SUMOylation of Dorsal attenuates Toll/NF-κB signaling.

In Drosophila, Toll/NF-κB signaling plays key roles in both animal development and in host defense. The activation, intensity, and kinetics of Toll signaling are regulated by posttranslational modifications such as phosphorylation, SUMOylation, or ubiquitination that target multiple proteins in the Toll/NF-κB cascade. Here, we have generated a CRISPR-Cas9 edited Dorsal (DL) variant that is SUMO conjugation resistant. Intriguingly, embryos laid by dlSCR mothers overcome dl haploinsufficiency and complete the developmental program. This ability appears to be a result of higher transcriptional activation by DLSCR. In contrast, SUMOylation dampens DL transcriptional activation, ultimately conferring robustness to the dorso-ventral program. In the larval immune response, dlSCR animals show an increase in crystal cell numbers, stronger activation of humoral defense genes, and high cactus levels. A mathematical model that evaluates the contribution of the small fraction of SUMOylated DL (1-5%) suggests that it acts to block transcriptional activation, which is driven primarily by DL that is not SUMO conjugated. Our findings define SUMO conjugation as an important regulator of the Toll signaling cascade, in both development and host defense. Our results broadly suggest that SUMO attenuates DL at the level of transcriptional activation. Furthermore, we hypothesize that SUMO conjugation of DL may be part of a Ubc9-dependent mechanism that restrains Toll/NF-κB signaling.

Caspar, an adapter for VAPB and TER94, modulates the progression of ALS8 by regulating IMD/NFκB-mediated glial inflammation in a Drosophila model of human disease.

Amyotrophic lateral sclerosis (ALS) is a fatal, late-onset, progressive motor neurodegenerative disorder. A key pathological feature of the disease is the presence of heavily ubiquitinated protein inclusions. Both the unfolded protein response and the ubiquitin-proteasome system appear significantly impaired in patients and animal models of ALS. We have studied cellular and molecular mechanisms involved in ALS using a vesicle-associated membrane protein-associated protein B (VAPB/ALS8) Drosophila model [Moustaqim-Barrette, A., Lin, Y.Q., Pradhan, S., Neely, G.G., Bellen, H.J. and Tsuda, H. (2014) The ALS 8 protein, VAP, is required for ER protein quality control. Hum. Mol. Genet., 23, 1975-1989], which mimics many systemic aspects of the human disease. Here, we show that VAPB, located on the cytoplasmic face of the endoplasmic reticulum membrane, interacts with Caspar, an orthologue of human fas associated factor 1 (FAF1). Caspar, in turn, interacts with transitional endoplasmic reticulum ATPase (TER94), a fly orthologue of ALS14 (VCP/p97, valosin-containing protein). Caspar overexpression in the glia extends lifespan and also slows the progression of motor dysfunction in the ALS8 disease model, a phenomenon that we ascribe to its ability to restrain age-dependent inflammation, which is modulated by Relish/NFκB signalling. Caspar binds to VAPB via an FFAT motif, and we find that Caspar's ability to negatively regulate NFκB signalling is not dependent on the VAPB:Caspar interaction. We hypothesize that Caspar is a key molecule in the pathogenesis of ALS. The VAPB:Caspar:TER94 complex appears to be a candidate for regulating both protein homeostasis and NFκB signalling, with our study highlighting a role for Caspar in glial inflammation. We project human FAF1 as an important protein target to alleviate the progression of motor neuron disease.

SUMO conjugation regulates immune signalling.

Post-translational modifications (PTMs) are critical drivers and attenuators for proteins that regulate immune signalling cascades in host defence. In this review, we explore functional roles for one such PTM, the small ubiquitin-like modifier (SUMO). Very few of the SUMO conjugation targets identified by proteomic studies have been validated in terms of their roles in host defence. Here, we compare and contrast potential SUMO substrate proteins in immune signalling for flies and mammals, with an emphasis on NFκB pathways. We discuss, using the few mechanistic studies that exist for validated targets, the effect of SUMO conjugation on signalling and also explore current molecular models that explain regulation by SUMO. We also discuss in detail roles of evolutionary conservation of mechanisms, SUMO interaction motifs, crosstalk of SUMO with other PTMs, emerging concepts such as group SUMOylation and finally, the potentially transforming roles for genome-editing technologies in studying the effect of PTMs.

Lamin A/C and Emerin depletion impacts chromatin organization and dynamics in the interphase nucleus.

BACKGROUND: Nuclear lamins are type V intermediate filament proteins that maintain nuclear structure and function. Furthermore, Emerin - an interactor of Lamin A/C, facilitates crosstalk between the cytoskeleton and the nucleus as it also interacts with actin and Nuclear Myosin 1 (NM1). RESULTS: Here we show that the depletion of Lamin A/C or Emerin, alters the localization of the nuclear motor protein - Nuclear Myosin 1 (NM1) that manifests as an increase in NM1 foci in the nucleus and are rescued to basal levels upon the combined knockdown of Lamin A/C and Emerin. Furthermore, Lamin A/C-Emerin co-depletion destabilizes cytoskeletal organization as it increases actin stress fibers. This further impinges on nuclear organization, as it enhances chromatin mobility more toward the nuclear interior in Lamin A/C-Emerin co-depleted cells. This enhanced chromatin mobility was restored to basal levels either upon inhibition of Nuclear Myosin 1 (NM1) activity or actin depolymerization. In addition, the combined loss of Lamin A/C and Emerin alters the otherwise highly conserved spatial positions of chromosome territories. Furthermore, knockdown of Lamin A/C or Lamin A/C-Emerin combined, deregulates expression levels of a candidate subset of genes. Amongst these genes, both KLK10 (Chr.19, Lamina Associated Domain (LAD+)) and MADH2 (Chr.18, LAD-) were significantly repressed, while BCL2L12 (Chr.19, LAD-) is de-repressed. These genes differentially reposition with respect to the nuclear envelope. CONCLUSIONS: Taken together, these studies underscore a remarkable interplay between Lamin A/C and Emerin in modulating cytoskeletal organization of actin and NM1 that impinges on chromatin dynamics and function in the interphase nucleus.

Adaptation Through Lifestyle Switching Sculpts the Fitness Landscape of Evolving Populations: Implications for the Selection of Drug-Resistant Bacteria at Low Drug Pressures.

Novel genotypes evolve under selection through mutations in pre-existing genes. However, mutations have pleiotropic phenotypic effects that influence the fitness of emerging genotypes in complex ways. The evolution of antimicrobial resistance is mediated by selection of mutations in genes coding for antibiotic-target proteins. Drug-resistance is commonly associated with a fitness cost due to the impact of resistance-conferring mutations on protein function and/or stability. These costs are expected to prohibit the selection of drug-resistant mutations at low drug pressures. Using laboratory evolution of rifampicin resistance in Escherichia coli, we show that when exposed intermittently to low concentration (0.1 × minimal inhibitory concentration) of rifampicin, the evolution of canonical drug resistance was indeed unfavorable. Instead, these bacterial populations adapted by evolving into small-colony variants that displayed enhanced pellicle-forming ability. This shift in lifestyle from planktonic to pellicle-like was necessary for enhanced fitness at low drug pressures, and was mediated by the genetic activation of the fim operon promoter, which allowed expression of type I fimbriae. Upon continued low drug exposure, these bacteria evolved exclusively into high-level drug-resistant strains through mutations at a limited set of loci within the rifampicin-resistance determining region of the rpoB gene. We show that our results are explained by mutation-specific epistasis, resulting in differential impact of lifestyle switching on the competitive fitness of different rpoB mutations. Thus, lifestyle-alterations that are selected at low selection pressures have the potential to modify the fitness effects of mutations, change the genetic structure, and affect the ultimate fate of evolving populations.