The promoter of filamentation (POF1) protein from <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> is an ATPase involved in the protein quality control process

Background

The gene YCL047C, which has been renamed promoter of filamentation gene (POF1), has recently been described as a cell component involved in yeast filamentous growth. The objective of this work is to understand the molecular and biological function of this gene.

Results

Here, we report that the protein encoded by the POF1 gene, Pof1p, is an ATPase that may be part of the Saccharomyces cerevisiae protein quality control pathway. According to the results, Δpof1 cells showed increased sensitivity to hydrogen peroxide, tert-butyl hydroperoxide, heat shock and protein unfolding agents, such as dithiothreitol and tunicamycin. Besides, the overexpression of POF1 suppressed the sensitivity of Δpct1, a strain that lacks a gene that encodes a phosphocholine cytidylyltransferase, to heat shock. In vitro analysis showed, however, that the purified Pof1p enzyme had no cytidylyltransferase activity but does have ATPase activity, with catalytic efficiency comparable to other ATPases involved in endoplasmic reticulum-associated degradation of proteins (ERAD). Supporting these findings, co-immunoprecipitation experiments showed a physical interaction between Pof1p and Ubc7p (an ubiquitin conjugating enzyme) in vivo.

Conclusions

Taken together, the results strongly suggest that the biological function of Pof1p is related to the regulation of protein degradation.

     

Economic evaluation of water loss saving due to the biological control of water hyacinth at New Year’s Dam,Eastern Cape province,South Africa

Water hyacinth Eichhornia crassipes is considered the most damaging aquatic weed in the world. However, few studies have quantified the impact of this weed economically and ecologically, and even fewer studies have quantified the benefits of its control. This paper focuses on water loss saving as the benefit derived from biological control of this plant between 1990 and 2013 at New Year’s Dam, Alicedale, Eastern Cape, South Africa. Estimates of water loss due to evapotranspiration from water hyacinth vary significantly; therefore, the study used three different rates, high, medium and low. A conservative raw agriculture value of R 0.26 per m³ was used to calculate the benefits derived by the water saved. The present benefit and cost values were determined using 10% and 5% discount rates. The benefit/cost ratio at the low evapotranspiration rate was less than one, implying that biological control was not economically viable but, at the higher evapotranspiration rates, the return justified the costs of biological control. However, at the marginal value product of water, the inclusion of the costs of damage to infrastructure, or the adverse effects of water hyacinth on biodiversity, would justify the use of biological control, even at the low transpiration rate.     

The abundance of an invasive freshwater snail Tarebia granifera (Lamarck, 1822) in the Nseleni River,South Africa

The invasive freshwater snail Tarebia granifera (Lamarck, 1822) was first reported in South Africa in 1999 and it has become widespread across the country, with some evidence to suggest that it reduces benthic macroinvertebrate biodiversity. The current study aimed to identify the primary abiotic drivers behind abundance patterns of T. granifera, by comparing the current abundance of the snail in three different regions, and at three depths, of the highly modified Nseleni River in KwaZulu-Natal, South Africa. Tarebia granifera was well established throughout the Nseleni River system, with an overall preference for shallow waters and seasonal temporal patterns of abundance. Although it is uncertain what the ecological impacts of the snail in this system are, its high abundances suggest that it should be controlled where possible and prevented from invading other systems in the region.     

Association between TAS2R38 gene polymorphisms and colorectal cancer risk: a case-control study in two independent populations of Caucasian origin

Molecular sensing in the lingual mucosa and in the gastro-intestinal tract play a role in the detection of ingested harmful drugs and toxins. Therefore, genetic polymorphisms affecting the capability of initiating these responses may be critical for the subsequent efficiency of avoiding and/or eliminating possible threats to the organism. By using a tagging approach in the region of Taste Receptor 2R38 (TAS2R38) gene, we investigated all the common genetic variation of this gene region in relation to colorectal cancer risk with a case-control study in a German population (709 controls and 602 cases) and in a Czech population (623 controls and 601 cases). We found that there were no significant associations between individual SNPs of the TAS2R38 gene and colorectal cancer in the Czech or in the German population, nor in the joint analysis. However, when we analyzed the diplotypes and the phenotypes we found that the non-taster group had an increased risk of colorectal cancer in comparison to the taster group. This association was borderline significant in the Czech population, (OR = 1.28, 95% CI 0.99–1.67; P_value = 0.058) and statistically significant in the German population (OR = 1.36, 95% CI 1.06–1.75; P_value = 0.016) and in the joint analysis (OR = 1.34, 95% CI 1.12–1.61; P_value = 0.001). In conclusion, we found a suggestive association between the human bitter tasting phenotype and the risk of CRC in two different populations of Caucasian origin.     

Metal-binding characteristics of the amino-terminal domain of ZntA: binding of lead is different compared to cadmium and zinc

ZntA from Escherichia coli, a P1-type ATPase, specifically transports Pb(II), Zn(II), and Cd(II). Most P1-type ATPases have an N-terminal domain that contains one or more copies of the conserved metal-binding motif, GXXCXXC. In ZntA, the N-terminal domain has approximately 120 residues with a single GXXCXXC motif, as well as four additional cysteine residues as part of the CCCDGAC motif. The metal-binding specificity and affinity of this domain in ZntA was investigated. Isolated proteins, N1-ZntA and N2-ZntA, containing residues 1-111 and 47-111 of ZntA, respectively, were characterized. N1-ZntA has both the CCCDGAC and GXXCXXC motifs, while N2-ZntA has only the GXXCXXC motif. ICP-MS measurements showed that N1-ZntA can bind both divalent metal ions such as Cd(II), Pb(II), and Zn(II) and monovalent metal ions such as Ag(I), with a stoichiometry of 1. N2-ZntA can bind Zn(II) and Cd(II) with a stoichiometry of 1 but not Pb(II). The affinity of N1-ZntA for Zn(II), Pb(II), and Cd(II) was measured by competition titration with metallochromic indicators. Association constants of approximately 10(8) M(-)(1) were obtained for Zn(II), Pb(II), and Cd(II) binding to N1-ZntA. To investigate whether the CCCDGAC sequence has an important role in binding specifically Pb(II), a mutant of ZntA, which lacked the first 46 residues, was constructed. This mutant, Delta46-ZntA, had the same activity as wtZntA with respect to Cd(II) and Zn(II). However, its activity with Pb(II) was similar to the mutant DeltaN-ZntA, which lacks the entire N-terminal domain (Mitra, B., and Sharma, R. (2001) Biochemistry 40, 7694-7699). Thus, binding of Pb(II) appears to involve different ligands, and possibly geometry, compared to Cd(II) and Zn(II).     

Monocarbonyl Analogs of Curcumin with Potential to Treat Colorectal Cancer

Curcumin has a plethora of biological properties, making this compound potentially effective in the treatment of several diseases, including cancer. However, curcumin clinical use is compromised by its poor pharmacokinetics, being crucial to find novel analogs with better pharmacokinetic and pharmacological properties. Here, we aimed to evaluate the stability, bioavailability and pharmacokinetic profiles of monocarbonyl analogs of curcumin. A small library of monocarbonyl analogs of curcumin 1a–q was synthesized. Lipophilicity and stability in physiological conditions were both assessed by HPLC-UV, while two different methods assessed the electrophilic character of each compound monitored by NMR and by UV-spectroscopy. The potential therapeutic effect of the analogs 1a–q was evaluated in human colon carcinoma cells and toxicity in immortalized hepatocytes. Our results showed that the curcumin analog 1e is a promising agent against colorectal cancer, with improved stability and efficacy/safety profile.     

The metal centers of particulate methane monooxygenase from Methylosinus trichosporium OB3b

Particulate methane monooxygenase (pMMO) is a membrane-bound metalloenzyme that oxidizes methane to methanol in methanotrophic bacteria. The nature of the pMMO active site and the overall metal content are controversial, with spectroscopic and crystallographic data suggesting the presence of a mononuclear copper center, a dinuclear copper center, a trinuclear center, and a diiron center or combinations thereof. Most studies have focused on pMMO from Methylococcus capsulatus (Bath). pMMO from a second organism, Methylosinus trichosporium OB3b, has been purified and characterized by spectroscopic and crystallographic methods. Purified M. trichosporium OB3b pMMO contains approximately 2 copper ions per 100 kDa protomer. Electron paramagnetic resonance (EPR) spectroscopic parameters indicate that type 2 Cu(II) is present as two distinct species. Extended X-ray absorption fine structure (EXAFS) data are best fit with oxygen/nitrogen ligands and reveal a Cu-Cu interaction at 2.52 A. Correspondingly, X-ray crystallography of M. trichosporium OB3b pMMO shows a dinuclear copper center, similar to that observed previously in the crystal structure of M. capsulatus (Bath) pMMO. There are, however, significant differences between the pMMO structures from the two organisms. A mononuclear copper center present in M. capsulatus (Bath) pMMO is absent in M. trichosporium OB3b pMMO, whereas a metal center occupied by zinc in the M. capsulatus (Bath) pMMO structure is occupied by copper in M. trichosporium OB3b pMMO. These findings extend previous work on pMMO from M. capsulatus (Bath) and provide new insight into the functional importance of the different metal centers.     

Proteolytic refolding of the HIV-1 capsid protein amino-terminus facilitates viral core assembly.

After budding, the human immunodeficiency virus (HIV) must 'mature' into an infectious viral particle. Viral maturation requires proteolytic processing of the Gag polyprotein at the matrix-capsid junction, which liberates the capsid (CA) domain to condense from the spherical protein coat of the immature virus into the conical core of the mature virus. We propose that upon proteolysis, the amino-terminal end of the capsid refolds into a beta-hairpin/helix structure that is stabilized by formation of a salt bridge between the processed amino-terminus (Pro1) and a highly conserved aspartate residue (Asp51). The refolded amino-terminus then creates a new CA-CA interface that is essential for assembling the condensed conical core. Consistent with this model, we found that recombinant capsid proteins with as few as four matrix residues fused to their amino-termini formed spheres in vitro, but that removing these residues refolded the capsid amino-terminus and redirected protein assembly from spheres to cylinders. Moreover, point mutations throughout the putative CA-CA interface blocked capsid assembly in vitro, core assembly in vivo and viral infectivity. Disruption of the conserved amino-terminal capsid salt bridge also abolished the infectivity of Moloney murine leukemia viral particles, suggesting that lenti- and oncoviruses mature via analogous pathways.