Wu Q et al. (2017) ACS Chem Biol "Hepta-Mutant Staphylococcus aureus Sortase A (SrtA7m) as a Tool for in Vivo Protein Labeling in Caenorhabditis elegans."

Ploegh HL, Truttmann MC, Wu Q

[ACS Chem Biol, 2017]

In vivo protein ligation is of emerging interest as a means of endowing proteins with new properties in a controlled fashion. Tools to site-specifically and covalently modify proteins with small molecules, peptides, or other proteins in living cells are few and far between. Here, we describe the development of a Staphylococcus aureus sortase (SrtA)-based protein ligation approach for site-specific conjugation of fluorescent dyes and ubiquitin (Ub) to modify proteins in Caenorhabditis elegans. Hepta-mutant SrtA (SrtA7m) expressed in C. elegans is functional and supports in vitro sortase reactions in a low-Ca(2+) environment. Feeding SrtA7m-expressing C. elegans with small peptide-based probes such as (Gly)3- biotin or (Gly)3-fluorophores enables in vivo target protein modification. SrtA7m also catalyzes the circularization of suitably modified linear target proteins in vivo and allows the installation of F-box domains on targets to induce their degradation in a ubiquitin-dependent manner. This is a noninvasive method to achieve in vivo protein labeling, protein circularization, and targeted degradation in C. elegans. This technique should improve our ability to monitor and alter the function of intracellular proteins in vivo.

Truttmann MC et al. (2018) Proc Natl Acad Sci U S A "Chaperone AMPylation modulates aggregation and toxicity of neurodegenerative disease-associated polypeptides."

Pincus D, Ploegh HL, Truttmann MC

[Proc Natl Acad Sci U S A, 2018]

Proteostasis is critical to maintain organismal viability, a process counteracted by aging-dependent protein aggregation. Chaperones of the heat shock protein (HSP) family help control proteostasis by reducing the burden of unfolded proteins. They also oversee the formation of protein aggregates. Here, we explore how AMPylation, a posttranslational protein modification that has emerged as a powerful modulator of HSP70 activity, influences the dynamics of protein aggregation. We find that adjustments of cellular AMPylation levels in <i>Caenorhabditis elegans</i> directly affect aggregation properties and associated toxicity of amyloid- (A), of a polyglutamine (polyQ)-extended polypeptide, and of -synuclein (-syn). Expression of a constitutively active <i>C. elegans</i> AMPylase FIC-1(E274G) under its own promoter expedites aggregation of A and -syn, and drastically reduces their toxicity. A deficiency in AMPylation decreases the cellular tolerance for aggregation-prone polyQ proteins and alters their aggregation behavior. Overexpression of FIC-1(E274G) interferes with cell survival and larval development, underscoring the need for tight control of AMPylase activity in vivo. We thus define a link between HSP70 AMPylation and the dynamics of protein aggregation in neurodegenerative disease models. Our results are consistent with a cytoprotective, rather than a cytotoxic, role for such protein aggregates.

van der Voorn L et al. (1988) Worm Breeder's Gazette "characterization of G-proteins in C elegans."

Ploegh HL, van der Voorn L, Plasterk RHA

[Worm Breeder's Gazette, 1988]

Guanine nucleotide-binding regulatory proteins (G-proteins) are a family of proteins involved in receptor-mediated signal transduction. G-proteins are heterotrimers, composed of alpha-, - and gamma- subunits. The alpha- subunit, which binds GTP, is specific for each G- protein, whereas common - and gamma- subunits may be found in some G- proteins. Subspecies of the alpha- subunit can be distinguished by their characteristic susceptibility to ADP-ribosylation catalyzed by cholera toxin and/or pertussis toxin. We carried out ADP-ribosylation on a crude membrane fraction prepared from C. elegans and found a polypeptide of 40 kD specifically labelled. Moreover, antisera raised in rabbits against purified G-proteins from human and bovine brain, when used in Western blots on extracts from C. elegans, likewise recognized a polypeptide of appr. 40 kD. Combined, these findings argue for the existence of conserved C. elegans G-protein alpha- subunit(s) with remarkable similarity to their bovine and human counterparts. In addition to the alpha-related polypeptides, the same antisera revealed the presence of a C. elegans-polypeptide with a mobility identical to that of authentic human or bovine -subunit. By screening a C. elegans library with a bovine -probe, 12 independent hybridizing clones were isolated. Partial sequencing of one of these clones indicated the presence of a gene highly homogogous to -sequences already known. Over a stretch of 64 amino acids, 83% similarity with the bovine and human -sequences is found (see box), confirming the identity of the C. elegans -subunit gene. Further characterization of the -clones and isolation of clones encoding the C. elegans alpha-subunits is underway. [See Figure 1]

Pie J et al. (2007) Mol Genet Metab "Molecular genetics of HMG-CoA lyase deficiency."

Casals N, Menao S, Lopez-Vinas E, Puisac B, Ramos FJ, Pie J, Casale C, Pie A, Hegardt FG, Gomez-Puertas P

[Mol Genet Metab, 2007]

3-Hydroxy-3-methylglutaryl-CoA lyase (HL) deficiency is a rare autosomal recessive genetic disorder that affects ketogenesis and l-leucine catabolism, which generally appears during the first year of life. Patients with HL deficiency have a reduced capacity to synthesize ketone bodies. The disease is caused by lethal mutations in the HL gene (HMGCL). To date, up to 30 variant alleles (28 mutations and 2 SNPs) in 93 patients have been reported, with a recognizable population-specific mutational spectrum. This disorder is frequent in Saudi Arabia and the Iberian Peninsula (Portugal and Spain), where two mutations (122G&gt;A and 109G&gt;A) have been identified in 87% and 94% of the cases, respectively. In most countries a few patients have a high level of allelic heterogeneity. The mutations are distributed along the gene sequences, although some clustering was observed in exon 2, conforming a possible hot spot. Recently, the crystal structures of the human and two bacterial HL have been published. These experimentally obtained structures confirmed the overall architecture, previously predicted by our group and others using bioinformatic approaches, which shows the (betaalpha)8-barrel structure of the enzyme. In addition, the crystals confirmed the presence of an additional COOH domain containing important structures and residues for enzyme functionality and oligomerization processes. Here, we review all HMGCL mis-sense mutations identified to date, and their implication in enzyme structure and function is discussed. We found that genotype-phenotype correlations are difficult to establish because the evolution of the disease seems more related to the causes of hypoglycaemia (fasting or acute illness) than to a particular genotype.

van der Voorn L et al. (1990) J Mol Biol "Characterization of a G-protein beta-subunit gene from the nematode Caenorhabditis elegans."

Plasterk RHA, van der Voorn L, Gebbink M, Ploegh HL

[J Mol Biol, 1990]

The gene encoding the beta-subunit of guanine nucleotide binding regulatory proteins (G-proteins) has been cloned from the nematode Caenorhabditis elegans. The predicted 340 amino acid sequence matches the highly conserved amino acid sequences of previously isolated G-protein beta-subunits from mammals and Drosophila. The coding region of the C. elegans beta-subunit gene, which has been mapped to the C. elegans chromosome II, is interrupted by eight introns. Southern analysis indicates that C. elegans has only one beta-subunit gene. A 2.8 kb (1 kb = 10(3) bases or base-pairs) transcript derived from this gene could be detected.

Truttmann MC et al. (2016) PLoS Genet "The Caenorhabditis elegans Protein FIC-1 Is an AMPylase That Covalently Modifies Heat-Shock 70 Family Proteins, Translation Elongation Factors and Histones."

Guo X, Ploegh HL, Cruz VE, Truttmann MC, Engert C, Schwartz TU

[PLoS Genet, 2016]

Protein AMPylation by Fic domain-containing proteins (Fic proteins) is an ancient and conserved post-translational modification of mostly unexplored significance. Here we characterize the Caenorhabditis elegans Fic protein FIC-1 in vitro and in vivo. FIC-1 is an AMPylase that localizes to the nuclear surface and modifies core histones H2 and H3 as well as heat shock protein 70 family members and translation elongation factors. The three-dimensional structure of FIC-1 is similar to that of its human ortholog, HYPE, with 38% sequence identity. We identify a link between FIC-1-mediated AMPylation and susceptibility to the pathogen Pseudomonas aeruginosa, establishing a connection between AMPylation and innate immunity in C. elegans.

Truttmann MC et al. (2016) Proc Natl Acad Sci U S A "Unrestrained AMPylation targets cytosolic chaperones and activates the heat shock response."

Truttmann MC, Krakowiak J, Ploegh HL, Zheng X, Pincus D, Hanke L, Grootveld M, Damon JR

[Proc Natl Acad Sci U S A, 2016]

Protein AMPylation is a conserved posttranslational modification with emerging roles in endoplasmic reticulum homeostasis. However, the range of substrates and cell biological consequences of AMPylation remain poorly defined. We expressed human and Caenorhabditis elegans AMPylation enzymes-huntingtin yeast-interacting protein E (HYPE) and filamentation-induced by cyclic AMP (FIC)-1, respectively-in Saccharomyces cerevisiae, a eukaryote that lacks endogenous protein AMPylation. Expression of HYPE and FIC-1 in yeast induced a strong cytoplasmic Hsf1-mediated heat shock response, accompanied by attenuation of protein translation, massive protein aggregation, growth arrest, and lethality. Overexpression of Ssa2, a cytosolic heat shock protein (Hsp)70, was sufficient to partially rescue growth. In human cell lines, overexpression of active HYPE similarly induced protein aggregation and the HSF1-dependent heat shock response. Excessive AMPylation also abolished HSP70-dependent influenza virus replication. Our findings suggest a mode of Hsp70 inactivation by AMPylation and point toward a role for protein AMPylation in the regulation of cellular protein homeostasis beyond the endoplasmic reticulum.

Queliconi BB et al. (2013) Free Radic Biol Med "Bicarbonate modulates oxidative and functional damage in ischemia-reperfusion."

Augusto O, Kowaltowski AJ, Vaz SM, Brookes PS, Marazzi TB, Queliconi BB, Nehrke K

[Free Radic Biol Med, 2013]

The carbon dioxide/bicarbonate (CO(2)/HCO(3)(-)) pair is the main biological pH buffer. However, its influence on biological processes, and in particular redox processes, is still poorly explored. Here we study the effect of CO(2)/HCO(3)(-) on ischemic injury in three distinct models (cardiac HL-1 cells, perfused rat heart, and Caenorhabditis elegans). We found that, although various concentrations of CO(2)/HCO(3)(-) do not affect function under basal conditions, ischemia-reperfusion or similar insults in the presence of higher CO(2)/HCO(3)(-) resulted in greater functional loss associated with higher oxidative damage in all models. Because the effect of CO(2)/HCO(3)(-) was observed in all models tested, we believe this buffer is an important determinant of oxidative damage after ischemia-reperfusion.

Vandenberg LN et al. (2013) Commun Integr Biol "It's never too early to get it Right: A conserved role for the cytoskeleton in left-right asymmetry."

Lemire JM, Vandenberg LN, Levin M

[Commun Integr Biol, 2013]

For centuries, scientists and physicians have been captivated by the consistent left-right (LR) asymmetry of the heart, viscera, and brain. A recent study implicated tubulin proteins in establishing laterality in several experimental models, including asymmetric chemosensory receptor expression in C. elegans neurons, polarization of HL-60 human neutrophil-like cells in culture, and asymmetric organ placement in Xenopus. The same mutations that randomized asymmetry in these diverse systems also affect chirality in Arabidopsis, revealing a remarkable conservation of symmetry-breaking mechanisms among kingdoms. In Xenopus, tubulin mutants only affected LR patterning very early, suggesting that this axis is established shortly after fertilization. This addendum summarizes and extends the knowledge of the cytoskeleton's role in the patterning of the LR axis. Results from many species suggest a conserved role for the cytoskeleton as the initiator of asymmetry, and indicate that symmetry is first broken during early embryogenesis by an intracellular process.

Singh, Ramya et al. (2015) International Worm Meeting "CUP-2, a Derlin protein, promotes Notch signalling in C. elegans."

Hansen, Dave, Racher, Hilary, Kowalchuk, Kevin, Wang, Xin, Singh, Ramya, Sutton, Tiffany

[International Worm Meeting, 2015]

The C. elegans germ line has grown to become an important model system to study the molecular mechanisms regulating the decision made by stem cells to self-renew (proliferate) or to differentiate. The Notch protein, GLP-1, is the principal regulator for promoting self-renewal in the germ line (Austin & Kimble, 1987). This study identifies CUP-2, a Derlin family protein, as a player in regulating the self-renewal vs. differentiation decision. Our results suggest that CUP-2 promotes self-renewal by promoting Notch signalling. cup-2 is shown to promote Notch signalling in both the germ line and the vulva. Loss of CUP-2 activity is able to suppress germline over-proliferation that is due to increased Notch signalling. However, cup-2(0) is unable to suppress a GLP-1 independent germ line tumour, which suggests that CUP-2's influence on proliferation is Notch signalling dependent. This is the first known example of a Derlin protein's influence on Notch signalling. Other studies on CUP-2 have focused on its role as a Derlin in targeting misfolded proteins from the ER (Lilley & Ploegh, 2004) and plasma membrane (Schaheen et al., 2009; Dang et al., 2011) for cytosolic degradation. We believe that CUP-2 may be involved in regulating levels of properly folded Notch receptors on the plasma membrane of cells. .