SUMO paralogue–specific functions revealed through systematic analysis of human knockout cell lines and gene expression data

The small ubiquitin-related modifiers (SUMOs) regulate nearly every aspect of cellular function, from gene expression in the nucleus to ion transport at the plasma membrane. In humans, the SUMO pathway has five SUMO paralogues with sequence homologies that range from 45% to 97%. SUMO1 and SUMO2 are the most distantly related paralogues and also the best studied. To what extent SUMO1, SUMO2, and the other paralogues impart unique and nonredundant effects on cellular functions, however, has not been systematically examined and is therefore not fully understood. For instance, knockout studies in mice have revealed conflicting requirements for the paralogues during development and studies in cell culture have relied largely on transient paralogue overexpression or knockdown. To address the existing gap in understanding, we first analyzed SUMO paralogue gene expression levels in normal human tissues and found unique patterns of SUMO1–3 expression across 30 tissue types, suggesting paralogue-specific functions in adult human tissues. To systematically identify and characterize unique and nonredundant functions of the SUMO paralogues in human cells, we next used CRISPR-Cas9 to knock out SUMO1 and SUMO2 expression in osteosarcoma (U2OS) cells. Analysis of these knockout cell lines revealed essential functions for SUMO1 and SUMO2 in regulating cellular morphology, promyelocytic leukemia (PML) nuclear body structure, responses to proteotoxic and genotoxic stress, and control of gene expression. Collectively, our findings reveal nonredundant regulatory roles for SUMO1 and SUMO2 in controlling essential cellular processes and provide a basis for more precise SUMO-targeting therapies.     

Clade classification of monolignol biosynthesis gene family members reveals target genes to decrease lignin in Lolium perenne

In monocots, lignin content has a strong impact on the digestibility of the cell wall fraction. Engineering lignin biosynthesis requires a profound knowledge of the role of paralogues in the multigene families that constitute the monolignol biosynthesis pathway. We applied a bioinformatics approach for genome‐wide identification of candidate genes in Lolium perenne that are likely to be involved in the biosynthesis of monolignols. More specifically, we performed functional subtyping of phylogenetic clades in four multigene families: 4CL, COMT, CAD and CCR. Essential residues were considered for functional clade delineation within these families. This classification was complemented with previously published experimental evidence on gene expression, gene function and enzymatic activity in closely related crops and model species. This allowed us to assign functions to novel identified L. perenne genes, and to assess functional redundancy among paralogues. We found that two 4CL paralogues, two COMT paralogues, three CCR paralogues and one CAD gene are prime targets for genetic studies to engineer developmentally regulated lignin in this species. Based on the delineation of sequence conservation between paralogues and a first analysis of allelic diversity, we discuss possibilities to further study the roles of these paralogues in lignin biosynthesis, including expression analysis, reverse genetics and forward genetics, such as association mapping. We propose criteria to prioritise paralogues within multigene families and certain SNPs within these genes for developing genotyping assays or increasing power in association mapping studies. Although L. perenne was the target of the analyses presented here, this functional subtyping of phylogenetic clades represents a valuable tool for studies investigating monolignol biosynthesis genes in other monocot species.     

Discovering new enzymes and metabolic pathways: conversion of succinate to propionate by Escherichia coli

The Escherichia coli genome encodes seven paralogues of the crotonase (enoyl CoA hydratase) superfamily. Four of these have unknown or uncertain functions; their existence was unknown prior to the completion of the E. coli genome sequencing project. The gene encoding one of these, YgfG, is located in a four-gene operon that encodes homologues of methylmalonyl CoA mutases (Sbm) and acyl CoA transferases (YgfH) as well as a putative protein kinase (YgfD/ArgK). We have determined that YgfG is methylmalonyl CoA decarboxylase, YgfH is propionyl CoA:succinate CoA transferase, and Sbm is methylmalonyl CoA mutase. These reactions are sufficient to form a metabolic cycle by which E. coli can catalyze the decarboxylation of succinate to propionate, although the metabolic context of this cycle is unknown. The identification of YgfG as methylmalonyl CoA decarboxylase expands the range of reactions catalyzed by members of the crotonase superfamily.     

beta-oxidation - strategies for the metabolism of a wide variety of acyl-CoA esters

Living organisms are exposed to a number of different fatty acids and their various derivatives arising either via endogenous synthesis or from exogenous sources. These hydrophobic compounds can play specific metabolic, structural or endocrinic functions in the organisms before their elimination, which can be metabolism to CO(2) or to more polar lipid metabolites allowing their excretion. Quantitatively, one of the major pathways metabolizing fatty acids is beta-oxidation, which consists of a set of four reactions operating at the carbons 2 or 3 of acyl-CoA esters and shortening of the acyl-chain. To allow the beta-oxidation of acyl groups with various steric variants to proceed, different strategies have been developed. These strategies include evolution of beta-oxidation enzymes as paralogues showing specificity with respect to either chain-length or modified acyl-chain, metabolic compartmentalization in eukaryotic cells, controlling of substrate transport across membranes, development of auxiliary enzyme systems, acquisition of enzymes with adaptive active sites and recruiting and optimizing enzymes from non-homologous sources allowing them to catalyze a parallel set of reactions with different substrate specificities.     

Identification and biochemical characterization of two functional CMP-sialic acid synthetases in Danio rerio

Sialic acids (Sia) form the nonreducing end of the bulk of cell surface-expressed glycoconjugates. They are, therefore, major elements in intercellular communication processes. The addition of Sia to glycoconjugates requires metabolic activation to CMP-Sia, catalyzed by CMP-Sia synthetase (CMAS). This highly conserved enzyme is located in the cell nucleus in all vertebrates investigated to date, but its nuclear function remains elusive. Here, we describe the identification and characterization of two Cmas enzymes in Danio rerio (dreCmas), one of which is exclusively localized in the cytosol. We show that the two cmas genes most likely originated from the third whole genome duplication, which occurred at the base of teleost radiation. cmas paralogues were maintained in fishes of the Otocephala clade, whereas one copy got subsequently lost in Euteleostei (e.g. rainbow trout). In zebrafish, the two genes exhibited a distinct spatial expression pattern. The products of these genes (dreCmas1 and dreCmas2) diverged not only with respect to subcellular localization but also in substrate specificity. Nuclear dreCmas1 favored N-acetylneuraminic acid, whereas the cytosolic dreCmas2 showed highest affinity for 5-deamino-neuraminic acid. The subcellular localization was confirmed for the endogenous enzymes in fractionated zebrafish lysates. Nuclear entry of dreCmas1 was mediated by a bipartite nuclear localization signal, which seemed irrelevant for other enzymatic functions. With the current demonstration that in zebrafish two subfunctionalized cmas paralogues co-exist, we introduce a novel and unique model to detail the roles that CMAS has in the nucleus and in the sialylation pathways of animal cells.     

Divergence in spatial expression patterns and in response to stimuli of tandem-repeat paralogues encoding a novel class of proline-rich proteins in Oryza sativa

Gene duplication has been recognized as a major route to supply raw sequences for genome novelty in evolution, but the mechanism underlying the retention of duplicate genes is not fully understood yet. Divergence in spatial expression patterns was investigated here and in response to stimuli of the four members of a rice proline-rich protein gene family (OsPRP1), which encode a class of proline-rich proteins. The four paralogues are tandemly organized within a 20 kbp range of chromosome 10 without any interval of other open reading frames and with a median K(S) value of 0.474. These paralogues showed little similarity in their regulatory regions but high conservation in coding regions. Search of an intergenomic cis-element database predicted their promoter regions with divergent cis-element fingerprints. Further expression analyses involving different tissues/organs and nine types of stimuli by a promoter::GUS-fusion strategy revealed that the four paralogues were expressed mainly in vascular cylinders of different organs and showed diversity in tissue/organ specificity and in response to these stimuli, with some overlapping expression. Furthermore, these data show that OsPRP1.2 appeared to inherit most of the functions from their multifunctional progenitor, whereas the other three genes diverged after duplication events. Thus, the retention of paralogues in a multigene family seems to require a more complicated diversification process than originally thought. In addition, the promoter::GUS strategy is a powerful way to explore function divergence of a tandem-repeat gene family.     

A bacterial elongation factor G homologue exclusively functions in ribosome recycling in the spirochaete Borrelia burgdorferi

Translation elongation factor G (EF‐G) in bacteria plays two distinct roles in different phases of the translation system. EF‐G catalyses the translocation of tRNAs on the ribosome in the elongation step, as well as the dissociation of the post‐termination state ribosome into two subunits in the recycling step. In contrast to this conventional view, it has very recently been demonstrated that the dual functions of bacterial EF‐G are distributed over two different EF‐G paralogues in human mitochondria. In the present study, we show that the same division of roles of EF‐G is also found in bacteria. Two EF‐G paralogues are found in the spirochaete Borrelia burgdorferi, EF‐G1 and EF‐G2. We demonstrate that EF‐G1 is a translocase, while EF‐G2 is an exclusive recycling factor. We further demonstrate that B. burgdorferi EF‐G2 does not require GTP hydrolysis for ribosome disassembly, provided that translation initiation factor 3 (IF‐3) is present in the reaction. These results indicate that two B. burgdorferi EF‐G paralogues are close relatives to mitochondrial EF‐G paralogues rather than the conventional bacterial EF‐G, in both their phylogenetic and biochemical features.     

The Evolution of Ribosomal DNA: Divergent Paralogues and Phylogenetic Implications

Although nuclear ribosomal DNA (rDNA) repeats evolve together through concerted evolution, some genomes contain a considerable diversity of paralogous rDNA. This diversity includes not only multiple functional loci but also putative pseudogenes and recombinants. We examined the occurrence of divergent paralogues and recombinants in Gossypium, Nicotiana, Tripsacum, Winteraceae, and Zea ribosomal internal transcribed spacer (ITS) sequences. Some of the divergent paralogues are probably rDNA pseudogenes, since they have low predicted secondary structure stability, high substitution rates, and many deamination-driven substitutions at methylation sites. Under standard PCR conditions, the low stability paralogues amplified well, while many high-stability paralogues amplified poorly. Under highly denaturing PCR conditions (i.e., with dimethylsulfoxide), both low- and high-stability paralogues amplified well. We also found recombination between divergent paralogues. For phylogenetics, divergent ribosomal paralogues can aid in reconstructing ancestral states and thus serve as good outgroups. Divergent paralogues can also provide companion rDNA phylogenies. However, phylogeneticists must discriminate among families of divergent paralogues and recombinants or suffer from muddled and inaccurate organismal phylogenies.     

Function-dependent clustering of orthologues and paralogues of cyclophilins

The 18 kDa archetypal cyclosporin-A binding protein, cyclophilin-A, has multiple paralogues in the human genome. Only 18 of those paralogues have been detected as mRNAs or proteins whose masses vary from 18 to 354 kDa, whereas the functional significance of the open reading frames (ORFs) encoding other paralogues of cyclophilin-A remains unknown. The genomes of Drosophila melanogaster, Caenorhabditis elegans, Arabidopsis thaliana, Schizosaccharomyces pombe, and Saccharomyces cerevisiae encode different numbers of the cyclophilin paralogues, some of which are orthologous to the human cyclophilins. A library of novel algorithms was developed and used for computation of the conservation levels for hydrophobicity and bulkiness profiles, and amino acid compositions (AACs) of 303 aligned sequences of cyclophilins. The majority of the paralogues and orthologues encoded in these 6 genomes differ considerably from each other. Some of the orthologues and paralogues have high correlation coefficients (CCFs) for pairwise compared hydrophobicity and bulkiness profiles, and whose AACs differ to a low degree. Convergence of these three properties of the polypeptide chain and apparent conservation of the typical sequence hallmarks and parameters allowed for the clustering of the functionally related orthologues and paralogues of the cyclophilins. The clustering method allowed for sorting out the cyclophilins into several distinct classes. Analyses of the overlapping clusters of sequences permitted delineation of some hypothetical pathways that might have led to the creation of certain paralogues of cyclophilins in the eukaryotic genomes.     

Both alpha-haemolysin determinants contribute to full virulence of uropathogenic Escherichia coli strain 536

Uropathogenic Escherichia coli strain 536 possesses two intact copies of the alpha-haemolysin determinant localised on distinct pathogenicity islands. The coding regions of the two hlyCABD operons are conserved; however, upstream sequences are entirely dissimilar. Consequently, expression of the encoded toxin molecules in vitro is highly different. On the other hand, the contribution of the individual determinants to the strain's virulence is the same. Isogenic mutants lacking individual hly determinants have a similar increase in LD50 value in a mouse model of urinary tract infection. Mouse lung toxicity as well as in vitro assays reveals a significant decrease in acute cytotoxicity of both mutants in comparison to the parent wild-type strain; however, the two hly mutants do not significantly differ from each other in these respects. Single channel recordings show no difference in electrophysiological characteristics of the pores formed by the individual HlyA molecules on synthetic planar lipid membranes. Nor do the paralogues have any target cell preference in an in vitro cytotoxicity assay. Our data suggest that the two hly paralogues encode identical toxin functions; however, due to different regulation of expression, they participate at distinct stages of the infectious process. Interestingly, the unrelated uropathogenic E. coli strain J96 shares the same two hly alleles, suggesting that acquisition of the two paralogues accorded a selective evolutionary advantage.     

Molecular annotation of ketol-acid reductoisomerases from Streptomyces reveals a novel amino acid biosynthesis interlock mediated by enzyme promiscuity

The 6-phosphogluconate dehydrogenase superfamily oxidize and reduce a wide range of substrates, making their functional annotation challenging. Ketol-acid reductoisomerase (KARI), encoded by the ilvC gene in branched-chain amino acids biosynthesis, is a promiscuous reductase enzyme within this superfamily. Here, we obtain steady-state enzyme kinetic parameters for 10 IlvC homologues from the genera Streptomyces and Corynebacterium, upon eight selected chemically diverse substrates, including some not normally recognized by enzymes of this superfamily. This biochemical data suggested a Streptomyces biosynthetic interlock between proline and the branched-chain amino acids, mediated by enzyme substrate promiscuity, which was confirmed via mutagenesis and complementation analyses of the proC, ilvC1 and ilvC2 genes in Streptomyces coelicolor. Moreover, both ilvC orthologues and paralogues were analysed, such that the relationship between gene duplication and functional diversification could be explored. The KARI paralogues present in S. coelicolor and Streptomyces lividans, despite their conserved high sequence identity (97%), were shown to be more promiscuous, suggesting a recent functional diversification. In contrast, the KARI paralogue from Streptomyces viridifaciens showed selectivity towards the synthesis of valine precursors, explaining its recruitment within the biosynthetic gene cluster of valanimycin. These results allowed us to assess substrate promiscuity indices as a tool to annotate new molecular functions with metabolic implications.     

Tissue- and paralogue-specific functions of acyl-CoA-binding proteins in lipid metabolism in Caenorhabditis elegans

ACBP (acyl-CoA-binding protein) is a small primarily cytosolic protein that binds acyl-CoA esters with high specificity and affinity. ACBP has been identified in all eukaryotic species, indicating that it performs a basal cellular function. However, differential tissue expression and the existence of several ACBP paralogues in many eukaryotic species indicate that these proteins serve distinct functions. The nematode Caenorhabditis elegans expresses seven ACBPs: four basal forms and three ACBP domain proteins. We find that each of these paralogues is capable of complementing the growth of ACBP-deficient yeast cells, and that they exhibit distinct temporal and tissue expression patterns in C. elegans. We have obtained loss-of-function mutants for six of these forms. All single mutants display relatively subtle phenotypes; however, we find that functional loss of ACBP-1 leads to reduced triacylglycerol (triglyceride) levels and aberrant lipid droplet morphology and number in the intestine. We also show that worms lacking ACBP-2 show a severe decrease in the β-oxidation of unsaturated fatty acids. A quadruple mutant, lacking all basal ACBPs, is slightly developmentally delayed, displays abnormal intestinal lipid storage, and increased β-oxidation. Collectively, the present results suggest that each of the ACBP paralogues serves a distinct function in C. elegans.     

An Atg4B mutant hampers the lipidation of LC3 paralogues and causes defects in autophagosome closure

In the process of autophagy, a ubiquitin-like molecule, LC3/Atg8, is conjugated to phosphatidylethanolamine (PE) and associates with forming autophagosomes. In mammalian cells, the existence of multiple Atg8 homologues (referred to as LC3 paralogues) has hampered genetic analysis of the lipidation of LC3 paralogues. Here, we show that overexpression of an inactive mutant of Atg4B, a protease that processes pro-LC3 paralogues, inhibits autophagic degradation and lipidation of LC3 paralogues. Inhibition was caused by sequestration of free LC3 paralogues in stable complexes with the Atg4B mutant. In mutant overexpressing cells, Atg5- and ULK1-positive intermediate autophagic structures accumulated. The length of these membrane structures was comparable to that in control cells; however, a significant number were not closed. These results show that the lipidation of LC3 paralogues is involved in the completion of autophagosome formation in mammalian cells. This study also provides a powerful tool for a wide variety of studies of autophagy in the future.     

Distinct in vivo dynamics of vertebrate SUMO paralogues

There are three mammalian SUMO paralogues: SUMO-1 is approximately 45% identical to SUMO-2 and SUMO-3, which are 96% identical to each other. It is currently unclear whether SUMO-1, -2, and -3 function in ways that are unique, redundant, or antagonistic. To address this question, we examined the dynamics of individual SUMO paralogues by using cell lines that stably express each of the mammalian SUMO proteins fused to the yellow fluorescent protein (YFP). Whereas SUMO-2 and -3 showed very similar distributions throughout the nucleoplasm, SUMO-1 was uniquely distributed to the nuclear envelope and to the nucleolus. Photobleaching experiments revealed that SUMO-1 dynamics was much slower than SUMO-2 and -3 dynamics. Additionally, the mobility of SUMO paralogues differed between subnuclear structures. Finally, the timing and distributions were dissimilar between paralogues as cells exited from mitosis. SUMO-1 was recruited to nuclear membrane as nuclear envelopes reformed in late anaphase, and accumulated rapidly into the nucleus. SUMO-2 and SUMO-3 localized to chromosome earlier and accumulated gradually during telophase. Together, these findings demonstrate that mammalian SUMO-1 shows patterns of utilization that are clearly discrete from the patterns of SUMO-2 and -3 throughout the cell cycle, arguing that it is functionally distinct and specifically regulated in vivo.     

Vps26A and Vps26B subunits define distinct retromer complexes

The trimeric Vps29-Vps35-Vps26 sub-complex of retromer mediates retrograde transport of transmembrane proteins from endosomes to the trans-Golgi network. Our group has recently identified a Vps26 paralogue, Vps26B, which is able to suppress the expression of Vps26A when exogenously expressed in mammalian cells and defines a distinct retromer complex (Vps26B-retromer) in vivo and in vitro. In this study, we use HEK293 cells stably expressing either Vps26A-myc or Vps26B-myc to address the role of retromer cargo transport and subcellular localization of the two core retromer complexes as defined by the two mammalian-specific Vps26 paralogues. Vps26B-retromer, like Vps26A-retromer, associates with TBC1D5 and GOLPH3. In contrast, no interaction between Vps26B-retromer and cation-independent mannose 6-phosphate receptor (CI-M6PR) was detected, leading to a degradation of this receptor and an increase in cathepsin D secretion. Colocalization of Vps26 paralogues with different endosomally located Rab proteins shows prolonged association of Vps26B-retromer with maturing endosomes relative to Vps26A-retromer. Interestingly, the cycling of CI-M6PR is restored upon deletion of the variable Vps26B C-terminal region indicating that this region is directly responsible for the differential function of the two paralogues. In summary, we show that the two distinct retromer complexes defined by different Vps26 paralogues are not functionally equivalent and that the Vps26B C-terminal region can control cargo selection of the Vps26B-retromer.