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Small ubiquitin-related modifiers (SUMOs) function as post-translational protein modifications and regulate nearly every aspect of cellular function. While a single ubiquitin protein is expressed across eukaryotic organisms, multiple SUMO paralogues with distinct biomolecular properties have been identified in plants and vertebrates. Five SUMO paralogues have been characterized in humans, with SUMO1, SUMO2 and SUMO3 being the best studied. SUMO2 and SUMO3 share 97% protein sequence homology (and are thus referred to as SUMO2/3) but only 47% homology with SUMO1. To date, thousands of putative sumoylation substrates have been identified thanks to advanced proteomic techniques, but the identification of SUMO1- and SUMO2/3-specific modifications and their unique functions in physiology and pathology are not well understood. The SUMO2/3 paralogues play an important role in proteostasis, converging with ubiquitylation to mediate protein degradation. This function is achieved primarily through SUMO-targeted ubiquitin ligases (STUbLs), which preferentially bind and ubiquitylate poly-SUMO2/3 modified proteins. Effects of the SUMO1 paralogue on protein solubility and aggregation independent of STUbLs and proteasomal degradation have also been reported. Consistent with these functions, sumoylation is implicated in multiple human diseases associated with disturbed proteostasis, and a broad range of pathogenic proteins have been identified as SUMO1 and SUMO2/3 substrates. A better understanding of paralogue-specific functions of SUMO1 and SUMO2/3 in cellular protein quality control may therefore provide novel insights into disease pathogenesis and therapeutic innovation. This review summarizes current understandings of the roles of sumoylation in protein quality control and associated diseases, with a focus on the specific effects of SUMO1 and SUMO2/3 paralogues. [ABSTRACT FROM AUTHOR]

A decade has passed since the first reported connection between RAP80 and BRCA1 in DNA double-strand break repair. Despite the initial identification of RAP80 as a factor localizing BRCA1 to DNA double-strand breaks and potentially promoting homologous recombination, there is increasing evidence that RAP80 instead suppresses homologous recombination to fine-tune the balance of competing DNA repair processes during the S/G phase of the cell cycle. RAP80 opposes homologous recombination by inhibiting DNA end-resection and sequestering BRCA1 into the BRCA1-A complex. Ubiquitin and SUMO modifications of chromatin at DNA double-strand breaks recruit RAP80, which contains distinct sequence motifs that recognize ubiquitin and SUMO. Here, we review RAP80's role in repressing homologous recombination at DNA double-strand breaks and how this role is facilitated by its ability to bind ubiquitin and SUMO modifications. [ABSTRACT FROM AUTHOR]

R36 --> 652.1 --> Protein quality control (PQC) maintains proteostasis in cells, whereas PQC dysregulation is found in diseases such as cancers, cardiovascular diseases, and neurodegenerative disorders. Over the past few decades, remarkable progress has been made in understanding compartmentalized PQC pathways that operate inside the nucleus and the endoplasmic reticulum (ER). The small ubiquitin‐related modifiers (SUMOs), for example, were recently found to participate in a nuclear PQC pathway that involves selective sumoylation of misfolded proteins by the SUMO E3 ligase PML, and subsequent recognition and ubiquitylation by the SUMO‐targeted ubiquitin E3 ligase RNF4 (Guo et al., Mol Cell, 2014). Roles of SUMOs in cytoplasmic PQC, however, have not previously been reported. Using yeast mutants and human knockout cell lines, our lab has recently identified a conserved SUMO‐dependent pathway for degradation of a cytosolic, misfolded protein model substrate. The substrate consists of GFP fused to an N‐terminal nuclear export signal (NES) and a C‐terminal hydrophobic degron known as CL1 (NES‐GFP‐CL1). Using protein stability assays, we observed a decrease in the turnover rate of NES‐GFP‐CL1 in a yeast SUMO mutant strain and a human U2OS SUMO1 knockout cell line. Further exploration using nuclear and ER‐localized substrates revealed that this pathway is specific for soluble, cytosolic proteins. In addition, analysis of a U2OS SUMO2 knockout cell line revealed that the observed PQC pathway is specific for the SUMO1 paralogue. This suggests a distinct mechanism from the previously characterized nuclear PML‐SUMO‐RNF4 pathway, which targets misfolded proteins modified by SUMO2/3 polymeric chains. Using cellular fractionation studies, we observed an increase in levels of insoluble NES‐GFP‐CL1 specifically in SUMO1 knockout cells compared to wild type cells. We therefore propose a model where SUMO1 modification of misfolded cytosolic proteins promotes their turnover by enhancing solubility and preventing aggregation. Results from our studies reveal a novel cytosolic PQC regulatory network and provide insights into roles for sumoylation in PQC‐associated diseases. [ABSTRACT FROM AUTHOR]

R3750 --> 792.7 --> Mitochondria are dynamic powerhouses of cells and their fission and fusion must be tightly regulated for normal cell function. Mitochondrial fission is mediated by the GTPase, Drp1, and mis‐regulation of Drp1 leads to mitochondrial hyper‐fragmentation, a known marker of disease. It has been reported that Drp1 is modified by SUMO1 and SUMO2/3, however, the mechanisms of Drp1 regulation by individual SUMO paralogs remain to be fully understood. Here, we have used CRISPR/Cas9 derived SUMO1 and SUMO2 knockout (KO) cell lines, to perform a systematic investigation of paralog‐specific effects on mitochondrial maintenance and function. In contrast to expectations, we observed multiple mitochondrial defects specifically in SUMO2 KO, but not SUMO1 KO, cells. SUMO2 KO cells had reduced mitochondrial activity based on results of MTT assays. By immunofluorescence microscopy, we also observed an increase in mitochondrial fragmentation in SUMO2 KO cells. Paradoxically, increased fragmentation occurred despite reduced levels of Drp1 protein expression and sequestration of Drp1 in large cytosolic foci. Taken together, our findings indicate that SUMO2 plays a non‐redundant, paralog‐specific role in regulating mitochondrial function, in part through effects on Drp1. We anticipate that a more detailed understanding of the molecular effects of SUMO2 on Drp1 may lead to novel therapeutic approaches to treat or prevent ischemic injury, neurodegeneration, and other mitochondrial‐related maladies. [ABSTRACT FROM AUTHOR]

Highlights: [•] Genetically equal stem cells compete for niche occupancy in multiple tissues. [•] Stochastic competition for niche access is important for stem cell fitness. [•] Mutated stem cells can gain a competitive advantage or disadvantage. [•] Stem cells with a competitive advantage can cause diseases or niche defects. [ABSTRACT FROM AUTHOR]

A major challenge in nuclear organization is the packaging of DNA into dynamic chromatin structures that can respond to changes in the transcriptional requirements of the cell. Posttranslational protein modifications, of histones and other chromatin-associated factors, are essential regulators of chromatin dynamics. In this Review, we summarize studies demonstrating that posttranslational modification of proteins by small ubiquitin-related modifiers (SUMOs) regulates chromatin structure and function at multiple levels and through a variety of mechanisms to influence gene expression and maintain genome integrity. [ABSTRACT FROM AUTHOR]

Abstract: Stem cells sustain tissue regeneration by their remarkable ability to replenish the stem cell pool and to generate differentiating progeny. Signals from local microenvironments, or niches, control stem cell behavior. In the Drosophila testis, a group of somatic support cells called the hub creates a stem cell niche by locally activating the Janus Kinase-Signal Transducer and Activator of Transcription (JAK-STAT) pathway in two adjacent types of stem cells: germline stem cells (GSCs) and somatic cyst stem cells (CySCs). Here, we find that ken and barbie (ken) is autonomously required for the self-renewal of CySCs but not GSCs. Furthermore, Ken misexpression in the CySC lineage induces the cell-autonomous self-renewal of somatic cells as well as the nonautonomous self-renewal of germ cells outside the niche. Thus, Ken, like Stat92E and its targets ZFH1 () and Chinmo (), is necessary and sufficient for CySC renewal. However, ken is not a JAK-STAT target in the testis, but instead acts in parallel to Stat92E to ensure CySC self-renewal. Ken represses a subset of Stat92E targets in the embryo () suggesting that Ken maintains CySCs by repressing differentiation factors. In support of this hypothesis, we find that the global JAK-STAT inhibitor Protein tyrosine phosphatase 61F (Ptp61F) is a JAK-STAT target in the testis that is repressed by Ken. Together, our work demonstrates that Ken has an important role in the inhibition of CySC differentiation. Studies of ken may inform our understanding of its vertebrate orthologue B-Cell Lymphoma 6 ( BCL6 ) and how misregulation of this oncogene leads to human lymphomas. [Copyright &y& Elsevier]

The ability of stem cells to divide asymmetrically to produce both self-renewing and differentiating daughter cells sustains many adult tissues, but germline stem cells (GSCs) are unique among stem cells as they perpetuate the genome of the species. The cellular and molecular mechanisms regulating most mammalian stem cells in their endogenous local microenvironments, or niches, are quite challenging to study. However, studies of stem cell niches such as those found in the Drosophila gonads have proven very useful. In these tissues, GSCs are housed in a readily identifiable niche, and the ability to genetically manipulate these cells and their neighbors has uncovered several fundamental mechanisms that are relevant to stem cells more generally. Here, we summarize recent work on the regulation of GSCs in the Drosophila testis niche by intercellular signals, and on the intracellular mechanisms that cooperate with these signals to ensure the survival of the germline. This review focuses on GSCs within the adult Drosophila testis; somatic stem cells in this tissue are reviewed by Zoller and Schulz in this issue. For a review of the testis niche as a whole, see de Cuevas and Matunis, and for more comprehensive reviews of the Drosophila testis, refer to Fuller and Davies and Fuller. [ABSTRACT FROM AUTHOR]

Abstract: The nuclear envelope is a double lipid bilayer that physically separates the functions of the nucleus and the cytoplasm of eukaryotic cells. Regulated transport of molecules between the nucleus and the cytoplasm is essential for normal cell metabolism and is mediated by large protein complexes, termed nuclear pore complexes (NPCs), which span the inner and outer membranes of the nuclear envelope. Significant progress has been made in the past 10 years in identifying the protein composition of NPCs and the basic molecular mechanisms by which these complexes facilitate the selective exchange of molecules between the nucleus and the cytoplasm. However, many fundamentally important questions about the functions of NPCs, the specific functions of individual NPC-associated proteins, and the assembly and disassembly of NPCs, remain unanswered. This review describes approaches for isolating and characterizing nuclear envelopes and NPC-associated proteins from mammalian cells. It is anticipated that these procedures can be used as a starting point for further molecular and biochemical analysis of the mammalian nuclear envelope, NPCs, and NPC-associated proteins. [Copyright &y& Elsevier]

Although the ability of engrafted stem cells to regenerate tissue has received much attention, the molecular mechanisms controlling regeneration are poorly understood. In the Drosophila male germline, local activation of the Janus kinase-signal transducer and activator of transcription (Jak-STAT) pathway maintains stem cells; germline stem cells lacking Jak-STAT signaling differentiate into spermatogonia without self-renewal. By conditionally manipulating Jak-STAT signaling, we find that spermatogonia that have initiated differentiation and are undergoing limited mitotic (transitamplifying) divisions can repopulate the niche and revert to stem cell identity. Thus, in the appropriate microenvironment, transit-amplifying cells dedifferentiate, becoming functional stem cells during tissue regeneration. [ABSTRACT FROM AUTHOR]

Small ubiquitin-related modifiers (SUMOs) are proteins that are posttranslationally conjugated to other cellular proteins, particularly those that localize and function in the nucleus. Enzymes regulating SUMO modification localize in part to nuclear pore complexes (NPCs), indicating that modification of some proteins may occur as they are translocated between the nucleus and the cytoplasm. Substrates that are regulated by SUMO modification at NPCs, however, have not been previously identified. Among the most abundant cargos transported through NPCs are the heterogeneous nuclear ribonucleoproteins (hnRNPs). HnRNPs are involved in various aspects of mRNA biogenesis, including regulation of pre-mRNA splicing and nuclear export. Here, we demonstrate that two subsets of hnRNPs, the hnRNP C and M proteins, are substrates for SUMO modification. We demonstrate that the hnRNP C proteins are modified by SUMO at a single lysine residue, K237, and that SUMO modification at this site decreases their binding to nucleic acids. We also show that Nup358, a SUMO E3 ligase associated with the cytoplasmic fibrils of NPCs, enhances the SUMO modification of the hnRNP C and M proteins. Based on our findings, we propose that SUMO modification of the hnRNP C and M proteins may occur at NPCs and facilitate the nucleocytoplasmic transport of mRNAs. [ABSTRACT FROM AUTHOR]

Nuclear pore complexes (NPCs) are large protein structures spanning the double membrane of the eukaryotic nucleus that serve as sites for translocation of macromolecules between the nucleus and the cytoplasm. The vertebrate NPC has recently been found to comprise ∼30 distinct proteins, collectively referred to as nucleoporins. Studies over the past several years have demonstrated that individual nucleoporins have unique roles in regulating NPC function and the nucleocytoplasmic transport of proteins and RNAs. The unique functions of individual nucleoporins have been made most clear through their associations with specific human diseases. Here, we highlight the relationships between individual nucleoporins and disease, with particular emphasis given to ALADIN, a nucleoporin linked to a genetically heritable human disease known as triple A syndrome. [Copyright &y& Elsevier]

Triple A syndrome is a human autosomal recessive disorder characterized by an unusual array of tissue-specific defects. Triple A syndrome arises from mutations in a WD-repeat protein of unknown function called ALADIN (also termed Adracalin or AAAS). We showed previously that ALADIN localizes to nuclear pore complexes (NPCs), large multiprotein assemblies that are the sole sites of nucleocytoplasmic transport. Here, we present evidence indicating that NPC targeting is essential for the function of ALADIN. Characterization of mutant ALADIN proteins from triple A patients revealed a striking effect of these mutations on NPC targeting. A variety of disease-associated missense, nonsense, and frameshift mutations failed to localize to NPCs and were found predominantly in the cytoplasm. Microscopic analysis of cells from a triple A patient revealed no morphological abnormalities of the nuclei, nuclear envelopes, or NPCs. Importantly, these findings indicate that defects in NPC function, rather than structure, give rise to triple A syndrome. We propose that ALADIN plays a cell type-specific role in regulating nucleocytoplasmic transport and that this function is essential for the proper maintenance and/or development of certain tissues. Our findings provide a foundation for understanding the molecular basis of triple A syndrome and may lead to unique insights into the role of nucleocytoplasmic transport in adrenal function and neurodevelopment. [ABSTRACT FROM AUTHOR]

Stem cells, which regenerate tissue by producing differentiating cells, also produce cells that renew the stem cell population. Signals from regulatory microenvironments (niches) are thought to cause stem cells to retain self-renewing potential. However, the molecular characterization of niches remains an important goal. In Drosophila testes, germ line and somatic stem cells attach to a cluster of support cells called the hub. The hub specifically expresses Unpaired, a ligand activating the JAK-STAT (Janus—signal transducer and activator of transcription) signaling cascade. Without JAK-STAT signaling, germ line stem cells differentiate but do not self-renew. Conversely, ectopic JAK-STAT signaling greatly expands both stem cell populations. We conclude that the support cells of the hub signal to adjacent stem cells by activation of the JAK-STAT pathway, thereby defining a niche for stem cell self-renewal. [ABSTRACT FROM AUTHOR]

The adult Drosophila testis contains a well-defined niche created by a cluster of hub cells, which secrete signals that maintain adjacent germline stem cells and somatic cyst stem cells (CySCs). Hub cells are normally quiescent in adult flies but can exit quiescence, delaminate from the hub and convert into CySCs after ablation of all CySCs. The opposite event, CySC conversion into hub cells, was proposed to occur under physiological conditions, but the frequency of this event is debated. Here, to probe further the question of whether or not hub cells can be regenerated, we developed methods to genetically ablate some or all hub cells. Surprisingly, when flies were allowed to recover from ablation, the missing hub cells were not replaced. Hub cells did not exit quiescence after partial ablation of hub cells, and labeled cells from outside the hub did not enter the hub during or after ablation. Despite its ability to exit quiescence in response to CySC ablation, we conclude that the hub in the adult Drosophila testis does not have a mechanism to replenish missing hub cells. [ABSTRACT FROM AUTHOR]

Focuses on a study which looked at the RanGAP1 protein involved in the regulating of nucleocytoplasmic transport, with reference to cytoplasmic vertebrates. Identification of RanGAP1; Information on RanGAP1 in vertebrates; Discussion on the NPC-associated form of RanGAP1; Methodology used in the study; Results from the study.

Investigates the modulation of the partitioning of the Ran-GT-Pase-activating protein RanGAP1 between the cytosol and the nuclear pore complex through a ubiquitin-like modification. Bidirectional transport of proteins and ribonucleoproteins; Mitotic spindle apparatus during mitosis; Implications for protein regulation.

The PML protein, identified first as part of the oncogenic PML­RARα chimera in acute promyelocytic leukemia (APL), concentrates within discrete subnuclear structures, corresponding to some types of nuclear bodies. These structures are disrupted in APL cells, and retinoic acid (RA) can trigger their reorganization, correlating with its therapeutic effect in this type of leukemia. Recently, arsenic trioxide (As2O3) was identified as a potent antileukemic agent which, similarly to RA, induces complete remissions in APL patients. Here we show that, in APL cells, As2O3 triggers rapid degradation of PML—RARα and provokes the restoration of intact nuclear bodies. In non-APL cells, the ubiquitin-like protein SUMO-1 is covalently attached to a subset of wild-type PML in a reversible and phosphorylation-dependent manner. The unmodified form of PML is found in the soluble nucleoplasmic fraction, whereas the SUMO-1-polymodified forms of PML are compartmentalized exclusively in the PML nuclear bodies. As2O3 administration strikingly increases the pool of SUMO-1—PML conjugates that, subsequently, accumulate in enlarged nuclear bodies. In contrast to PML—RARα, the overall amount of PML seems to remain unaltered up to 36 h following As2O3 treatment. These findings indicate that the conjugation of PML with SUMO-1 modulates its intracellular localization and suggest that post-translational modification by SUMO-1 may be more generally involved than previously suspected in the targeting of proteins to distinct subcellular structures. They provide additional evidence that the role of ‘ubiquitin-like’ post-translational modification is not limited to a degradation signal. [ABSTRACT FROM AUTHOR]

The article discusses the study conducted by S. Watanabe which identifies that the protein demethylase JMJD1C is the first selective branch-factor required for BRCA1-dependent HR-mediated repair. It examines how JMJD1C affect RAP80-BRCA1 recruitment to double-strand breaks (DSBs). It states that the earliest proteins detected at DSBs is the mediator of DNA-damage checkpoint 1 (MDC1). It also mentions that JMJD1C enhance interactions between MDC1 and RNF8.

The activation and targeted localization of ubiquitin E2 conjugating enzymes could provide a point for regulating ubiquitin-dependent cell functions. Supporting this view, the ubiquitin charging and activation of a class of E2 enzymes has been directly linked to their nuclear import. [ABSTRACT FROM AUTHOR]

In adult gonads, disruption of somatic sexual identity leads to defective gametogenesis and infertility. However, the underlying mechanisms by which somatic signals regulate germline cells to achieve proper gametogenesis remain unclear. In our previous study, we introduced the chinmoSex Transformation (chinmoST) mutant Drosophila testis phenotype as a valuable model for investigating the mechanisms underlying sex maintenance. In chinmoST testes, depletion of the Janus Kinase-Signal Transducer and Activator of Transcription downstream effector Chinmo from somatic cyst stem cells (CySCs) feminizes somatic cyst cells and arrests germline differentiation. Here, we use single-cell RNA sequencing to uncover chinmoST -specific cell populations and their transcriptomic changes during sex transformation. Comparative analysis of intercellular communication networks between wild-type and chinmoST testes revealed disruptions in several soma-germline signaling pathways in chinmoST testes. Notably, the insulin signaling pathway exhibited significant enhancement in germline stem cells (GSCs). Chinmo cleavage under targets and tagmentation (CUT&Tag) assay revealed that Chinmo directly regulates two male sex determination factors, doublesex (dsx) and fruitless (fru), as well as Ecdysone-inducible gene L2 (ImpL2), a negative regulator of the insulin signaling pathway. Further genetic manipulations confirmed that the impaired gametogenesis observed in chinmoST testes was partly contributed by dysregulation of the insulin signaling pathway. In summary, our study demonstrates that somatic sex maintenance promotes normal spermatogenesis through Chinmo-mediated conserved sex determination and the insulin signaling pathway. Our work offers new insights into the complex mechanisms of somatic stem cell sex maintenance and soma-germline communication at the single-cell level. Additionally, our discoveries highlight the potential significance of stem cell sex instability as a novel mechanism contributing to testicular tumorigenesis. [ABSTRACT FROM AUTHOR]

Midbrain dopamine neurons impact neural processing in the prefrontal cortex (PFC) through mesocortical projections. However, the signals conveyed by dopamine projections to the PFC remain unclear, particularly at the single-axon level. Here, we investigated dopaminergic axonal activity in the medial PFC (mPFC) during reward and aversive processing. By optimizing microprism-mediated two-photon calcium imaging of dopamine axon terminals, we found diverse activity in dopamine axons responsive to both reward and aversive stimuli. Some axons exhibited a preference for reward, while others favored aversive stimuli, and there was a strong bias for the latter at the population level. Long-term longitudinal imaging revealed that the preference was maintained in reward- and aversive-preferring axons throughout classical conditioning in which rewarding and aversive stimuli were paired with preceding auditory cues. However, as mice learned to discriminate reward or aversive cues, a cue activity preference gradually developed only in aversive-preferring axons. We inferred the trial-by-trial cue discrimination based on machine learning using anticipatory licking or facial expressions, and found that successful discrimination was accompanied by sharper selectivity for the aversive cue in aversive-preferring axons. Our findings indicate that a group of mesocortical dopamine axons encodes aversive-related signals, which are modulated by both classical conditioning across days and trial-by-trial discrimination within a day. [ABSTRACT FROM AUTHOR]

The article offers information on PTEN protein regarding tumor suppression. It mentions that PTEN protein is a lipid phosphatase that dephosphorylates phosphatidylinositol-3,4,5-triphosphate (PIP3). According to a study, PTEN is modified by the small ubiquitin-related modifier (SUMO). It includes that sumoylation facilitates cooperative binding of PTEN to the plasma membrane.

An intimate relationship exists between the transcriptional coregulator Daxx, SUMO, and PML nuclear bodies. In this issue, provide structural insights into how phosphorylation of Daxx increases its affinity toward SUMOs and functional insights into how enhanced SUMO binding affects Daxx-PML interactions, PML nuclear body localization, and Daxx-mediated repression of genes encoding for antiapoptotic factors. [Copyright &y& Elsevier]

Two articles in a recent issue of Molecular Cell () demonstrate that noncovalent interactions between the SUMO moieties of SUMO-modified PML, and SUMO binding motifs on PML and other PML nuclear-body-associated proteins, affect the assembly of PML nuclear bodies and the recruitment of proteins in and out of these subnuclear structures. [Copyright &y& Elsevier]

The article focuses on the small ubiquitin-like modifier (SUMO) ligase. Ubiquitin-like protein modifiers (Ubls) regulate a host of processes in eukaryotic cells. Ubiquitin, the original Ubl, signals proteolysis and diverse non-proteolytic outcomes. Functional breadth is also characteristic of SUMO. SUMOylation was discovered because it is required to localize RanGAP1, a regulator of nucleocytoplasmic transport, to nuclear pore complexes. Studies of the SUMOylation of RanGAP1 and other substrates have revealed distinguishing features. SUMOylation, unlike ubiquitylation, is frequently site specific. SUMOylation of RanGAPl is frequently observable with just E1 and Ubc9 (the SUMO E2).

The study of stem cells holds immense promise for furthering our understanding of processes such as embryonic development, adult aging, and tumor formation. This is due to their remarkable ability to self-renew, to produce more stem cells and to differentiate into one or more specialized cell types. The mechanism by which stem cells decide either to remain in the niche or to leave it should be a major player in the balancing act between stem cell self-renewal and differentiation. Researchers explore this mechanism in the germline stem cell niche of the Drosophila testis. They find that the stem cells themselves control this process directly by orienting the plane of their division. Surprisingly, this orientation is established by an apparently new method of asymmetric cell division, which could potentially be used in other systems where external signals dictate cell fate.

The molecular events and targets regulated by the RAD6 pathway, which mediates postreplication DNA repair, have remained elusive. Now, ubiquitin and SUMO modification of proliferating cell nuclear antigen (PCNA) is shown to be induced by DNA damage and linked to components of the RAD6 pathway. [Copyright &y& Elsevier]

Misfolded proteins are recognized and degraded through protein quality control (PQC) pathways, which are essential for maintaining proteostasis and normal cellular functions. Defects in PQC can result in disease, including cancer, cardiovascular disease, and neurodegeneration. The small ubiquitin-related modifiers (SUMOs) were previously implicated in the degradation of nuclear misfolded proteins, but their functions in cytoplasmic PQC are unclear. Here, in a systematic screen of SUMO protein mutations in the budding yeast Saccharomyces cerevisiae, we identified a mutant allele (Smt3-K38A/K40A) that sensitizes cells to proteotoxic stress induced by amino acid analogs. Smt3-K38A/K40A mutant strains also exhibited a defect in the turnover of a soluble PQC model substrate containing the CL1 degron (NES-GFP-Ura3-CL1) localized in the cytoplasm, but not the nucleus. Using human U2OS SUMO1- and SUMO2-KO cell lines, we observed a similar SUMOdependent pathway for degradation of the mammalian degron-containing PQC reporter protein, GFP-CL1, also only in the cytoplasm but not the nucleus. Moreover, we found that turnover of GFP-CL1 in the cytoplasm was uniquely dependent on SUMO1 but not the SUMO2 paralogue. Additionally, we showed that turnover of GFP-CL1 in the cytoplasm is dependent on the AAA-ATPase, Cdc48/p97. Cellular fractionation studies and analysis of a SUMO1-GFP-CL1 fusion protein revealed that SUMO1 promotes cytoplasmic misfolded protein degradation by maintaining substrate solubility. Collectively, our findings reveal a conserved and previously unrecognized role for SUMO1 in regulating cytoplasmic PQC and provide valuable insights into the roles of sumoylation in PQCassociated diseases. [ABSTRACT FROM AUTHOR]

Sexual identity is continuously maintained in specific differentiated cell types long after sex determination occurs during development. In the adult Drosophila testis, the putative transcription factor Chronologically inappropriate morphogenesis (Chinmo) acts with the canonical male sex determinant DoublesexM (DsxM) to maintain themale identity of somatic cyst stemcells and their progeny. Herewe find that ectopic expression of chinmo is sufficient to induce a male identity in adult ovarian somatic cells, but it acts through a DsxM - independent mechanism. Conversely, the feminization of the testis somatic stem cell lineage caused by loss of chinmo is enhanced by expression of the canonical female sex determinant DsxF, indicating that chinmo acts in parallel with the canonical sex determination pathway to maintain the male identity of testis somatic cells. Consistent with this finding, ectopic expression of female sex determinants in the adult testis disrupts tissue morphology. The miRNA let-7 downregulates chinmo in many contexts, and ectopic expression of let-7 in the adult testis is sufficient to recapitulate the chinmo loss-of-function phenotype, but we find no apparent phenotypes upon removal of let-7 in the adult ovary or testis. Our finding that chinmo is necessary and sufficient to promote a male identity in adult gonadal somatic cells suggests that the sexual identity of somatic cells can be reprogrammed in the adult Drosophila ovary as well as in the testis. [ABSTRACT FROM AUTHOR]

Stem cells are necessary for the maintenance of many adult tissues. Signals within the stem cell microenvironment, or niche, regulate the self-renewal and differentiation capability of these cells. Misregulation of these signals through mutation or damage can lead to overgrowth or depletion of different stem cell pools. In this review, we focus on the Drosophila testis and ovary, both of which contain well-defined niches, as well as the mouse testis, which has become a more approachable stem cell system with recent technical advances. We discuss the signals that regulate gonadal stem cells in their niches, how these signals mediate self-renewal and differentiation under homeostatic conditions, and how stress, whether from mutations or damage, can cause changes in cell fate and drive stem cell competition. [ABSTRACT FROM AUTHOR]

Tissue-specific stem cells are thought to resist environmental insults better than their differentiating progeny, but this resistance varies from one tissue to another, and the underlying mechanisms are not well-understood. Here, we use the Drosophila testis as a model system to study the regulation of cell death within an intact niche. This niche contains sperm-producing germline stem cells (GSCs) and accompanying somatic cyst stem cells (or CySCs). Although many signals are known to promote stem cell self-renewal in this tissue, including the highly conserved JAK–STAT pathway, the response of these stem cells to potential death-inducing signals, and factors promoting stem cell survival, have not been characterized. Here we find that both GSCs and CySCs resist cell death better than their differentiating progeny, under normal laboratory conditions and in response to potential death-inducing stimuli such as irradiation or starvation. To ask what might be promoting stem cell survival, we characterized the role of the anti-apoptotic gene Drosophila inhibitor of apoptosis 1 ( diap1) in testis stem cells. DIAP1 protein is enriched in the GSCs and CySCs and is a JAK–STAT target. diap1 is necessary for survival of both GSCs and CySCs, and ectopic up-regulation of DIAP1 in somatic cyst cells is sufficient to non-autonomously rescue stress-induced cell death in adjacent differentiating germ cells (spermatogonia). Altogether, our results show that niche signals can promote stem cell survival by up-regulation of highly conserved anti-apoptotic proteins, and suggest that this strategy may underlie the ability of stem cells to resist death more generally. [ABSTRACT FROM AUTHOR]

Summary Local signals maintain adult stem cells in many tissues. Whether the sexual identity of adult stem cells must also be maintained was not known. In the adult Drosophila testis niche, local Jak-STAT signaling promotes somatic cyst stem cell (CySC) renewal through several effectors, including the putative transcription factor Chronologically inappropriate morphogenesis (Chinmo). Here, we find that Chinmo also prevents feminization of CySCs. Chinmo promotes expression of the canonical male sex determination factor DoublesexM (Dsx M ) within CySCs and their progeny, and ectopic expression of DsxM in the CySC lineage partially rescues the chinmo sex transformation phenotype, placing Chinmo upstream of Dsx M . The Dsx homolog DMRT1 prevents the male-to-female conversion of differentiated somatic cells in the adult mammalian testis, but its regulation is not well understood. Our work indicates that sex maintenance occurs in adult somatic stem cells and that this highly conserved process is governed by effectors of niche signals. PaperClip [ABSTRACT FROM AUTHOR]

R4641 --> 486.3 --> Replication‐dependent histone genes encode the only non‐polyadenylated mRNAs in mammals. Histone pre‐mRNAs, which do not contain introns, therefore require only one processing step that involves 3' end recognition and cleavage. Both histone gene expression and pre‐mRNA processing take place in specialized nuclear organelles known as histone locus bodies. Recent studies have revealed that polyadenylation of histone mRNAs does occur, however, under specific physiological conditions. These include during normal terminal cellular differentiation, tumorigenesis, and in disease patients with Aicardi–Goutières syndrome. Despite these links between polyadenylation of histone mRNAs, human physiology, and disease, the triggers, the mechanisms, and the consequences of histone mRNA polyadenylation remain poorly defined. In recent studies of small ubiquitin‐related modifier (SUMO) knockout cell lines, we have obtained evidence that SUMO1 and SUMO2 play roles in regulating histone pre‐mRNA 3' end processing and polyadenylation. This evidence includes alterations in histone locus body size and structure in SUMO1 and SUMO2 knockout cells, as well as mislocalization of key pre‐mRNA processing factors required for normal processing. Collectively, our findings provide new insights into control of histone pre‐mRNA processing that involves posttranslational protein modifications by SUMO1 and SUMO2. [ABSTRACT FROM AUTHOR]

Adult stem cells are maintained in niches, specialized microenvironments that regulate their self-renewal and differentiation. In the adult Drosophila testis stem cell niche, somatic hub cells produce signals that regulate adjacent germline stem cells (GSCs) and somatic cyst stem cells (CySCs). Hub cells are normally quiescent, but after complete genetic ablation of CySCs, they can proliferate and transdifferentiate into new CySCs. Here we find that Epidermal growth factor receptor (EGFR) signaling is upregulated in hub cells after CySC ablation and that the ability of testes to recover from ablation is inhibited by reduced EGFR signaling. In addition, activation of the EGFR pathway in hub cells is sufficient to induce their proliferation and transdifferentiation into CySCs. We propose that EGFR signaling, which is normally required in adult cyst cells, is actively inhibited in adult hub cells to maintain their fate but is repurposed to drive stem cell regeneration after CySC ablation. [ABSTRACT FROM AUTHOR]

In replicating yeast, lysine 63-linked polyubiquitin (polyUb) chains are extended from the ubiquitin moiety of monoubiquitinated proliferating cell nuclear antigen (monoUb-PCNA) by the E2-E3 complex of (Ubcl3-Mms2)-Rad5. This promotes errorfree bypass of DNA damage lesions. The unusual ability of Ubcl3-Mms2 to synthesize unanchored Lys63-linked polyUb chains in vitro allowed us to resolve the individual roles that it and Rad5 play in the catalysis and specificity of PCNA polyubiquitination. We found that Rad5 stimulates the synthesis of free polyUb chains by Ubcl3-Mms2 in part by enhancing the reactivity of the Ubc13∼Ub thiolester bond. Polyubiquitination of monoUb-PCNA was further enhanced by interactions between the N-terminal domain of Rad5 and PCNA. Thus, Rad5 acts both to align monoUb-PCNA with Ub-charged Ubc13 and to stimulate Ub transfer onto Lys63 of a Ub acceptor. We also found that Rad5 interacts with PCNA independently of the number of monoubiquitinated subunits in the trimer and that it binds to both unmodified and monoUb-PCNA with similar affinities. These findings indicate that Rad5-mediated recognition of monoUb-PCNA in vivo is likely to depend upon interactions with additional factors at stalled replication forks. [ABSTRACT FROM AUTHOR]

Abstract: Identifying new targets for SUMO and understanding the function of protein SUMOylation are largely limited by low level of SUMOylation. It was found recently that Ubc9, the SUMO E2 conjugating enzyme, is covalently modified by SUMO at a lysine 14 in the N-terminal alpha helix, and that SUMO-modified Ubc9 has enhanced conjugation activity for certain target proteins containing a SUMO-interacting motif (SIM). Here, we show that, compared to intact Ubc9, the SUMO-Ubc9 fusion protein has higher conjugating activity for SIM-containing targets such as Sp100 and human cytomegalovirus IE2. Assays using an IE2 SIM mutant revealed the requirement of SIM for the enhanced IE2 SUMOylation by SUMO-Ubc9. In pull-down assays with cell extracts, the SUMO-Ubc9 fusion protein bound to more diverse cellular proteins and interacted with some SIM-containing proteins with higher affinities than Ubc9. Therefore, the devised SUMO-Ubc9 fusion will be useful for identifying SIM-containing SUMO targets and producing SUMO-modified proteins. [Copyright &y& Elsevier]

Abstract: Stem cells are defined by the fact that they both self-renew, producing additional stem cells, and generate lineal descendants that differentiate into distinct functional cell types. In Drosophila, a small germline stem cell population is influenced by a complex microenvironment, the stem cell niche, which itself includes a somatic stem cell population. While stem cells are unique, their immediate descendants retain considerable stem cell character as they mitotically amplify prior to differentiation and can be induced to de-differentiate into stem cells. Despite their importance, very few genes are known that are expressed in the stem cells or their early amplifying daughters. We present here whole-genome microarray expression analysis of testes specifically enriched for stem cells, their amplifying daughters, and their niche. These studies have identified a number of loci with highly specific stem cell expression and provide candidate downstream targets of Jak/Stat self-renewal signaling. Furthermore, functional analysis for two genes predicted to be enriched has enabled us to define novel regulators of the germline lineage. The gene list generated in this study thus provides a potent resource for the investigation of stem cell identity and regulation from functional as well as evolutionary perspectives. [Copyright &y& Elsevier]

Abstract: SUMOs (small ubiquitin-related modifiers) are eukaryotic proteins that are covalently conjugated to other proteins and thereby regulate a wide range of important cellular processes. The molecular mechanisms by which SUMO modification influences the functions of most target proteins and cellular processes, however, remain poorly defined. A major obstacle to investigating the effects of SUMO modification is the availability of a system for selectively inducing the modification or demodification of an individual protein. To address this problem, we have developed a procedure using the rapamycin heterodimerizer system. This procedure involves co-expression of rapamycin-binding domain fusion proteins of SUMO and candidate SUMO substrates in living cells. Treating cells with rapamycin induces a tight association between SUMO and a single SUMO substrate, thereby allowing specific downstream effects to be analyzed. Using RanGAP1 as a model SUMO substrate, the heterodimerizer system was used to investigate the molecular mechanism by which SUMO modification targets RanGAP1 from the cytoplasm to nuclear pore complexes (NPCs). Our results revealed a dual role for Ubc9 in targeting RanGAP1 to NPCs: In addition to conjugating SUMO-1 to RanGAP1, Ubc9 is also required to form a stable ternary complex with SUMO-1 modified RanGAP1 and Nup358. As illustrated by our studies, the rapamycin heterodimerizer system represents a novel tool for studying the molecular effects of SUMO modification. [Copyright &y& Elsevier]

A common inherited cause of renal failure, autosomal dominant polycystic kidney disease results from mutations in either of two genes, PKD1 and PKD2, which encode polycystin-1 and polycystin-2, respectively . Polycystin-2 has distant homology to TRP cation channels and associates directly with polycystin-1 . The normal functions of polycystins are poorly understood, although recent studies indicate that they are concentrated in the primary cilia of a variety of cell types . In this report we identified a polycystin-2 homolog in Drosophila melanogaster; this homolog localized to the distal tip of the sperm flagella. A targeted mutation in this gene, almost there (amo), caused nearly complete male sterility. The amo males produced and transferred normal amounts of motile sperm to females, but mutant sperm failed to enter the female sperm storage organs, a prerequisite for fertilization. The finding that Amo functions in sperm flagella supports a common and evolutionarily conserved role for polycystin-2 proteins in both motile and nonmotile axonemal-containing structures. [Copyright &y& Elsevier]

The PIAS (protein inhibitors of activated STAT) family of proteins were first discovered as inhibitors of activated signal transducers and activators of transcription (STATs). More recently these proteins have been shown to function as E3 ligases that promote the SUMO modification of a number of transcription regulators. We have investigated the relationship between the effects of PIAS proteins on STAT1 transcriptional activity and the ability of the PIAS proteins to function as SUMO E3 ligases. We demonstrate that STAT1 is a substrate for SUMO modification and that PIASx-α, but not PIAS1, functions as an E3 ligase to promote STAT1 modification. In addition, we have mapped the major site for SUMO modification on STAT1 to lysine 703. This lysine residue is in close proximity to the regulatory tyrosine residue at position 701, whose phosphorylation mediates STAT1 activation in response to cytokine signaling. Mutation of lysine 703 to arginine abolishes SUMO modification of STAT1 both in vitro and in vivo. However, this mutation does not affect the activation of STAT1 or the ability of either PIAS1 or PIASx-α to function as an inhibitor of STAT1-mediated transcription activation. Our findings demonstrate that inhibition of STAT1 by PIAS proteins does not require SUMO modification of STAT1 itself. SUMO modification of STAT1 may nonetheless be functionally important given the close proximity between the SUMO modification site and tyrosine 701. [ABSTRACT FROM AUTHOR]

Reports on the structural characterization of a complex between ubiquitin-conjugating enzyme Ubc9 and RanGAP1 to determine the molecular basis for E2-dependent transfer mechanisms and to elaborate interactions utilized by Ubc9 for substrate recognition. Regulation of nuclear transport, stress response and signal transduction in eukaryotes by the small ubiquitin-like modifier SUMO.

Keratin 17 (KRT17; K17), a non-lamin intermediate filament protein, was recently found to occur in the nucleus. We report here on K17-dependent differences in nuclear morphology, chromatin organization, and cell proliferation. Human tumor keratinocyte cell lines lacking K17 exhibit flatter nuclei relative to normal. Re-expression of wild-type K17, but not a mutant form lacking an intact nuclear localization signal (NLS), rescues nuclear morphology in KRT17-null cells. Analyses of primary cultures of skin keratinocytes from a mouse strain expressing K17 with a mutated NLS corroborated these findings. Proteomics screens identified K17-interacting nuclear proteins with known roles in gene expression, chromatin organization and RNA processing. Key histone modifications and LAP2β (an isoform encoded by TMPO) localization within the nucleus are altered in the absence of K17, correlating with decreased cell proliferation and suppression of GLI1 target genes. Nuclear K17 thus impacts nuclear morphology with an associated impact on chromatin organization, gene expression, and proliferation in epithelial cells. [ABSTRACT FROM AUTHOR]

Sumoylation regulates a wide range of essential cellular functions through diverse mechanisms that remain to be fully understood. Using S. cerevisiae, a model organism with a single essential SUMO gene (SMT3), we developed a library of >250 mutant strains with single or multiple amino acid substitutions of surface or core residues in the Smt3 protein. By screening this library using plate-based assays, we have generated a comprehensive structure-function based map of Smt3, revealing essential amino acid residues and residues critical for function under a variety of genotoxic and proteotoxic stress conditions. Functionally important residues mapped to surfaces affecting Smt3 precursor processing and deconjugation from protein substrates, covalent conjugation to protein substrates, and non-covalent interactions with E3 ligases and downstream effector proteins containing SUMO-interacting motifs. Lysine residues potentially involved in formation of polymeric chains were also investigated, revealing critical roles for polymeric chains, but redundancy in specific chain linkages. Collectively, our findings provide important insights into the molecular basis of signaling through sumoylation. Moreover, the library of Smt3 mutants represents a valuable resource for further exploring the functions of sumoylation in cellular stress response and other SUMO-dependent pathways. [ABSTRACT FROM AUTHOR]