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Several β-barrel-type channels are involved in the translocation or assembly of outer membrane proteins of bacteria or endosymbiotically derived organelles. Here we analyzed the functional units of the β-barrel polypeptide transporter Toc75 (translocon in outer envelope of chloroplasts) of the outer envelope of chloroplasts and of a protein, alr2269, from Nostoc PCC7120 with homology to Toc75, both proteins having a similar domain organization. We demonstrated that the N-terminal region functions as a recognition and complex assembly unit, whereas the C terminus forms the β-bartel-type pore. The pore region is, in turn, modulated by the N terminus of the proteins. The protein from Nostoc PCC7120, which shares a common ancestor with Toc75, is able to recognize precursor proteins destined for chloroplasts. In contrast, the recognition of peripheral translocon subunits by Toc75 is a novel feature acquired through evolution. [ABSTRACT FROM AUTHOR]

Although survival rates for patients with newly diagnosed multiple myeloma (NDMM) have improved over recent decades, multiple myeloma (MM) remains without a cure for most. There is increasing consensus that achievement of deep remissions, especially minimal residual disease negativity (MRD −), in frontline treatment is crucial and translates into improved survival. The standard of care (SOC) for NDMM consists at minimum of a triplet regimen of therapies, with or without an autologous stem cell transplant, or a doublet regimen for certain ineligible, particularly frail patients who may have specific limitations. Recently, anti-CD38 monoclonal antibodies (mAbs), such as daratumumab (Dara) or isatuximab (Isa), have been integrated into frontline SOC regimens. Seeking to further deepen and prolong responses, several clinical trials have commenced investigating the addition of anti-CD38 mAbs to carfilzomib, lenalidomide, and dexamethasone (KRd). These quadruplet regimens (Isa/Dara-KRd) are being evaluated in the context of evolving treatment considerations for the heterogeneous population of patients with NDMM. In clinical trials, the addition of Isa/Dara to KRd achieved high rates of deep responses and MRD − . Favorable outcomes were observed in patients with NDMM independent of age, transplant eligibility, and cytogenetic risk, while these treatments did not result in unexpected or emergent safety risks. The efficacy observed with intensified, yet well-tolerated therapy may offer further development of risk- and response-adapted therapy for individualized patient needs. This review summarizes the clinical outcomes of quadruplet-based therapy with Isa/Dara-KRd in NDMM. [ABSTRACT FROM AUTHOR]

Background: Obatoclax is a clinical stage drug candidate that has been proposed to target and inhibit prosurvival members of the Bcl-2 family, and thereby contribute to cancer cell lethality. The insolubility of this compound, however, has precluded the use of many classical drug-target interaction assays for its study. Thus, a direct demonstration of the proposed mechanism of action, and preferences for individual Bcl-2 family members, remain to be established. Methods: Employing modified proteins and lipids, we recapitulated the constitutive association and topology of mitochondrial outer membrane Mcl-1 and Bak in synthetic large unilamellar liposomes, and measured bakdependent bilayer permeability. Additionally, cellular and tumor models, dependent on Mcl-1 for survival, were employed. Results: We show that regulation of bilayer permeabilization by the tBid - Mcl-1 - Bak axis closely resemblesthe tBid - Bcl-XL - Bax model. Obatoclax rapidly and completely partitioned into liposomal lipid but also rapidly exchanged between liposome particles. In this system, obatoclax was found to be a direct and potent antagonist of liposome-bound Mcl-1 but not of liposome-bound Bcl-XL, and did not directly influence Bak. A 2.5 molar excess of obatoclax relative to Mcl-1 overcame Mcl-1-mediated inhibition of tBid-Bak activation. Similar results were found for induction of Bak oligomers by Bim. Obatoclax exhibited potent lethality in a cellmodel dependent on Mcl-1 for viability but not in cells dependent on Bcl-XL. Molecular modeling predicts that the 3-methoxy moiety of obatoclax penetrates into the P2 pocket of the BH3 binding site of Mcl-1. A desmethoxy derivative of obatoclax failed to inhibit Mcl-1 in proteoliposomes and did not kill cells whose survival depends on Mcl-1. Systemic treatment of mice bearing Tsc2+/- Em-myc lymphomas (whose cells depend on Mcl-1 for survival) with obatoclax conferred a survival advantage compared to vehicle alone (median 31 days vs 22 days, respectively; p=0.003). In an Akt-lymphoma mouse model, the anti-tumor effects of obatoclax synergized with doxorubicin. Finally, treatment of the multiple myeloma KMS11 cell model (dependent on Mcl-1 for survival) with dexamethasone induced Bim and Bim-dependent lethality. As predicted for an Mcl-1 antagonist, obatoclax and dexamethasone were synergistic in this model. Conclusions: Taken together, these findings indicate that obatoclax is a potent antagonist of membranerestricted Mcl-1. Obatoclax represents an attractive chemical series to generate second generation Mcl-1 inhibitors. [ABSTRACT FROM AUTHOR]

We showed previously that the strong PHO5 promoter is less dependent on chromatin cofactors than the weaker coregulated PHO8 promoter. In this study we asked if chromatin remodeling at the even stronger PHO84 promoter was correspondingly less cofactor dependent. The repressed PHO84 promoter showed a short hypersensitive region that was flanked upstream and downstream by a positioned nucleosome and contained two transactivator Pho4 sites. Promoter induction generated an extensive hypersensitive and histone-depleted region, yielding two more Pho4 sites accessible. This remodeling was strictly Pho4 dependent, strongly dependent on the remodelers Snf2 and Ino80 and on the histone acetyltransferase Gcn5, and more weakly on the acetyltransferase Rtt109. Importantly, remodeling of each of the two positioned nucleosomes required Snf2 and Ino80 to different degrees. Only remodeling of the upstream nucleosome was strictly dependent on Snf2. Further, remodeling of the upstream nucleosome was more dependent on Ino80 than remodeling of the downstream nucleosome. Both nucleosomes differed in their intrinsic stabilities as predicted in silico and measured in vitro. The causal relationship between the different nucleosome stabilities and the different cofactor requirements was shown by introducing destabilizing mutations in vivo. Therefore, chromatin cofactor requirements were determined by intrinsic nucleosome stabilities rather than correlated to promoter strength. [ABSTRACT FROM AUTHOR]

β-Barrel-shaped channels of the Omp85 family are involved in the translocation or assembly of proteins of bacterial, mitochondrial, and plastidic outer membranes. We have compared these proteins to understand the evolutionary development of the translocators. We have demonstrated that the proteins from proteobacteria and mitochondria have a pore diameter that is at least five times smaller than found for the Omp85 in cyanobacteria and plastids. This finding can explain why Omp85 from cyanobacteria (but not the homologous protein from proteobacteria) was remodeled to become the protein translocation pore after endosymbiosis. Further, the pore-forming region of the Omp85 proteins is restricted to the C terminus. Based on a phylogenetic analysis we have shown that the pore-forming domain displays a different evolutionary relationship than the N-terminal domain. In line with this, the affinity of the N-terminal domain to the C-terminal region of the Omp85 from plastids and- cyanobacteria differs, even though the N-terminal domain is involved in gating of the pore in both groups. We have further shown that the N-terminal domain of nOmp85 takes part in homo-oligomerization. Thereby, the differences in the phylogeny of the two domains are explained by different functional constraints acting on the regions. The pore-forming domain, however, is further divided into two functional regions, where the distal C terminus itself forms a dimeric pore. Based on functional and phylogenetic analysis, we suggest an evolutionary scenario that explains the origin of the contemporary translocon. [ABSTRACT FROM AUTHOR]

Proteins of the Omp85 family are involved in the insertion of β-barrel shaped outer membrane proteins in bacteria and mitochondria, and-at least-in the transfer of preproteins across the chloroplast outer envelope. In general these proteins consist of up to five N-terminal 'polypeptide transport associated' (POTRA) domains and a C-terminal, membrane embedded β-barrel domain. In Arabidopsis thaliana two plastidic gene families coding for Omp85-like proteins exist, namely the Toc75-III and the Toc75-V/Oep80 sub-family. The latter is composed of three genes, of which two do not contain POTRA domains. These are annotated as P39 and P36. However, P36 resulted from a very recent gene duplication of P39 and appears to be specific to Arabidopsis thaliana. Furthermore, we show that P39 is specifically expressed in vein tissues, while P36 is expressed at early and late developmental stages. T-DNA insertion in P36 causes a mild phenotype with reduced starch accumulation in chloroplasts of sepals pointing towards a yet to be described plastid function. [ABSTRACT FROM AUTHOR]

Background: Obatoclax is a clinical stage drug candidate that has been proposed to target and inhibit prosurvival members of the Bcl-2 family, and thereby contribute to cancer cell lethality. The insolubility of this compound, however, has precluded the use of many classical drug-target interaction assays for its study. Thus, a direct demonstration of the proposed mechanism of action, and preferences for individual Bcl-2 family members, remain to be established. Methods: Employing modified proteins and lipids, we recapitulated the constitutive association and topology of mitochondrial outer membrane Mcl-1 and Bak in synthetic large unilamellar liposomes, and measured bakdependent bilayer permeability. Additionally, cellular and tumor models, dependent on Mcl-1 for survival, were employed. Results: We show that regulation of bilayer permeabilization by the tBid - Mcl-1 - Bak axis closely resemblesthe tBid - Bcl-XL - Bax model. Obatoclax rapidly and completely partitioned into liposomal lipid but also rapidly exchanged between liposome particles. In this system, obatoclax was found to be a direct and potent antagonist of liposome-bound Mcl-1 but not of liposome-bound Bcl-XL, and did not directly influence Bak. A 2.5 molar excess of obatoclax relative to Mcl-1 overcame Mcl-1-mediated inhibition of tBid-Bak activation. Similar results were found for induction of Bak oligomers by Bim. Obatoclax exhibited potent lethality in a cellmodel dependent on Mcl-1 for viability but not in cells dependent on Bcl-XL. Molecular modeling predicts that the 3-methoxy moiety of obatoclax penetrates into the P2 pocket of the BH3 binding site of Mcl-1. A desmethoxy derivative of obatoclax failed to inhibit Mcl-1 in proteoliposomes and did not kill cells whose survival depends on Mcl-1. Systemic treatment of mice bearing Tsc2(+) (/) (-) Em-myc lymphomas (whose cells depend on Mcl-1 for survival) with obatoclax conferred a survival advantage compared to vehicle alone (median 31 days vs 22 days, respectively; p=0.003). In an Akt-lymphoma mouse model, the anti-tumor effects of obatoclax synergized with doxorubicin. Finally, treatment of the multiple myeloma KMS11 cell model (dependent on Mcl-1 for survival) with dexamethasone induced Bim and Bim-dependent lethality. As predicted for an Mcl-1 antagonist, obatoclax and dexamethasone were synergistic in this model. Conclusions: Taken together, these findings indicate that obatoclax is a potent antagonist of membranerestricted Mcl-1. Obatoclax represents an attractive chemical series to generate second generation Mcl-1 inhibitors. [ABSTRACT FROM AUTHOR]

Mitochondria and chloroplasts are of endosymbiotic origin. Their integration into cells entailed the development of protein translocons, partially by recycling bacterial proteins. We demonstrate the evolutionary conservation of the translocon component Tic22 between cyanobacteria and chloroplasts. Tic22 in Anabaena sp. PCC 7120 is essential. The protein is localized in the thylakoids and in the periplasm and can be functionally replaced by a plant orthologue. Tic22 physically interacts with the outer envelope biogenesis factor Omp85 in vitro and in vivo, the latter exemplified by immunoprecipitation after chemical cross-linking. The physical interaction together with the phenotype of a tic22 mutant comparable with the one of the omp85 mutant indicates a concerted function of both proteins. The three-dimensional structure allows the definition of conserved hydrophobic pockets comparable with those of ClpS or BamB. The results presented suggest a function of Tic22 in outer membrane biogenesis. [ABSTRACT FROM AUTHOR]