Are insect immune suppressors driving cellular uptake reactions?

Many insect parasitoids that deposit their eggs inside immature stages of other insect species inactivate the cellular host defence to protect the growing embryo from encapsulation. Suppression of encapsulation by polydnavirus-encoded immune-suppressors correlates with specific alterations in hemocytes, mainly cytoskeletal rearrangements and actin-cytoskeleton breakdown. We have previously shown that the Cotesia rubecula polydnavirus gene product CrV1 causes immune suppression when injected into the host hemocoel. CrV1 is taken up by hemocytes although no receptors have been found to bind the protein. Instead CrV1 uptake depends on dimer formation, which is required for interacting with lipophorin, suggesting a CrV1-lipophorin complex internalisation by hemocytes. Since treatment of hemocytes with oligomeric lectins and cytochalasin D can mimic the effects of CrV1, we propose that some dimeric and oligomeric adhesion molecules are able to cross-link receptors on the cell surface and depolymerise actin by leverage-mediated clearance reactions in the hemolymph.     

De novo DNA methyltransferases: oncogenes, tumor suppressors, or both?

Aberrant promoter DNA hypermethylation of tumor suppressor genes is a hallmark of cancer. This alteration is largely dependent on the action of de novo DNA methyltransferases (DNMTs) early during tumor progression, which supports the oncogenic role for these enzymes. However, recent research has identified several inactivating mutations of de novo DNMTs in various types of tumor. In addition, it has been shown that loss of de novo DNA methylation activity at advanced tumor stages leads to the promoter DNA demethylation-dependent expression of specific oncogenes. These new data support the notion that de novo DNMTs also have an important role in the maintenance of DNA methylation and suggest that, in addition to acting as oncogenes, they also behave as tumor suppressors. This potential dual role might have clinical implications, as DNMTs are currently considered bona fide targets in cancer therapy.     

MicroRNAs: Oncogenes,tumor suppressors or master regulators of cancer heterogeneity?

The realization that microRNAs are intimately linked to cancer pathogenesis has spawned an explosion of research activity in recent years. Their presence is not merely predictive of tumor origin and behavior, they are causally linked to the emergence and development of cancer by acting as oncogenes or tumor suppressors. The understanding of the functional consequences of altered microRNA expression in cancer is progressing rapidly, even though the prediction of microRNA targets is still a hit and miss process. MicroRNAs may not act primarily by strongly reducing the expression of a few prominent cancer-regulatory genes, but by influencing the properties of the network of which these regulators are a central part. By coordinately regulating many genes, microRNAs are exquisitely suited to act as stabilizers of networks and to prevent extreme variations in phenotype due to intrinsic and extrinsic disturbances. Many advanced tumors show defects in microRNA expression and processing, which could increase phenotypic variability within tumors. This allows small subsets of cells with altered characteristics to emerge, which can have grave consequences as typically a small fraction of tumor cells is responsible for metastasis and treatment resistance, and ultimately treatment failure. Investigating microRNAs from the perspective of master regulators of network stability in cancer calls for new experimental approaches and may help to understand causes of cancer heterogeneity and disease progression.     

Multiple global suppressors of protein stability defects facilitate the evolution of extended-spectrum TEM β-lactamases

The introduction of extended-spectrum cephalosporins and β-lactamase inhibitors has driven the evolution of extended-spectrum β-lactamases (ESBLs) that possess the ability to hydrolyze these drugs. The evolved TEM ESBLs from clinical isolates of bacteria often contain substitutions that occur in the active site and alter the catalytic properties of the enzyme to provide an increased hydrolysis of extended-spectrum cephalosporins or an increased resistance to inhibitors. These active-site substitutions often result in a cost in the form of reduced enzyme stability. The evolution of TEM ESBLs is facilitated by mutations that act as global suppressors of protein stability defects in that they allow the enzyme to absorb multiple amino acid changes despite incremental losses in stability associated with the substitutions. The best-studied example is the M182T substitution, which corrects protein stability defects and is commonly found in TEM ESBLs or inhibitor-resistant variants from clinical isolates. In this study, a genetic selection for second-site mutations that could partially restore function to a severely destabilized primary mutant enabled the identification of A184V, T265M, R275Q, and N276D, which are known to occur in TEM ESBLs from clinical isolates, as suppressors of TEM-1 protein stability defects. Further characterization demonstrated that these substitutions increased the thermal stability of TEM-1 and were able to correct the stability defects of two different sets of destabilizing mutations. The acquisition of compensatory global suppressors of stability costs associated with active-site mutations may be a common mechanism for the evolution of novel protein function.     

The inositol polyphosphate 5-phosphatases: traffic controllers, waistline watchers and tumour suppressors?

Phosphoinositide signals regulate cell proliferation, differentiation, cytoskeletal rearrangement and intracellular trafficking. Hydrolysis of PtdIns(4,5)P2 and PtdIns(3,4,5)P3, by inositol polyphosphate 5-phosphatases regulates synaptic vesicle recycling (synaptojanin-1), hematopoietic cell function [SHIP1(SH2-containing inositol polyphosphate 5-phosphatase-1)], renal cell function [OCRL (oculocerebrorenal syndrome of Lowe)] and insulin signalling (SHIP2). We present here a detailed review of the characteristics of the ten mammalian 5-phosphatases. Knockout mouse phenotypes and underexpression studies are associated with significant phenotypic changes, indicating non-redundant roles, despite, in many cases, overlapping substrate specificity and tissue expression. The extraordinary complexity in the control of phosphoinositide signalling continues to be revealed.     

Can yeast be used to study mitochondrial diseases? Biolistic tRNA mutants for the analysis of mechanisms and suppressors

Base substitutions equivalent to those causing human pathologies have been introduced in yeast mitochondrial tRNA genes. These mutants can be utilized as flexible tools to investigate the molecular aspects of mitochondrial diseases and identify correcting genes. We show that for all studied tRNA mutations (including an homoplasmic one in tRNA^Val) the severity of phenotypes follows the same trend in four different nuclear backgrounds. Correcting genes include TUF1 and genes encoding aminoacyl-tRNA synthetase. The effect of suppressors was analyzed by Northern blot. Mutated leucyl-tRNA synthetase with highly reduced catalytic activity maintains full suppressing effect, thus suggesting a chaperone-like and/or stabilizing function.     

A novel RNA-binding motif in omnipotent suppressors of translation termination, ribosomal proteins and a ribosome modification enzyme?

Using computer methods for database search, multiple alignment, protein sequence motif analysis and secondary structure prediction, a putative new RNA-binding motif was identified. The novel motif is conserved in yeast omnipotent translation termination suppressor SUP1, the related DOM34 protein and its pseudogene homologue; three groups of eukaryotic and archaeal ribosomal proteins, namely L30e, L7Ae/S6e and S12e; an uncharacterized Bacillus subtilis protein related to the L7A/S6e group; and Escherichia coli ribosomal protein modification enzyme RimK. We hypothesize that a new type of RNA-binding domain may be utilized to deliver additional activities to the ribosome.     

Cytogenetic analysis of variegation suppressors and a dominant temperature-sensitive lethal in region 23–26 of chromosome 2L in Drosophila melanogaster

Three suppressor loci for position-effect variegation, one dominant temperature-sensitive (DTS), three Minute genes, and two recessive visible mutants (ed, tkv) have been cytogenetically localized by using duplications and deficiencies in regions 23-25 of chromosome arm 2L of Drosophila melanogaster. Two of the suppressor loci studied proved to represent haplo-abnormal genes localized in regions 23A6-23F6 and 24E2-25A1, respectively. The third one is a strong triplo-abnormal suppressor mapping in 25F4-26B9 which affects white variegation in wm4h when present in three doses. The l(2)2DTS mutation, which belongs to a group of noncomplementing dominant temperature-sensitive mutations, is localized in the 25A4-B1 region. Furthermore, two Minute genes have been localized in region 24 that are included in Df(2L)M11 and can be separated employing translocation (Y;2)P8 (24E2-4): M(2)LS2 in 24D3-4-24E2-4, and M(2)z in 24E4-5-24F5-7. A third Minute gene (M(2)S1) is localized in 25C3-8-25C9-D1. The usefulness of the isolated chromosomal rearrangements for further genetic studies of region 23-26 is discussed.     

Identification of protein kinase disruptions as suppressors of the calcium sensitivity of S. cerevisiae Δptp2 Δmsg5 protein phosphatase double disruptant

The double disruptant of the S. cerevisiae protein phosphatase (PPase) genes, PTP2 (phosphotyrosine-specific PPase) and MSG5 (phosphotyrosine and phosphothreonine/serine-PPase) causes calcium-sensitive growth (Ca^s). Previous study using Fluorescent-activated cell sorting (FACS) analysis showed that this growth defect with calcium occurs at G1–S transition in the cell cycle. We discovered that six non-essential protein kinase (PKase) disruptions (Δbck1, Δmkk1, Δslt2/Δmpk1, Δmck1, Δssk2 and Δyak1) suppressed the Ca^s-phenotype of the Δptp2 Δmsg5 double disruptant. Bck1p, Mkk1p and Slt2p are components of the mitogen-activated protein kinase (MAPK) cascade of cell wall integrity pathway (Slt2 pathway), and Mck1p is its down regulator. Ssk2p is the MAPK kinase kinase of the high-osmolarity glycerol (HOG) pathway, while Yak1p is a negative regulator for the cAMP-dependent PKA pathway. FACS analysis revealed that only the disruption of Δssk2 and Δyak1 but not Δbck1, Δmkk1, Δslt2 and Δmck1 was able to suppress the delayed G1–S transition, suggesting that suppression of the growth defect is not always accompanied by suppression of the G1–S transition delay. The discovery of these PKases as suppressors revealed that in addition to the previously anticipated Slt2 pathway, HOG, Yak1p and Mck1p regulatory pathways may also be involved in the calcium sensitivity of the Δptp2 Δmsg5 double disruptant.     

lamB mutations in E. coli K12: Growth of λ host range mutants and effect of nonsense suppressors

Summary Over sixty EMS induced mutations affecting gene lamB, presumably the structural gene for the receptor in Escherichia coli K12, were examined for growth of host range mutants and effect of nonsense suppressors. By the first criterion the mutations could be grouped in three classes. Bacteria with class I mutations allow growth of mutants with extended host range (noted h) of the type already described (Appleyard, Mac Gregor and Baird, 1956). Bacteria with class II mutations allow growth of h mutants with still more extended host range (noted hh ^*). No host range mutants of could be found which would grow on bacteria with class III mutations. Using nonsense suppressors it was found that class I and II consist of missense mutations, while class III consists of nonsense mutations. Exceptions are likely to exist (especially in class III) but were not found among the mutations tested. These observations are briefly discussed in terms of outer membrane protein integration and of phage receptor interaction.     

Studies on suppressors of <Emphasis Type="Italic">sav2/shade avoidance 2</Emphasis> revealed altered interaction at the interface of αβ-tubulin intradimer affects microtubule dynamics

Microtubules are highly dynamic cellular structures that are required for many biological processes. Cortical microtubules in plant play crucial roles during cell expansion. Its proper dynamics are required for plant growth and responses to environmental stimuli. Arabidopsis mutants, such as sav2/tub4 ^P287L , display a variety of growth defects, including short and twisting hypocotyls in dark and shade. Both microtubule organization and dynamics are altered in sav2. Here, we have identified a suppressor of sav2 (sus2), which surprisingly contains a missense mutation in another β tubulin gene, TUB6. The mutation results in a L246F substitution in TUB6. It locates at the interface of αβ-intradimer. This mutation partially suppressed the swirling microtubule arrangement in sav2 hypocotyl cells, leading to the partial rescue of sav2 phenotypes. As the mutant behaves as a semi-dominant mutation and the CFP-labeled tub6^L246F can incorporate into microtubules, we propose that the incorporation of tub6^L246F interferes with the normal function of microtubules. tub6 ^L246F single mutant is hypersensitive to drugs disrupting microtubule dynamics, such as colchicine, suggesting the mutation may affect microtubule dynamics. Moreover, we found the colchicine hypersensitivity of tub6^L246F can be suppressed by tub4^P287L, while tub6^L246F interferes with the rescuing effect of EB1 on sav2. As P287 locates around M-loop, which is involved in interactions between microtubule protofilaments, we propose that altered interactions at αβ-intradimer interface may affect microtubule dynamics through M-loop mediated interactions between microtubule protofilaments.