The emerging regulatory potential of SCFMet30 -mediated polyubiquitination and proteolysis of the Met4 transcriptional activator

The yeast SCFMet30 ubiquitin ligase plays a critical role in cell division by regulating the Met4 transcriptional activator of genes that control the uptake and assimilation of sulfur into methionine and S-adenosyl-methionine. The initial view on how SCFMet30 performs its function has been driven by the assumption that SCFMet30 acts exclusively as Met4 inhibitor when high levels of methionine drive an accumulation of cysteine. We revisit this model in light of the growing evidence that SCFMet30 can also activate Met4. The notion that Met4 can be inhibited or activated depending on the sulfur metabolite context is not new, but for the first time both aspects have been linked to SCFMet30, creating an interesting regulatory paradigm in which polyubiquitination and proteolysis of a single transcriptional activator can play different roles depending on context. We discuss the emerging molecular basis and the implications of this new regulatory phenomenon

Effects of Environmental Factors on Sensitivity of Cryptococcus Neoformans to Fluconazole and Amphotericin B

Cryptococcus neoformans is a leading cause of fungal meningitis in immunocompromized populations. Amphotericin B (AMB) and fluconazole (FLC) are common anticryptococcal agents. AMB treatment leads to severe side-effects. In contrast, FLC-based therapy is relatively safe, although C. neoformans often develops resistance to this drug. C. neoformans must adapt to the challenging environment of the human host. Environmental effects on potency of AMB and FLC and development of drug resistance remain poorly characterized. Here, the effects of nutrients, temperature and antioxidants on susceptibility of C. neoformans towards FLC and AMB were investigated. Limited nutrients led to a decrease and an increase of sensitivity towards FLC and AMB, respectively. Co-treatment with various antioxidants also demonstrated reciprocal effects on susceptibility towards FLC and AMB. In contrast, elevated temperature increased the efficacy of both drugs, although the effect on FLC was more drastic as compared to that of AMB. In addition, temperatures of 37◦C and above prevented development of FLC resistance. Our study pointed to a critical role of the environment on susceptibility towards AMB and FLC and revealed reciprocal effects towards these antifungal drugs, reflecting contrasting modes of action of AMB and FLC

Increase of reactive oxygen species contributes to growth inhibition by fluconazole in \u3cem\u3eCryptococcus neoformans\u3c/em\u3e

Background Cryptococcus neoformans, a basidiomycetous yeast, is a fungal pathogen that can colonize the lungs of humans causing pneumonia and fungal meningitis in severely immunocompromised individuals. Recent studies have implied that the antifungal drug fluconazole (FLC) can induce oxidative stress in C. neoformans by increasing the production of reactive oxygen species (ROS), as presence of the antioxidant ascorbic acid (AA) could reverse the inhibitory effects of FLC on C. neoformans. However, in Candida albicans, AA has been shown to stimulate the expression of genes essential for ergosterol biosynthesis. Hence, the contribution of ROS in FLC-mediated growth inhibition remains unclear. Results In order to determine whether counteracting ROS generated by FLC in C. neoformans can contribute to diminishing inhibitory effects of FLC, we tested three other antioxidants in addition to AA, namely, pyrrolidine dithiocarbamate (PDTC), retinoic acid (RA), and glutathione (GSH). Our data confirm that there is an increase in ROS in the presence of FLC in C. neoformans. Importantly, all four antioxidants reversed FLC-mediated growth inhibition of C. neoformans to various extents. We further verified the involvement of increased ROS in FLC-mediated growth inhibition by determining that ROS-scavenging proteins, metallothioneins (CMT1 and CMT2), contribute to growth recovery by PDTC and AA during treatment with FLC. Conclusion Our study suggests that ROS contributes to FLC-mediated growth inhibition and points to a complex nature of antioxidant-mediated growth rescue in the presence of FLC

Recruitment of the human Cdt1 replication licensing protein by the loop domain of Hec1 is required for stable kinetochore–microtubule attachment

Cdt1, a protein critical for replication origin licensing in G1 phase is degraded during S phase but re-accumulates in G2 phase. We now demonstrate that human Cdt1 has a separable essential mitotic function. Cdt1 localizes to kinetochores during mitosis through interaction with the Hec1 component of the Ndc80 complex. G2-specific depletion of Cdt1 arrests cells in late prometaphase due to abnormally unstable kinetochore-microtubule (kMT) attachments and Mad1-dependent spindle assembly checkpoint activity. Cdt1 binds a unique loop extending from the rod domain of Hec1 that we show is also required for kMT attachment. Mutation of the loop domain prevents Cdt1 kinetochore localization and arrests cells in prometaphase. Super-resolution fluorescence microscopy indicates that Cdt1 binding to the Hec1 loop domain promotes a microtubule-dependent conformational change in the Ndc80 complex in vivo. These results support the conclusion that Cdt1 binding to Hec1 is essential for an extended Ndc80 configuration and stable kinetochore microtubule attachment

The emerging regulatory potential of SCF-mediated polyubiquitination and proteolysis of the Met4 transcriptional activator-0

Assembly between Met4 and cofactors (A), and that the dissociation of Met4 homodimers by SCF(B, ) is necessary to stabilize and/or rearrange the protein-protein interactions within the Met4/Met28/Cbf1 complex, triggering its proper assembly (B). The low abundance of SCFsuggests that proteolysis of the remaining Met4 molecule occupying SCF(B, ) is necessary for SCFrecycling (B, ). As a result, 'two stepping' with SCFcould be necessary for each round of Met4 activity at a promoter (all steps involving SCFare marked in blue). The stabilizing effect of cofactors on the SCF-Met4 interaction ('tight complex', note exposure of the UIM domain in Met4) allows Met4 inhibition by polyubiquitination only (C), unless methionine (D) or cysteine (D) is available to destabilize the tight interaction between Met4 and SCF, allowing Met4 proteolysis. Disassembly of the Met4 complex by the proteasome could link activation of methionine biosynthesis to cell division by releasing the DNA binding cofactors Cbf1 and Met31/32 from promoters (D), which, at least in the case of Cbf1, would make it available for its cell division role. Dissociation of Met4 prior to its proteolysis could protect the cofactors and SCFfrom effects of the proteasome-mediated disassembly (D), preventing Cbf1 and/or Met31/32 release. See text for details.<p><b>Copyright information:</b></p><p>Taken from "The emerging regulatory potential of SCF-mediated polyubiquitination and proteolysis of the Met4 transcriptional activator"</p><p>http://www.celldiv.com/content/3/1/11</p><p>Cell Division 2008;3():11-11.</p><p>Published online 25 Jul 2008</p><p>PMCID:PMC2526995.</p><p></p

Recruitment of the human Cdt1 replication licensing protein by the loop domain of Hec1 is required for stable kinetochore–microtubule attachment

Cdt1, a protein critical for replication origin licensing in G1 phase is degraded during S phase but re-accumulates in G2 phase. We now demonstrate that human Cdt1 has a separable essential mitotic function. Cdt1 localizes to kinetochores during mitosis through interaction with the Hec1 component of the Ndc80 complex. G2-specific depletion of Cdt1 arrests cells in late prometaphase due to abnormally unstable kinetochore-microtubule (kMT) attachments and Mad1-dependent spindle assembly checkpoint activity. Cdt1 binds a unique loop extending from the rod domain of Hec1 that we show is also required for kMT attachment. Mutation of the loop domain prevents Cdt1 kinetochore localization and arrests cells in prometaphase. Super-resolution fluorescence microscopy indicates that Cdt1 binding to the Hec1 loop domain promotes a microtubule-dependent conformational change in the Ndc80 complex in vivo. These results support the conclusion that Cdt1 binding to Hec1 is essential for an extended Ndc80 configuration and stable kinetochore microtubule attachment