtubule attachments in early mitosis and that, TD-60 links RalA GTPase function to the CPC during mitosis.

The Integrated Stress Response (ISR) is a conserved eukaryotic translational and transcriptional program implicated in mammalian metabolism, memory and immunity. Although mainly considered to be a protective mechanism, prolonged and severe ISR can result in cell death. The ISR is activated by diverse stress pathways converged on phosphorylation of the alpha subunit of eukaryotic translation initiation factor 2 (eIF2) that inhibits the guanine nucleotide exchange activity of its partner eIF2B and attenuates overall rates of protein synthesis. Numerous mutations in eIF2B are linked to a fatal neurodegenerative disease of vanishing white matter. A new chemical inhibitor of the ISR (ISRIB), a bis-O-arylglycolamide, can reverse the attenuation of mRNA translation by phosphorylated eIF2 protecting mice from prion-induced neurodegeneration and traumatic brain injury. The work presented in this dissertation describes identification of mammalian eIF2B as a cellular target of ISRIB by implementing biochemical, biophysical, structural and chemogenetic methods. The herein reported cryo-electron microscopy-based structure of eIF2B uncovers a novel allosteric site on the translation factor capturing the ISRIB-binding pocket at the interface between its β and δ regulatory subunits. The extensive CRISPR/ Cas9-based screen for ISRIB-resistant and analogue-sensitive phenotypes revealed residues on the eIF2B dimer interface important for ISRIB binding. Based on the results reported in this dissertation along with the similar findings of others the potential molecular basis of ISRIB action, and its implication for the regulation of eIF2B's activity is broadly discussed. The identification of the ISRIB binding pocket away from the known interaction sites between eIF2B and eIF2 is also put into the context of a possible molecular mechanism of eIF2B's guanine exchange inhibition by phosphorylated eIF2. The work described in this dissertation provides new insight into the translational regulation and points to the importance of fine-tuning the activity of translation factors by small chemical molecules.

Dynamic regulation of the actin cytoskeleton is required for synapses to form and maintain their shape. The actin cytoskeleton is regulated by Rho GTPases in response to genetic and extracellular signals. Rho GTPases are regulated by guanine nucleotide exchange factors and GTPase activating proteins (GAPs). Syd-1 is a protein that has been identified as necessary for synapse formation in worms, with similar proteins in flies, and mice. Little is known about the molecular mechanism by which Syd-1 is acting. While genetic techniques are great tools for examining synapse development, they are limited by their inability to consider the molecular nature of the protein product. By studying the biochemical nature of synaptic proteins, we can begin to understand their function with a new level of clarity. Syd-1 has a predicted Rho GAP domain; however it is thought to be inactive. The activity of the fly protein, Dsyd-1, has never been examined although it has been speculated that it is inactive in all invertebrates. Recently the mouse version was reported to have Rho GAP activity. By performing GTPase activity assays on purified proteins, I found the GAP domain of Dsyd-1 increased the GTPase activity of Rac-1 and Cdc42 but not RhoA. Members of our lab found the activity of Dsyd-1 is necessary for proper synapse formation both at the Drosophila neuromuscular junction as well as in R7 neurons. In Caenorhabditis elegans, Syd-1 was found to interact with presynaptic protein RSY-1. Since RSY-1 is evolutionarily conserved, I tested whether or not RSY-1 has a similar effect on R neurons in Drosophila. I also isolated mRNA from R neurons and evaluated the possibility of analyzing mutant neurons using comparative transcriptomics. This dissertation includes previously unpublished coauthored material.

Zwischen Blutkompartiment und umliegenden Interstitium besteht eine Barriere, die durch eine einzelne Schicht aus Endothelzellen gebildet wird. Essentiell für diese Barriere, deren Funktion in der Begrenzung des Austausches von Flüssigkeit und gelösten Stoffen liegt, sind interzelluläre Junktionen, welche die Endothelzellen miteinander verbinden. Durch eine gestörte Funktion und Regulation der Endothelbarriere entstehen beim Menschen verschiedene Pathologien wie zum Beispiel Ödeme, hämorrhagischer Schlaganfall und vaskuläre Malformationen. Es ist bekannt, dass cAMP die Endothelbarriere zum Teil durch Aktivierung der kleinen GTPase Rac1 stabilisiert. Trotz der großen medizinischen Relevanz dieses Signalweges, sind die damit einhergehenden Effekte auf die interzellulären Kontakte auf ultrastruktureller Ebene weitgehend unbekannt. In mikrovaskulären Endothelzellkulturen kam es ähnlich wie in intakten Mikrogefäßen zur Stärkung der Barrierefunktion. So resultierte sowohl nach Behandlung mit Forskolin und Rolipram (F/R), welche zur Steigerung der intrazellulären cAMP-Spiegel führen, als auch nach Zugabe von 8-(4-chlorophenylthio)-2´-O-methyladenosin-3´,5´-cyclic monophosphorothioate (O-Me-cAMP), einem selektiven Aktivator des cAMP nachgeschalteten Epac/Rap1-Signalweges, ein Anstieg des TER; außerdem konnte durch beide Substanzen (F/R und O-Me-cAMP) die Aktivierung von Rac1 induziert werden. Desweiteren wurde eine verstärkte Intensität und Linearisierung des Immunfluoreszenzsignals der Zelljunktionsproteine VE-Cadherin und Claudin5 entlang der Zellgrenzen beobachtet. In der ultrastrukturellen Analyse der interzellulären Kontaktzonen-Architektur zeigte sich unter F/R- oder O-Me-cAMP-Exposition ein signifikanter Anstieg an komplexen Interdigitationen. Diese komplexen Strukturen waren dadurch charakterisiert, dass sich die Membranen benachbarter Zellen, die durch zahlreiche endotheliale Junktionen stabilisiert wurden, über vergleichsweise lange Distanzen eng aneinanderlegten, so dass ein deutlich verlängerter Interzellularspalt resultierte. Die Inhibition der Rac1-Aktivierung durch NSC-23766 verminderte die Barrierefunktion und blockierte effektiv die O-Me-cAMP-vermittelte Barrierestabilisierung und Reorganisation der Kontaktzone einschließlich der Junktionsproteine. Demgegenüber konnte die F/R-vermittelte Barrierestabilisierung durch NSC-23766 nicht beeinträchtigt werden. Parallel dazu durchgeführte Experimente mit makrovaskulären Endothelien zeigten, dass es in diesem Zelltyp unter Bedingungen erhöhter cAMP-Konzentrationen weder zur Rac1-Aktivierung noch zur Barrierestärkung oder Kontaktzonen-Reorganisation kam. Diese Ergebnisse deuten darauf hin, dass in mikrovaskulären Endothelien Rac1-vermittelte Änderungen der Kontaktzonen-Morphologie zur cAMP-induzierten Barrierestabilisierung beitragen. / Evidence exists that cAMP stabilizes the endothelial barrier in part via activation of the small GTPase Rac1. However, despite the high medical relevance of this signaling pathway, the mechanistic effects on intercellular contacts on the ultrastructural level are largely unknown. In microvascular endothelial cell monolayers, in which increased cAMP strengthened barrier properties similar to intact microvessels in vivo, both forskolin and rolipram (F/R) to increase cAMP and 8-(4-chlorophenylthio)-2´-O-methyladenosine-3´,5´-cyclic monophosphorothioate (O-Me-cAMP) to stimulate exchange protein directly activated by cAMP/Ras proximate-1 (Epac/Rap1) signaling enhanced transendothelial electrical resistance (TER) and induced activation of Rac1. Concurrently, augmented immunofluorescence intensity and linearization of signals at cell borders were observed for intercellular junction proteins VE-cadherin and claudin5. Ultrastructural analysis of the intercellular contact zone morphology documented that exposure to F/R or O-Me-cAMP led to a significant increase in the proportion of contacts displaying complex interdigitations of cell borders in which membranes of neighboring cells were closely apposed over comparatively long distances and which were stabilized by numerous intercellular junctions. Interference with Rac1 activation by NSC-23766 completely abolished both barrier stabilization and contact zone reorganization in response to O-Me-cAMP whereas F/R-mediated barrier enhancement was not affected by NSC-23766. In parallel experiments using macrovascular endothelium, increased cAMP failed to induce Rac1 activation, barrier enhancement and contact zone reorganization. These results indicate that in microvascular endothelium Rac1-mediated alterations in contact zone architecture contributes to cAMP-induced barrier stabilization.

Protein biosynthesis is a core process in all living organisms. Assembly of the protein chain from aminoacids is catalysed by the ribosome, ancient and extremely complex macromolecular machine. Several different classes of accessory molecules are involved in translation, and one set of them, called translational GTPases (trGTPases), was in the focus of this work. In this thesis properties of two trGTPases– EF-G and eRF3 - were studied by means of direct biochemical experiments. EF-G is a bacterial trGTPase involved in two steps of translation: translocation and ribosomal recycling. Translocation is a process of the ribosomal movement along the mRNA, and recycling as the step when upon completion of the protein ribosome is released from the mRNA via splitting in two ribosomal subunits. We found that off the ribosome EF-G has similar affinities to GDP and GTP, and thus given the predominance of the latter in the cell, EF-G should be present mostly in the complex with GTP. However, binding to the ribosome increases factors affinity to GTP drastically, ensuring that it is in the GTP-bound state. GDP can not promote neither translocation, not recycling, and GDPNP can not promote recycling. It can, however, promote translocation, but in so doing it results in an intermediate ribosomal state and translocation process can be reversed by addition of GDP, which is not the case for the EF-G•GTP-catalyzed reaction. The second trGTPase we investigated is eukaryotic termination factor eRF3. This protein together with another factor, eRF1, is involved translation termination, which is release of the synthesized protein from the ribosome. We demonstrateed, that eRF3 alone has basically no propensity to bind GTP and thus resides in the GDP-bound state. Complex formation between eRF1 and eRF3 promotes GTP binding by the latter, resulting in the formation of the ternary complex eRF1•eRF3•GTP, which in turn is catalyzing the termination event. Experimental investigations of trGTPases where rationalized within a generalized thermodynamical framework, accommoding the existent experimental observations, both structural and biochemical.

Rho GTPases are critical regulators of actin cytoskeletal dynamics. The three most well characterized Rho GTPases, Rac1, RhoA and Cdc42 share a common inhibitor, RhoGDI. It is only recently becoming clear how upstream signals cause the selective release of individual Rho GTPases from RhoGDI. For example, our laboratory showed that diacylglycerol kinase zeta (DGKz), which converts diacylglycerol (DAG) to phosphatidic acid (PA), activates PAK1-mediated RhoGDI phosphorylation on Ser-101/174, causing selective Rac1 release and activation. Phosphorylation of RhoGDI on Ser-34 by PKCa has recently been demonstrated to selectively release RhoA, promoting RhoA activation. Here, I show DGKz is required for optimal RhoA activation and RhoGDI Ser-34 phosphorylation. Both were substantially reduced in DGKz-null fibroblasts and occurred independently of DGKz activity, but required a function DGKz PDZ-binding motif. In contrast, Rac1 activation required DGKz-derived PA, but not PDZ-interactions, indicating DGKz regulates these Rho GTPases by two distinct regulatory complexes. Interestingly, RhoA bound directly to the DGKz C1A domain, the same region known to bind Rac1. By direct interactions with RhoA and PKCa, DGKz was required for the efficient co-precipitation of these proteins, suggesting it is important to assemble a signalling complex that functions as a RhoA-specific RhoGDI dissociation complex. Consequently, cells lacking DGKz exhibited decreased RhoA signalling downstream and disrupted stress fibers. Moreover, DGKz loss resulted in decreased stress fiber formation following the expression of a constitutively active RhoA mutant, suggesting it is also important for RhoA function following activation. This is consistent with the ability of DGKz to bind both active and inactive RhoA conformations. Collectively, these findings suggest DGKz is central to two distinct Rho GTPase activation complexes, each having different requirements for DGKz activity and PDZ interactions, and might regulate the balance of Rac1 and RhoA activity during dynamic changes to the actin cytoskeleton.

Baz/PAR3 is a key regulator of epithelial cell polarity (ECP). To identify proteins functioning with Baz, I completed a baz genetic interaction screen by localizing 15 GFP-tagged candidates. Then I tested the role of a top candidate, dASAP (Drosophila Arf GTPase-activating protein with SH3 domain, Ankyrin repeat and PH domain), in Drosophila ECP. To determine whether dASAP might interact with polarity players, I defined the localization of dASAP throughout embryogenesis with GFP-tagged proteins and an anti-dASAP antibody. To study how loss of dASAP function affects ECP, I generated a deletion allele by imprecise P-element excision. To evaluate how each of the six domains of dASAP contributes to its localization and functions, I generated constructs deleting each domain. I found associations between dASAP, actin and the apical domain. The six domains may act redundantly to localize dASAP, although interactions between domains may affect the degree of membrane association.

Baz/PAR3 is a key regulator of epithelial cell polarity (ECP). To identify proteins functioning with Baz, I completed a baz genetic interaction screen by localizing 15 GFP-tagged candidates. Then I tested the role of a top candidate, dASAP (Drosophila Arf GTPase-activating protein with SH3 domain, Ankyrin repeat and PH domain), in Drosophila ECP. To determine whether dASAP might interact with polarity players, I defined the localization of dASAP throughout embryogenesis with GFP-tagged proteins and an anti-dASAP antibody. To study how loss of dASAP function affects ECP, I generated a deletion allele by imprecise P-element excision. To evaluate how each of the six domains of dASAP contributes to its localization and functions, I generated constructs deleting each domain. I found associations between dASAP, actin and the apical domain. The six domains may act redundantly to localize dASAP, although interactions between domains may affect the degree of membrane association.