Mitotic phosphorylation of the anaphase-promoting complex inhibitory subunit Mnd2 is necessary for efficient progression through meiosis i

The yeast anaphase-promoting complex (APC) subunit Mnd2 is necessary for maintaining sister chromatid cohesion in prophase I of meiosis by inhibiting premature ubiquitination and subsequent degradation of substrates by the APC(Ama1) ubiquitin ligase. In a proteomics screen for post-translational modifications on the APC, we discovered that Mnd2 is phosphorylated during mitosis in a cell cycle-dependent manner. We identified and characterized the sites of mitotic Mnd2 phosphorylation during the cell cycle. Collective mutation of Mnd2 phosphorylation sites to alanine had no effect on vegetative growth but a striking effect (>85% reduction) on the percentage of tetrad-forming cells compared with the wild type strain. Similar to the MND2 deletion strain, cells harboring the alanine mutant that did not form spores arrested after premeiotic S phase with a single undivided nucleus and low levels of the APC(Ama1) meiotic substrate, Clb5, relative to wild type cells. In contrast, collective mutation of Mnd2 phosphorylation sites to aspartic acid resulted in partial suppression of the sporulation defect. No differences were observed in the binding between each Mnd2 isoform and the APC in vitro. However, in vivo, we observed a gradient in the abundance of APC-associated Mnd2 in each strain that was proportional to the observed differences in sporulation and Clb5 levels. Taken together, these data suggest that mitotic phosphorylation of Mnd2 is necessary for APC-mediated progression beyond the first meiotic nuclear division.     

Serotonin increases phosphorylation of synaptic 4EBP through TOR, but eukaryotic initiation factor 4E levels do not limit somatic cap-dependent translation in aplysia neurons

The target of rapamycin (TOR) plays an important role in memory formation in Aplysia californica. Here, we characterize one of the downstream targets of TOR, the eukaryotic initiation factor 4E (eIF4E) binding protein (4EBP) from Aplysia. Aplysia 4EBP contains the four critical phosphorylation sites regulated by TOR as well as an N-terminal RAIP motif and a C-terminal TOS site. Aplysia 4EBP was hypophosphorylated in synaptosomes, and serotonin addition caused a rapamycin-sensitive increase in 4EBP phosphorylation both in synaptosomes and in isolated neurites. Aplysia 4EBP was regulated in a fashion similar to that of mammalian 4EBPs, binding to eIF4E when dephosphorylated and releasing eIF4E after phosphorylation. Overexpression of 4EBP in the soma of Aplysia neurons caused a specific decrease in cap-dependent translation that was rescued by concomitant overexpression of eIF4E. However, eIF4E overexpression by itself did not increase cap-dependent translation, suggesting that increasing levels of free eIF4E by phosphorylating 4EBP is not important in regulating cap-dependent translation in the cell soma. Total levels of eIF4E were also regulated by 4EBP, suggesting that 4EBP can also act as an eIF4E chaperone. These studies demonstrate the conserved nature of 4EBP regulation and its role in cap-dependent translation and suggest differential roles of 4EBP phosphorylation in the soma and synapse.     

Protein translocation through a tunnel induces changes in folding kinetics: a lattice model study

Compaction of a nascent polypeptide chain inside the ribosomal exit tunnel, before it leaves the ribosome, has been proposed to accelerate the folding of newly synthesized proteins following their release from the ribosome. Thus, we used Kinetic Monte Carlo simulations of a minimalist on-lattice model to explore the effect that polypeptide translocation through a variety of channels has on protein folding kinetics. Our results demonstrate that tunnel confinement promotes faster folding of a well-designed protein relative to its folding in free space by displacing the unfolded state towards more compact structures that are closer to the transition state. Since the tunnel only forbids rarely visited, extended configurations, it has little effect on a "poorly designed" protein whose unfolded state is largely composed of low-energy, compact, misfolded configurations. The beneficial effect of the tunnel depends on its width; for example, a too-narrow tunnel enforces unfolded states with limited or no access to the transition state, while a too-wide tunnel has no effect on the unfolded state entropy. We speculate that such effects are likely to play an important role in the folding of some proteins or protein domains in the cellular environment and might dictate whether a protein folds co-translationally or post-translationally.     

Nonsecreted insulin-like growth factor binding protein-3 (IGFBP-3) can induce apoptosis in human prostate cancer cells by IGF-independent mechanisms without being concentrated in the nucleus

Insulin-like growth factor binding protein-3 (IGFBP-3), a secreted protein, has the intrinsic ability to induce apoptosis directly without binding insulin-like growth factors. Previous studies suggested that IGFBP-3 must be secreted to exert its biological functions. IGFBP-3 contains a nuclear localization signal (NLS), and exogenous IGFBP-3 is translocated into the nucleus, suggesting that both secretion and nuclear localization may play important roles in IGFBP-3 action. To address these questions, we fused yellow fluorescent protein (YFP) to mature IGFBP-3 lacking its signal peptide so that it would remain intracellular and mutated the C-terminal NLS of IGFBP-3, (228)KGRKR(232), to MDGEA. Following transfection of PC-3 human prostate cancer cells with these constructs, Western blots indicated that YFP-IGFBP-3 lacking a signal peptide was cell-associated and not present in the extracellular media. Moreover, the fusion protein was not N-glycosylated, indicating that it had not entered the secretory pathway. Confocal imaging showed that intracellular YFP-MDGEA-IGFBP-3 was predominantly cytoplasmic. Transient transfection of nonsecreted YFP-wild-type IGFBP-3 decreased cell viability, as assessed by staining with annexin V followed by flow cytometry. Induction of cell death was caspase-dependent, indicative of apoptosis. Apoptosis also was induced by the nonsecreted NLS mutant (YFP-MDGEA-IGFBP-3) alone and when the IGF-binding site also had been mutated. These results indicate that IGFBP-3 can induce apoptosis in an IGF-independent manner without being secreted or concentrated in the nucleus.     

Structure of the bovine natural resistance associated macrophage protein (NRAMP 1) gene and identification of a novel polymorphism.

The NRAMP 1 gene is a major candidate gene influencing the outcome of infections with intracellular pathogens in numerous species. NRAMP 1 is highly conserved in many mammalian species and the NRAMP 1 gene shows considerable conservation in structure between mice and humans. The association of NRAMP 1 gene polymorphisms with disease in cattle has been limited to a single microsatellite located within the 3'-non coding region of the bovine NRAMP 1 gene. In order to facilitate further studies on this important gene, we now report the nearly complete structure of the bovine NRAMP 1 gene, including sizes and positions of 13 introns relative to the bovine NRAMP 1 gene coding sequence and the DNA sequence of intron-exon junctions. Comparison of the bovine, murine and human NRAMP 1 gene structures revealed a high degree of conservation in intron placement, though the lengths of several introns were less-well conserved. In general, the greatest divergence in intron lengths occurred in regions of the NRAMP 1 gene displaying the lowest coding sequence conservation. In addition, mutations near intron-exon junctions could account for 25 of the 75 total amino acid differences between murine and bovine NRAMP 1. Using information gained through this study, it was possible to rapidly identify a novel polymorphism within the bovine NRAMP 1 gene intron X. This polymorphism was shown by direct DNA sequence analysis to consist of insertion of three guanine nucleotides at positions 37,40 and 98 relative to the intron X start point. Initial scans of several cattle breeds suggest that the two intron X alleles identified here are stable and widespread in the Bos taurus population.     

Clonal Expansion of Normal-Appearing Human Hepatocytes during Chronic Hepatitis B Virus Infection

Chronic hepatitis B virus (HBV) infections are associated with persistent immune killing of infected hepatocytes. Hepatocytes constitute a largely self-renewing population. Thus, immune killing may exert selective pressure on the population, leading it to evolve in order to survive. A gradual course of hepatocyte evolution toward an HBV-resistant state is suggested by the substantial decline in the fraction of infected hepatocytes that occurs during the course of chronic infections. Consistent with hepatocyte evolution, clones of >1,000 hepatocytes develop postinfection in the noncirrhotic livers of chimpanzees chronically infected with HBV and of woodchucks infected with woodchuck hepatitis virus (W. S. Mason, A. R. Jilbert, and J. Summers, Proc. Natl. Acad. Sci. U. S. A. 102:1139-1144, 2005; W. S. Mason et al., J. Virol. 83:8396-8408, 2009). The present study was carried out to determine (i) if extensive clonal expansion of hepatocytes also occurred in human HBV carriers, particularly in the noncirrhotic liver, and (ii) if clonal expansion included normal-appearing hepatocytes, not just hepatocytes that appear premalignant. Host DNA extracted from fragments of noncancerous liver, collected during surgical resection of hepatocellular carcinoma (HCC), was analyzed by inverse PCR for randomly integrated HBV DNA as a marker of expanding hepatocyte lineages. This analysis detected extensive clonal expansion of hepatocytes, as previously found in chronically infected chimpanzees and woodchucks. Tissue sections were stained with hematoxylin and eosin (H&E), and DNA was extracted from the adjacent section for inverse PCR to detect integrated HBV DNA. This analysis revealed that clonal expansion can occur among normal-appearing human hepatocytes.Transient hepatitis B virus (HBV) infections, which generally last <6 months, do not cause cirrhosis and cause only minor increases in the risk of hepatocellular carcinoma (HCC) (3, 46). Chronic infections, typically lifelong, can cause cirrhosis and HCC (3). Of the ∼350 million HBV carriers now alive, ca. 60 million will die prematurely of cirrhosis and/or HCC. Cirrhosis, which usually develops late in infection, is a significant risk factor for HCC. Early reports stated that most HCCs occur on a background of cirrhosis. However, later studies suggested that as many as 50% of HCCs may occur in noncirrhotic liver (4), that is, in patients in whom the progression of liver disease still appears rather mild. Thus, liver damage that appears severe by histologic examination is not a prerequisite for HCC.Interestingly, during chronic HBV infections there is, in the face of persistent viremia, a decline over time in the fraction of infected hepatocytes, from 100% to as little as a few percent (5, 12-14, 16, 17, 22, 23, 27, 34, 37, 38). Along with HCC, this is perhaps the most surprising and unexplained outcome of chronic infection. The timing of this decline has not been systematically studied, but it is presumably gradual, occurring over years or decades, and dependent on persistent, albeit low-level, killing of infected hepatocytes by antiviral cytotoxic T lymphocytes (CTLs) (20). It is believed that the liver is largely a closed, self-renewing population. Such a population might be expected to evolve under any strong or persistent selective pressure. In HBV-infected patients, the earliest and most persistent selective pressure is immune killing of infected hepatocytes, which should initially constitute the entire hepatocyte population. Persistent killing of HBV-infected hepatocytes could lead to clonal expansion of mutant or epigenetically altered hepatocytes that had lost the ability to support infection and that were not, therefore, targeted by antiviral CTLs.Such a selective pressure may explain why foci of altered hepatocytes (FAH) and HCC are typically virus negative (1, 6, 11, 26, 29, 31, 35, 40, 41, 44). Normal or preneoplastic hepatocytes (e.g., in FAH) that have evaded the host immune response should undergo clonal expansion, because their death rate is lower than that of surrounding hepatocytes, even if they do not have a higher growth rate. Indeed, clonal expansion of hepatocytes has been detected, in the absence of cirrhosis, in woodchucks chronically infected with woodchuck hepatitis virus (WHV) (19) and in chimpanzees chronically infected with HBV (21). The presence of discrete foci of normal-appearing but virus-negative hepatocytes in chronically infected woodchuck livers (39) suggested, but did not prove, that normal-appearing hepatocytes that had lost the ability to support virus replication might clonally expand.The purpose of the present study was, therefore, to determine if normal-appearing hepatocytes undergo clonal expansion. To address this issue, we focused on noncirrhotic livers, because hepatocyte appearance and organization in many cirrhotic nodules are often considered to indicate premalignancy (7, 24, 25, 44), and this, together with the cellular environment in the cirrhotic liver, may explain why as many as 50% of cirrhotic nodules have been found to be made up of clonally expanded hepatocytes (2, 18, 24, 25, 28, 44). In older HBV patients, cirrhosis, the result of cumulative scarring due to ongoing tissue injury, presumably produces an evolutionary pressure on the hepatocyte population due to restricted blood flow and altered hepatic architecture.Clonal expansion was detected by assaying for integrated HBV DNA by inverse PCR (19, 21). Because integration occurs at random sites in host DNA, each integration event provides a unique genetic marker for the cell in which it occurred, and for any daughter cells. Thus, the clonal expansion of these tagged hepatocytes can be measured by determining how many times a given virus-cell DNA junction is repeated in a liver fragment. Analysis of fragments of nontumorous liver from noncirrhotic HCC patients revealed that at least 1% of hepatocytes are present as clones of >1,000 cells. Examination of 5-μm-thick sections of paraffin-embedded livers from the same patients revealed that clonally expanded hepatocytes were present in liver sections lacking preneoplastic lesions or changes. Therefore, normal-appearing hepatocytes must have undergone clonal expansion. Although clonal expansion was detected by analysis of integrated HBV DNA, the expansion did not appear to be due to the site of integration of the viral DNA into host DNA.These results are consistent with the hypothesis that immune selection and the later emergence of liver cirrhosis, with altered lobular organization and restricted blood flow, may constitute the two major selective pressures on the hepatocyte population that culminate in hepatocellular carcinoma. More-direct proof of the role, if any, of immune selection in hepatocyte evolution and HCC will require, first of all, an assay with a greater ability to detect clonally expanded hepatocytes. The present approach is limited by a number of factors, including a need for integration near a particular restriction endonuclease cleavage site in host DNA and for conservation of particular viral sequences so that the integrated DNA can be amplified using the PCR primers chosen. These issues may explain why the fraction of clonally expanded hepatocytes reported here is much less than that suggested by histologic data showing that more than 50% of hepatocytes appear negative for virus replication in long-term carriers. Further dissection of this issue will also require localization and determination of the virologic status of hepatocyte clones present in tissue sections.     

A systems-biology analysis of feedback inhibition in the Sho1 osmotic-stress-response pathway

BACKGROUND: A common property of signal transduction systems is that they rapidly lose their ability to respond to a given stimulus. For instance in yeast, the mitogen-activated protein (MAP) kinase Hog1 is activated and inactivated within minutes, even when the osmotic-stress stimulus is sustained. RESULTS: Here, we used a combination of experimental and computational analyses to investigate the dynamic behavior of Hog1 activation in vivo. Computational modeling suggested that a negative-feedback loop operates early in the pathway and leads to rapid attenuation of Hog1 signaling. Experimental analysis revealed that the membrane-bound osmosensor Sho1 is phosphorylated by Hog1 and that phosphorylation occurs on Ser-166. Moreover, Sho1 exists in a homo-oligomeric complex, and phosphorylation by Hog1 promotes a transition from the oligomeric to monomeric state. A phosphorylation-site mutation (Sho1(S166E)) diminishes the formation of Sho1-oligomers, dampens activation of the Hog1 kinase, and impairs growth in high-salt or sorbitol conditions. CONCLUSIONS: These findings reveal a novel phosphorylation-dependent feedback loop leading to diminished cellular responses to an osmotic-stress stimulus.     

Drought constraints on C4 photosynthesis: stomatal and metabolic limitations in C3 and C4 subspecies of Alloteropsis semialata

The C4 photosynthetic pathway uses water more efficiently than the C3 type, yet biogeographical analyses show a decline in C4 species relative to C3 species with decreasing rainfall. To investigate this paradox, the hypothesis that the C4 advantage over C3 photosynthesis is diminished by drought was tested, and the underlying stomatal and metabolic mechanisms of this response determined. The effects of drought and high evaporative demand on leaf gas exchange and photosynthetic electron sinks in C3 and C4 subspecies of the grass Alloteropsis semialata were examined. Plant responses to climatic variation and soil drought were investigated using a common garden experiment with well-watered and natural rainfall treatments, and underlying mechanisms analysed using controlled drying pot experiments. Photosynthetic rates were significantly higher in the C4 than the C3 subspecies in the garden experiment under well-watered conditions, but this advantage was completely lost during a rainless period when unwatered plants experienced severe drought. Controlled drying experiments showed that this loss was caused by a greater increase in metabolic, rather than stomatal, limitations in C4 than in the C3 leaves. Decreases in CO2 assimilation resulted in lower electron transport rates and decreased photochemical efficiency under drought conditions, rather than increased electron transport to alternative sinks. These findings suggest that the high metabolic sensitivity of photosynthesis to severe drought seen previously in several C4 grass species may be an inherent characteristic of the C4 pathway. The mechanism may explain the paradox of why C4 species decline in arid environments despite high water-use efficiency.