Characterizing the differential distribution and targets of Sumo paralogs in the mouse brain

SUMOylation is an evolutionarily conserved and essential mechanism whereby Small Ubiquitin Like Modifiers, or SUMO proteins (Sumo in mice), are covalently bound to protein substrates in a highly dynamic and reversible manner. SUMOylation is involved in a variety of basic neurological processes including learning and memory, and central nervous system development, but is also linked with neurological disorders. However, studying SUMOylation in vivo remains challenging due to limited tools to study Sumo proteins and their targets in their native context. More complexity arises from the fact that Sumo1 and Sumo2 are ∼50% homologous, whereas Sumo2 and Sumo3 are nearly identical and indistinguishable with antibodies. While Sumo paralogues can compensate for one another’s loss, Sumo2 is highest expressed and only paralog essential for embryonic development making it critical to uncover roles specific to Sumo2 in vivo. To further examine the roles of Sumo2, and to begin to tease apart the redundancy and similarity between key Sumo paralogs, we generated (His6-)HA epitope-tagged Sumo2 knock-in mouse alleles, expanding the current Sumo knock-in mouse tool-kit comprising of the previously generated His6-HA-Sumo1 knock-in model. Using these HA-Sumo mouse lines, we performed whole brain imaging and mapping to the Allen Brain Atlas to analyze the relative distribution of the Sumo1 and Sumo2 paralogues in the adult mouse brain. We observed differential staining patterns between Sumo1 and Sumo2, including a partial localization of Sumo2 in nerve cell synapses of the hippocampus. Combining immunoprecipitation with mass spectrometry, we identified native substrates targeted by Sumo1 or Sumo2 in the mouse brain. We validated select hits using proximity ligation assays, further providing insight into the subcellular distribution of neuronal Sumo2-conjugates. These mouse models thus serve as valuable tools to study the cellular and biochemical roles of SUMOylation in the central nervous system

New lessons on TDP-43 from old N. furzeri killifish

Frontotemporal dementia and amyotrophic lateral sclerosis are fatal and incurable neurodegenerative diseases linked to the pathological aggregation of the TDP-43 protein. This is an essential DNA/RNA-binding protein involved in transcription regulation, pre-RNA processing, and RNA transport. Having suitable animal models to study the mechanisms of TDP-43 aggregation is crucial to develop treatments against disease. We have previously demonstrated that the killifish Nothobranchius furzeri offers the advantage of being the shortest-lived vertebrate with a clear aging phenotype. Here, we show that the two N. furzeri paralogs of TDP-43 share high sequence homology with the human protein and recapitulate its cellular and biophysical behavior. During aging, N. furzeri TDP-43 spontaneously forms insoluble intracellular aggregates with amyloid characteristics and colocalizes with stress granules. Our results propose this organism as a valuable new model of TDP-43-related pathologies making it a powerful tool for the study of disease mechanism

SUMOylation and calcium signalling: potential roles in the brain and beyond

Small ubiquitin-like modi er (SUMO) conjugation (or SUMOylation) is a post-translational protein modi cation implicated in alterations to protein expression, localization and func- tion. Despite a number of nuclear roles for SUMO being well characterized, this process has only started to be explored in relation to membrane proteins, such as ion channels. Cal- cium ion (Ca2+) signalling is crucial for the normal functioning of cells and is also involved in the pathophysiological mechanisms underlying relevant neurological and cardiovascu- lar diseases. Intracellular Ca2+ levels are tightly regulated; at rest, most Ca2+ is retained in organelles, such as the sarcoplasmic reticulum, or in the extracellular space, whereas depolarization triggers a series of events leading to Ca2+ entry, followed by extrusion and reuptake. The mechanisms that maintain Ca2+ homoeostasis are candidates for modulation at the post-translational level. Here, we review the effects of protein SUMOylation, including Ca2+ channels, their proteome and other proteins associated with Ca2+ signalling, on vital cellular functions, such as neurotransmission within the central nervous system (CNS) and in additional systems, most prominently here, in the cardiac system

Characterizing the SUMO Pathway in Human Health and Disease

The small ubiquitin-related modifier (SUMO) regulates nearly every aspect of cellular function, from gene expression in the nucleus to ion transport at the plasma membrane. As such, misregulation of SUMO pathway enzymes are implicated in human cancers, neurodegeneration and inflammatory diseases, among others. Despite this knowledge, many questions remain unanswered, including, how can one ~15kDa protein contribute to so many various and fundamentally different cellular functions and diseases? And, can we efficaciously target the SUMO pathway for treatment of associated diseases? If so, how might we identify the best patients to treat with such therapies? The work presented in this thesis uses a multi-faceted approach to address these questions. First, we dive into an exploration of the five SUMO paralogs (SUMO1-5) to understand how they can collectively regulate such diverse biological functions. One potential explanation is that the paralogs have unique and non-redundant cellular functions, though comprehensive evidence to support this was lacking. We therefore performed a systematic analysis of the literature, SUMO paralog expression in various human tissues, and CRISPR paralog knock-out cell lines, which each provided evidence for tissue and paralog-specific functions. Analysis of the knockout cell lines revealed non-redundant roles for the SUMO1 and SUMO2 paralogs in regulating responses to various cellular stressors, nuclear body integrity, gene expression, and cellular morphology. Collectively, this work defines unique roles for the paralogs in diverse cellular processes, thus shedding light on how one pathway can be implicated in various cellular functions and diseases, and simultaneously providing a foundation for the development of precise SUMO paralog-targeting therapies. To aid in the selection of patients for treatment with such therapies, we developed a bioinformatics workflow to analyze the expression levels of SUMO pathway enzymes in cancerous versus normal tissues. We present a published example of this workflow where we revealed that expression levels of the SENP1 SUMO protease are unchanged in pancreatic cancer, thus indicating that SENP1 is not a predictive biomarker for this particular disease. Together, with our work on paralog-specific functions, we have provided insights essential for realizing the full therapeutic potential of the SUMO pathway

CHARACTERIZING THE ROLES OF SUMOYLATION IN MITOSIS THROUGH SUBSTRATE IDENTIFICATION AND AN ANALYSIS OF THE SUMO ISOPEPTIDASES, SENP1 AND SENP2

Cell cycle regulation is essential for all organisms and has profound impacts on development, tissue regeneration, and when aberrant, results in cancer. Mitosis, the process of nuclear division, is highly regulated by posttranslational modifications including sumoylation. The SUMO family consists of three paralogs, SUMO-1, -2 and -3, which are covalently attached to the lysine residues of substrates and affects substrate localization, function, and/or protein-protein interactions. In this thesis, we hypothesized that dynamic SUMO modification and demodification is essential for mitotic progression and that deregulation of the sumoylation machinery can lead to mitotic defects resulting in cancer. We analyzed the function of sumoylation in mitosis by a two-pronged approach. First, we conducted a mass spectrometry study to identify the proteins sumoylated in mitosis, which will provide a foundation for future studies identifying the molecular mechanisms of mitotic SUMO functions. Secondly, we characterized two SUMO deconjugating enzymes, SENP1 and SENP2, to enhance our understanding for how sumoylation is regulated temporally and spatially in mitosis. We demonstrated that sumoylation is required for chromosome alignment through SENP2 overexpression studies. Furthermore, we demonstrated that desumoylation is required for a timely metaphase to anaphase transition through SENP1 siRNA knockdown analysis. Finally, we conducted a literature review to describe the functions of sumoylation in regulating chromatin structure, which may impact the mitotic functions of sumoylation in chromosome condensation and/or centromere structure. In its entirety, this thesis presents a foundation of mitotic SUMO substrates for further analysis and a mechanism of isopeptidase-mediated regulation of sumoylation in mitosis

17th Spanish Society for Developmental Biology Meeting: New Trends in Developmental Biology

The Spanish Society for Developmental Biology organized its 17th meeting in November 2020. The meeting, organized by CIC bioGUNE, the University of the Basque Country and the University of Cantabria, gathered about 280 registrants and received 132 scientific abstracts. Participants ranged from undergraduate to senior researchers, with a broad participation of Ph.D. students. The meeting was organized in 8 sessions: Growth and Scaling, Self-organization, Neurodevelopment, Genomes, Cell Biology, Development and Disease, Evo-Devo and Regeneration (Araújo et al., 2021). These sessions focused on the new tendencies in Developmental Biology research and, based on the science presented there, we organized this special issue on The 17th Edition of the Spanish Society for Developmental Biology Meeting: New Trends in Developmental Biology. This collection of articles gathers several scientific contributions in this area, featuring collaborative and interdisciplinary approaches among developmental biologists. With the focus on organogenesis and gene regulation, our selected content embraces novel discoveries on muscle development, regeneration and the transcriptional control and role of miRNAs in development, while highlighting advances in organogenesis and gonadal development

CHARACTERIZING THE REGULATION AND FUNCTION OF THE SUMO-SPECIFIC ISOPEPTIDASE SENP2

The small ubiquitin-related modifier (SUMO) protein is post-translationally and covalently attached to a multitude of other proteins, regulating a plethora of essential cellular functions in the nucleus and the cytoplasm. Recent evidence links SUMO to membrane-associated functions, however, the mechanism of SUMO regulation at membranes remains largely unknown. To look at SUMO regulation, we focused on characterizing the subcellular localizations and functions of the SUMO-specific isopeptidases, collectively known as SENPs, since they comprise the largest family of SUMO proteases and are major regulators of SUMO dynamics. SENPs share a conserved C-terminal catalytic domain, but have divergent N-terminal domains containing targeting signals that determine their unique subcellular localizations and substrate specificities. In this thesis, we characterized the N-terminal domain of the mammalian SUMO-specific protease SENP2. We found that SENP2 can directly interact with intracellular membranes via a unique N-terminal amphipathic α-helix. We also show that SENP2-membrane interaction is directly regulated by Karyopherin-α (Kap-α). Furthermore, we identified SENP2 interacting proteins using BioID, which revealed that SENP2 interacts with a subset of ER-, Golgi-, and inner nuclear membrane-associated proteins. We also developed a new technique to identify SENP2 substrates. Collectively, our findings demonstrate the critical role N-terminal targeting signals play in the differential regulation of SUMO proteases, and indicate that SENP2 may play a role in regulating sumoylation at membranes

Characterization of the Ribosomal Protein L22e Family in Drosophila melanogaster: Evidence for Functional Diversification of Duplicated Ribosomal Protein Genes

Gene duplication is a contributing factor to genome evolution in eukaryotes. With an additional copy, selective pressure is relieved, allowing for accumulation of genetic variation and possible development of new or altered functions. Ribosomal protein (Rp) genes are a common class of duplicated genes found throughout eukaryotes. Typically encoding highly similar or identical proteins at separate loci, duplicated Rps were originally thought to be redundant and to relieve the high demand for translation. However, recent reports in yeast have shown phenotypic differences between Rp paralogue knockouts, suggesting functional non-redundancy. Little effort has been devoted toward elucidating the function of Rp paralogues in eukaryotes other than in yeast. Furthermore, in yeast, paralogous Rps are typically highly identical, making studying gene function difficult without protein tagging. To explore whether duplicated Rp genes have redundant roles, we focused on the eukaryotic-specific RpL22e family in Drosophila melanogaster. The Drosophila RpL22e family consists of two members, the ancestral rpL22e and its duplicate rpL22e-like, which are 37% identical. Divergence is evident in the genomic sequence, codon usage, and protein sequence, but whether this results in novel functions has not been previously addressed and is the focus of this dissertation.It is widely known that the ancestral RpL22e is ubiquitous, but our data show that RpL22e-like expression is primarily restricted to the male germline and is a true ribosomal component. Further investigation shows that in testis tissue, RpL22e is primarily SUMOylated and phosphorylated. Only unmodified RpL22e co-sediments with the translation machinery in Drosophila S2 cells, leading to the interpretation that the majority of testis RpL22e is not part of the translation machinery and that paralogue functions are non-redundant. Immunohistochemical analysis further supports non-redundant paralogue roles, as RpL22e is primarily restricted to the nucleoplasm in the maturing meiotic germline; RpL22e-like is cytoplasmic in these cells. Additionally, there is an unequal requirement for RpL22e members in vivo, as only rpL22e is essential in the fly.Taking the data in this dissertation together, it is evident that the Drosophila RpL22e paralogues have diverged in function within the male germline. RpL22e assumes an additional and unique role compared to RpL22e-like

Identification and characterization of hoxa2 target genes by ChIP

Hox genes are evolutionarily conserved transcription factors which act to control important developmental pathways involved in morphogenesis of the embryo. Hoxa2 is expressed in the developing CNS in rhombomeres 2-7 in the presumptive hindbrain. During development Hoxa2 expression extends caudally throughout the spinal cord and persists into adulthood. Although previous analysis of Hoxa2 expression indicates its possible role in neuronal circuit specification and/or dorsal-ventral patterning within the spinal cord, the precise genetic pathways through which Hoxa2 affects spinal cord development have not been characterized. We have used immunoprecipitation of Hoxa2-target DNA complexes from chromatin preparations of E18 mouse spinal cord and hindbrain tissue to isolate in vivo downstream target genes of Hoxa2. Seven DNA fragments were isolated, sequenced and were shown to exhibit in vitro DNA binding by Hoxa2. A search of sequence databases for the target sequences revealed that of these, two displayed high identity with novel mouse genes: toll-associated serine protease (Tasp) and the murine homolog of the human dual specificity tyrosine phosphorylation regulated kinase 4 (Dyrk4). Also, two of the isolated clones are presumably bacterial sequences containing the canonical homeodomain binding site TAAT, and the remaining three clones have not yet been mapped in the mouse genome. A potential core Hoxa2 binding motif consisting of 5' CCATCA/T 3', which is based on a previously characterized Hoxa2-Pbx consensus sequence (Lampe et al., 2004), has been identified in both the Tasp and Dyrk4 intronic elements. Both Dyrk4 and Tasp mRNA have been detected within the developing mouse from E10-18 and in the adult CNS. Analysis by RT-PCR of Tasp expression in Hoxa2-/- newborn mice hindbrain and spinal cord tissues showed an upregulation of Tasp, and transient transfection experiments indicated that Hoxa2 may act as a transcriptional repressor of Tasp through an intronic regulatory element. Transfection studies using the intronic sequence of Dyrk4 indicated that it may function as an enhancer of transcription of Dyrk4 in the presence of Hoxa2. Both Dyrk4 and Tasp belong to large protein subfamilies whose members play a role in numerous developmental pathways in several organisms. Tasp, also known as HtrA3, interacts with TGFâ signaling molecules which are known to be key regulators of development, dorsoventral patterning and are involved in various neuronal pathways. Although the function of Dyrk4 is not known, many of its family members are involved in the regulation of transcription factors and signaling molecules via phosphorylation that are involved in neuronal pathways also. Hoxa2 may act in specifying neuronal subtypes and dorsoventral patterning in the CNS through down and upregulation of its downstream targets Dyrk4 and Tasp, respectively

Systematic Analysis of SUMO Paralogue-specific Functions and a Novel SUMO1-Dependent Pathway for Cytosolic Protein Quality Control

The small ubiquitin-related modifiers (SUMOs) are essential protein regulators that modulate nearly every aspect of cellular functions. Five SUMO paralogues have been identified in humans, with sequence homologies ranging from 45% to 97%. Among them, SUMO1 and SUMO2 are the least similar and also the best studied. However, to what extent SUMO1 and SUMO2 impart distinct and non-redundant cellular functions, has not been systematically examined and is therefore not well understood. To systematically identify and characterize paralogue-specific functions of SUMO proteins, we used CRISPR-Cas9 to individually knock out SUMO1 and SUMO2 expression in human osteosarcoma (U2OS) cells. Analysis of these knockout cell lines revealed non-redundant roles for SUMO1 and SUMO2 in regulating essential cellular functions, including cellular morphology, PML nuclear body integrity, response to cellular stresses, and control of gene expression. Using SUMO knockout cell lines and yeast strains expressing SUMO mutant proteins, we also identified a conserved SUMO-dependent pathway for degradation of protein quality control (PQC) model proteins containing the CL1 degron (GFP-Ura3-CL1 in yeast and GFP-CL1 in humans) that operates only in the cytosol but not the nucleus. Furthermore, we found that in humans, turnover of GFP-CL1 in the cytosol was uniquely dependent on SUMO1 but not SUMO2, revealing a previously unrecognized role for SUMO1 in regulating cytosolic PQC. PQC is essential for maintaining proteostasis and normal cellular functions, and therefore PQC perturbation proceeds numerous human diseases. Compared to the well-characterized PQC pathways within the endoplasmic reticulum and the nucleus, much less is known about the mechanisms that modulate cytosolic misfolded protein degradation. Findings reported in this thesis reveal a novel regulatory mechanism of cytosolic PQC, which contributes to a comprehensive view of the complicated cellular PQC network and provides novel insights into PQC-associated diseases