Lela Stefanovic

Destabilization of Yeast Micro- and Minisatellite DNA Sequences by Mutations Affecting a Nuclease Involved in Okazaki Fragment Processing (rad27) and DNA Polymerase δ (pol3-t)

ABSTRACT We examined the effects of mutations in the Saccharomyces cerevisiae RAD27 (encoding a nuclease involved in the processing of Okazaki fragments) and POL3 (encoding DNA polymerase δ) genes on the stability of a minisatellite sequence (20-bp repeats) and microsatellites (1- to 8-bp repeat units). Both therad27 and pol3-t mutations destabilized both classes of repeats, although the types of tract alterations observed in the two mutant strains were different. The tract alterations observed in rad27 strains were primarily additions, and those observed in pol3-t strains were primarily deletions. Measurements of the rates of repetitive tract alterations in strains with both rad27 and pol3-t indicated that the stimulation of microsatellite instability by rad27 was reduced by the effects of the pol3-t mutation. We also found that rad27 and pol3-01 (an allele carrying a mutation in the “proofreading” exonuclease domain of DNA polymerase δ) mutations were synthetically lethal.

Increased rates of genomic deletions generated by mutations in the yeast gene encoding DNA polymerase delta or by decreases in the cellular levels of DNA polymerase delta.

ABSTRACT In Saccharomyces cerevisiae, POL3 encodes the catalytic subunit of DNA polymerase δ. While yeastPOL3 mutant strains that lack the proofreading exonuclease activity of the polymerase have a strong mutator phenotype, little is known regarding the role of other Pol3p domains in mutation avoidance. We identified a number of pol3 mutations in regions outside of the exonuclease domain that have a mutator phenotype, substantially elevating the frequency of deletions. These deletions appear to reflect an increased frequency of DNA polymerase slippage. In addition, we demonstrate that reduction in the level of wild-type DNA polymerase results in a similar mutator phenotype. Lowered levels of DNA polymerase also result in increased sensitivity to the DNA-damaging agent methyl methane sulfonate. We conclude that both the quantity and the quality of DNA polymerase δ is important in ensuring genome stability.

Direct Hepatotoxic Effect of KC Chemokine in the Liver Without Infiltration of Neutrophils

KC is a mouse homolog of human chemokine gro-α (CXCL1), expression of which is increased in liver diseases. We show that activated, but not quiescent, hepatic stellate cells (HSCs) express KC. Hepatic stellate cells constitutively express the KC receptor, CXCR2. Addition of recombinant KC to HSCs undergoing activation in culture Increases secretion and processing of Type I collagen. Overexpression of endogenous KC in the mouse liver could be achieved by an intraperitoneal injection CCl4 followed after 24 hrs by an injection of recombinant KC into circulation. This protocol resulted in about a 14-fold increase in concentration of KC protein in the liver. Overexpression of KC was associated with upregulation of the mRNA for CXCR2 and MIP-2 and with necrosis and increased synthesis of Type I collagen. This suggests that KC has a direct hepatotoxic effect, which led to a massive liver necrosis after 48 hrs. No accumulation of neutrophils was seen in the livers as judged by histology and reverse transcriptase-polymerase chain reaction analysis of myeloperoxidase mRNA. Autostimulation of KC and CXCR2 expression by recombinant KC protein in the mice with preexisting liver injury indicates a positive feedback regulation. Such regulation and direct hepatotoxicity of KC with increased collagen synthesis represent novel findings about the role of KC/gro-α in liver pathology.

TRAM2 Protein Interacts with Endoplasmic Reticulum Ca2+ Pump Serca2b and Is Necessary for Collagen Type I Synthesis

ABSTRACT Cotranslational insertion of type I collagen chains into the lumen of the endoplasmic reticulum (ER) and their subsequent folding into a heterotrimeric helix is a complex process which requires coordinated action of the translation machinery, components of translocons, molecular chaperones, and modifying enzymes. Here we describe a role for the protein TRAM2 in collagen type I expression in hepatic stellate cells (HSCs) and fibroblasts. Activated HSCs are collagen-producing cells in the fibrotic liver. Quiescent HSCs produce trace amounts of type I collagen, while upon activation collagen synthesis increases 50- to 70-fold. Likewise, expression of TRAM2 dramatically increases in activated HSCs. TRAM2 shares 53% amino acid identity with the protein TRAM, which is a component of the translocon. However, TRAM2 has a C terminus with only a 15% identity. The C-terminal part of TRAM2 interacts with the Ca2+ pump of the ER, SERCA2b, as demonstrated in a Saccharomyces cerevisiae two-hybrid screen and by immunoprecipitations in human cells. TRAM2 also coprecipitates with anticollagen antibody, suggesting that these two proteins interact. Deletion of the C-terminal part of TRAM2 inhibits type I collagen synthesis during activation of HSCs. The pharmacological inhibitor of SERCA2b, thapsigargin, has a similar effect. Depletion of ER Ca2+ with thapsigargin results in inhibition of triple helical collagen folding and increased intracellular degradation. We propose that TRAM2, as a part of the translocon, is required for the biosynthesis of type I collagen by coupling the activity of SERCA2b with the activity of the translocon. This coupling may increase the local Ca2+ concentration at the site of collagen synthesis, and a high Ca2+ concentration may be necessary for the function of molecular chaperones involved in collagen folding.

Isolation and Characterization of Point Mutations in Mismatch Repair Genes That Destabilize Microsatellites in Yeast

ABSTRACT The stability of simple repetitive DNA sequences (microsatellites) is a sensitive indicator of the ability of a cell to repair DNA mismatches. In a genetic screen for yeast mutants with elevated microsatellite instability, we identified strains containing point mutations in the yeast mismatch repair genes, MSH2,MSH3, MLH1, and PMS1. Some of these mutations conferred phenotypes significantly different from those of null mutations in these genes. One semidominant MSH2mutation was identified. Finally we showed that strains heterozygous for null mutations of mismatch repair genes in diploid strains in yeast confer subtle defects in the repair of small DNA loops.

Coming together: liver fibrosis, collagen mRNAs and the RNA binding protein.

Liver fibrosis is characterized by the excessive and uncontrolled production of type I collagen by activated hepatic stellate cells (HSCs). Major complications of liver fibrosis are caused by the deposition of type I collagen; however, most current research in liver fibrosis is directed towards understanding the activation of HSCs rather than the mechanism of collagen synthesis. Current models of the synthesis of type I collagen postulate that the procollagen α1(I) and α2(I) polypeptides are synthesized separately, and independently post-translationally modified within the lumen of the endoplasmic reticulum. Two α1(I) peptides and one α2(I) peptide then find each other and fold into a triple helix. However, two facts contradict this simplified model. First, more than 99% of naturally synthesized type I collagen is comprised of heterotrimers of two α1(I) chains and one α2(I) chain; homotrimers of α1(I) chains almost never form [1]. However, collagen α1(I) homotrimers readily form triple helices in humans who have a complete absence of the α2(I) chain [2] and in knockout mice where the gene coding for α2(I) has been inactivated [3]. Therefore, α1(I) chains have the propensity for folding into a functional triple helix, and a certain fraction of homotrimers would form if the interactions between the α1(I) and α2(I) polypeptides are not strictly coordinated. Second, the rate of post-translational modifi cations and the rate of folding into triple helices are coupled, since the mutations that delay folding result in hypermodifications of the chains and severe forms of osteogenesis imperfecta [4,5]. Therefore, some coordination of synthesis of the procollagen α1(I) and α2(I) polypeptides must take place. It’s a competitive world In mammalian cells, there is an excess of mRNAs, beyond the capacity of the translational machinery. This means that multiple mRNAs compete for a limited number of ribosomes. Features at the 5 ́ untranslated region determine the competitiveness of an mRNA; secondary structures are inhibitory and the translation start codon must be in the optimal sequence context for efficient initiation [6]. In the 5 ́ untranslated regions of mRNAs encoding the collagen α1(I) and α2(I) polypeptides, there is a stemloop structure encompassing the translation start codon [7,8]. There has been an evolutionary pressure to maintain the 5 ́ stem loop and the sequence around the start codon was sacrificed to maintain the stem loop. Thus, it seems that mRNAs coding for type I collagen are designed to be ineffi ciently translated, containing a secondary structure and a bad start codon. How then, can activated HSCs synthesize large amounts of type I collagen protein from the mRNAs that are poor messages?

Characterization of binding of LARP6 to the 5’ stem-loop of collagen mRNAs: Implications for synthesis of type I collagen

Type I collagen is composed of 2 polypeptides, α1(I) and α2(I), which fold into triple helix. Collagen α1(I) and α2(I) mRNAs have a conserved stem-loop structure in their 5’ UTRs, the 5’SL. LARP6 binds the 5’SL to regulate type I collagen expression. We show that 5 nucleotides within the single stranded regions of 5’SL contribute to the high affinity of LARP6 binding. Mutation of individual nucleotides abolishes the binding in gel mobility shift assay. LARP6 binding to 5’SL of collagen α2(I) mRNA is more stable than the binding to 5’SL of α1(I) mRNA, although the equilibrium binding constants are similar. The more stable binding to α2(I) mRNA may favor synthesis of the heterotrimeric type I collagen. LARP6 needs 2 domains to contact 5’SL, the La domain and the RRM. T133 in the La domain is critical for folding of the protein, while loop 3 in the RRM is critical for binding 5’SL. Loop 3 is also involved in the interaction of LARP6 and protein translocation channel SEC61. This interaction is essential for type I collagen synthesis, because LARP6 mutant which binds 5’SL but which does not interact with SEC61, suppresses collagen synthesis in a dominant negative manner. We postulate that LARP6 directly targets collagen mRNAs to the SEC61 translocons to facilitate coordinated translation of the 2 collagen mRNAs. The unique sequences of LARP6 identified in this work may have evolved to enable its role in type I collagen biosynthesis.

Role of cytokine receptor-like factor 1 in hepatic stellate cells and fibrosis

AIM To elucidate the role of cytokine receptor-like factor 1 (CRLF1) in hepatic stellate cells and liver fibrosis. METHODS Rat hepatic stellate cells (HSCs) were isolated by Nykodenz gradient centrifugation and activated by culturing in vitro. Differentially expressed genes in quiescent and culture activated HSCs were identified using microarrays. Injections of carbon tetrachloride (CCl(4)) for 4 wk were employed to induce liver fibrosis. The degree of fibrosis was assessed by Sirius red staining. Adenovirus expressing CRLF1 was injected through tail vein into mice to achieve overexpression of CRLF1 in the liver. The same adenovirus was used to overexpress CRLF1 in quiescent HSCs cultured in vitro. Expression of CRLF1, CLCF1 and ciliary neurotrophic factor receptor (CNTFR) in hepatic stellate cells and fibrotic livers was analyzed by semi-quantitative reverse transcription-polymerase chain reaction and Western blotting. Expression of profibrotic cytokines and collagens was analyzed by the same method. RESULTS CRLF1 is a secreted cytokine with unknown function. Human mutations suggested a role in development of autonomous nervous system and a role of CRLF1 in immune response was implied by its similarity to interleukin (IL)-6. Here we show that expression of CRLF1 was undetectable in quiescent HSCs and was highly upregulated in activated HSCs. Likewise, expression of CRLF1 was very low in normal livers, but was highly upregulated in fibrotic livers, where its expression correlated with the degree of fibrosis. A cofactor of CLRF1, cardiotrophin-like cytokine factor 1 (CLCF1), and the receptor which binds CRLF1/CLCF1 dimer, the CNTFR, were expressed to similar levels in quiescent and activated HSCs and in normal and fibrotic livers, indicating a constitutive expression. Overexpression of CLRF1 alone in the normal liver did not stimulate expression of profibrotic cytokines, suggesting that the factor itself is not pro-inflammatory. Ectopic expression in quiescent HSCs, however, retarded their activation into myofibroblasts and specifically decreased expression of type III collagen. Inhibition of type III collagen expression by CRLF1 was also seen in the whole liver. Our results suggest that CLRF1 is the only component of the CRLF1/CLCF1/CNTFR signaling system that is inducible by a profibrotic stimulus and that activation of this system by CLRF1 may regulate expression of type III collagen in fibrosis. CONCLUSION By regulating activation of HSCs and expression of type III collagen, CRLF1 may have an ability to change the composition of extracellular matrix in fibrosis.