A rapid intracellular enrichment of alkylating payload is essential for melphalan flufenamide potency and mechanism of action.

Despite decades of development of treatments and the successful application of targeted therapies for multiple myeloma, clinical challenges remain for patients with relapsed/refractory disease. A drug designed for efficient delivery of an alkylating payload into tumor cells that yields a favorable therapeutic window can be an attractive choice. Herein we describe melphalan flufenamide (melflufen), a drug with a peptide carrier component conjugated to an alkylating payload, and its cellular metabolism. We further underline the fundamental role of enzymatic hydrolysis in the rapid and robust accumulation of alkylating metabolites in cancer cells and their importance for downstream effects. The formed alkylating metabolites were shown to cause DNA damage, both on purified DNA and on chromatin in cells, with both nuclear and mitochondrial DNA affected in the latter. Furthermore, the rapid intracellular enrichment of alkylating metabolites is shown to be essential for the rapid kinetics of the downstream intracellular effects such as DNA damage signaling and induction of apoptosis. To evaluate the importance of enzymatic hydrolysis for melflufen's efficacy, all four stereoisomers of the compound were studied in a systematic approach and shown to have a different pattern of metabolism. In comparison with melflufen, stereoisomers lacking intracellular accumulation of alkylating payloads showed cytotoxic activity only at significantly higher concentration, slower DNA damage kinetics, and different mechanisms of action to reach cellular apoptosis.

Ataxin-3 consolidates the MDC1-dependent DNA double-strand break response by counteracting the SUMO-targeted ubiquitin ligase RNF4.

The SUMO-targeted ubiquitin ligase RNF4 functions at the crossroads of the SUMO and ubiquitin systems. Here, we report that the deubiquitylation enzyme (DUB) ataxin-3 counteracts RNF4 activity during the DNA double-strand break (DSB) response. We find that ataxin-3 negatively regulates ubiquitylation of the checkpoint mediator MDC1, a known RNF4 substrate. Loss of ataxin-3 markedly decreases the chromatin dwell time of MDC1 at DSBs, which can be fully reversed by co-depletion of RNF4. Ataxin-3 is recruited to DSBs in a SUMOylation-dependent fashion, and in vitro it directly interacts with and is stimulated by recombinant SUMO, defining a SUMO-dependent mechanism for DUB activity toward MDC1. Loss of ataxin-3 results in reduced DNA damage-induced ubiquitylation due to impaired MDC1-dependent recruitment of the ubiquitin ligases RNF8 and RNF168, and reduced recruitment of 53BP1 and BRCA1. Finally, ataxin-3 is required for efficient MDC1-dependent DSB repair by non-homologous end-joining and homologous recombination. Consequently, loss of ataxin-3 sensitizes cells to ionizing radiation and poly(ADP-ribose) polymerase inhibitor. We propose that the opposing activities of RNF4 and ataxin-3 consolidate robust MDC1-dependent signaling and repair of DSBs.

A new non-catalytic role for ubiquitin ligase RNF8 in unfolding higher-order chromatin structure.

The ubiquitin ligases RNF8 and RNF168 orchestrate DNA damage signalling through the ubiquitylation of histone H2A and the recruitment of downstream repair factors. Here, we demonstrate that RNF8, but not RNF168 or the canonical H2A ubiquitin ligase RNF2, mediates extensive chromatin decondensation. Our data show that CHD4, the catalytic subunit of the NuRD complex, interacts with RNF8 and is essential for RNF8-mediated chromatin unfolding. The chromatin remodelling activity of CHD4 promotes efficient ubiquitin conjugation and assembly of RNF168 and BRCA1 at DNA double-strand breaks. Interestingly, RNF8-mediated recruitment of CHD4 and subsequent chromatin remodelling were independent of the ubiquitin-ligase activity of RNF8, but involved a non-canonical interaction with the forkhead-associated (FHA) domain. Our study reveals a new mechanism of chromatin remodelling-assisted ubiquitylation, which involves the cooperation between CHD4 and RNF8 to create a local chromatin environment that is permissive to the assembly of checkpoint and repair machineries at DNA lesions.

Should I stay or should I go: VCP/p97-mediated chromatin extraction in the DNA damage response.

The ordered assembly of DNA repair factors on chromatin has been studied in great detail, whereas we are only beginning to realize that selective extraction of proteins from chromatin plays a central role in the DNA damage response. Interestingly, the protein modifier ubiquitin not only regulates the well-documented recruitment of repair proteins, but also governs the temporally and spatially controlled extraction of proteins from DNA lesions. The facilitator of protein extraction is the ubiquitin-dependent ATPase valosin-containing protein (VCP)/p97 complex, which, through its segregase activity, directly extracts ubiquitylated proteins from chromatin. In this review, we summarize recent studies that uncovered this important role of VCP/p97 in the cellular response to genomic insults and discuss how ubiquitin regulates two intuitively counteracting activities at sites of DNA damage.

Illuminating the ubiquitin/proteasome system.

The ubiquitin/proteasome system (UPS) is responsible for the regulated processive degradation of proteins residing in the cytosol, nucleus, and endoplasmic reticulum. The two central players are ubiquitin, a small protein that is conjugated to substrates, and the proteasome, a large multi-subunit proteolytic complex that executes degradation of ubiquitylated proteins. Ubiquitylation and proteasomal degradation are highly dynamic processes. During the last decade, many researchers have started taking advantage of fluorescent proteins, which allow studying the dynamic nature of this system in the context of its natural environment: the living cell. In this review, we will summarize studies that have implemented this approach to examine the UPS and discuss novel insights in the dynamic organization of the UPS.

Novel signaling pathways promote a paracrine wave of prostacyclin-induced vascular smooth muscle differentiation.

The important athero-protective role of prostacyclin is becoming increasingly evident as recent studies have revealed adverse cardiovascular effects in mice lacking the prostacyclin receptor, in patients taking selective COX-2 inhibitors, and in patients in the presence of a dysfunctional prostacyclin receptor genetic variant. We have recently reported that this protective mechanism includes the promotion of a quiescent differentiated phenotype in human vascular smooth muscle cells (VSMC). Herein, we address the intriguing question of how localized endothelial release of the very unstable eicosanoid, prostacyclin, exerts a profound effect on the vascular media, often 30 cell layers thick. We report a novel PKA-, Akt-1- and ERK1/2-dependent prostacyclin-induced prostacyclin release that appears to play an important role in propagation of the quiescent, differentiated phenotype through adjacent arterial smooth muscle cells in the vascular media. Treating VSMC with the prostacyclin analog iloprost induced differentiation (contractile protein expression and contractile morphology), and also up-regulated COX-2 expression, leading to prostacyclin release by VSMC. This paracrine prostacyclin release, in turn, promoted differentiation and COX-2 induction in neighboring VSMC that were not exposed to iloprost. Using siRNA and pharmacologic inhibitors, we report that this positive feedback mechanism, prostacyclin-induced prostacyclin release, is mediated by cAMP/PKA signaling, ERK1/2 activation, and a novel prostacyclin receptor signaling pathway, inhibition of Akt-1. Furthermore, these pathways appear to be regulated by the prostacyclin receptor independently of one another. We conclude that prevention of de-differentiation and proliferation through a paracrine positive feedback mechanism is a major cardioprotective function of prostacyclin.

Cdc48-independent proteasomal degradation coincides with a reduced need for ubiquitylation.

Ubiquitin fusion degradation (UFD) substrates are delivered at the proteasome by a handover mechanism involving the ubiquitin-selective chaperone Cdc48 and the ubiquitin shuttle factor Rad23. Here, we show that introduction of a 20 amino acid peptide extension not only rendered degradation independent of Cdc48, in line with the model that this chaperone is involved in early unfolding events of tightly folded substrates, but at the same time relieved the need for efficient polyubiquitylation and the ubiquitin shuttle factor Rad23. Removal of the ubiquitylation sites in the N-terminal UFD signal made the degradation of this substrate strictly dependent on the peptide extension and also on Cdc48 and, importantly the presence of a functional ubiquitylation machinery. This suggests that the extension in the absence of N-terminal ubiquitylation sites is not properly positioned to engage the unfoldase machinery of the proteasome. Thus the need for efficient ubiquitylation and Cdc48 in facilitating proteasomal degradation are tightly linked but can be bypassed in the context of UFD substrates by the introduction of an unstructured extension. Our data suggest that polyubiquitin-binding complexes acting upstream of the proteasome, rather than the proteasome itself, can be primary determinants for the level of ubiquitylation required for protein degradation.

Phylogenetic relationship of the genus Gilbertella and related genera within the order Mucorales based on 5.8 S ribosomal DNA sequences.

The complete ITS (internal transcribed spacer) region coding the ITS1, the ITS2 and the 5.8S rDNA was amplified by polymerase chain reaction from two strains of Gilbertella persicaria, six strains in the Mucoraceae (Mucor piriformis, M. rouxii, M. circinelloides, Rhizomucor miehei, R. pusillus and R. tauricus) and four strains representing three species of the Choanephoraceae (Blakeslea trispora, Choanephora infundibulifera and Poitrasia circinans). Sequences of the amplified DNA fragments were determined and analysed. G. persicaria belongs to the monogeneric family (Gilbertellaceae), however, originally it was described as Choanephora persicaria. The goal of this study was to reveal the phylogenetic relationship among fungi belonging to Gilbertellaceae, Choanephoraceae and Mucoraceae. Our results support that the "intermediate" position of this family is between Choanephoraceae and Mucoraceae.

DDB2 promotes chromatin decondensation at UV-induced DNA damage.

Nucleotide excision repair (NER) is the principal pathway that removes helix-distorting deoxyribonucleic acid (DNA) damage from the mammalian genome. Recognition of DNA lesions by xeroderma pigmentosum group C (XPC) protein in chromatin is stimulated by the damaged DNA-binding protein 2 (DDB2), which is part of a CUL4A-RING ubiquitin ligase (CRL4) complex. In this paper, we report a new function of DDB2 in modulating chromatin structure at DNA lesions. We show that DDB2 elicits unfolding of large-scale chromatin structure independently of the CRL4 ubiquitin ligase complex. Our data reveal a marked adenosine triphosphate (ATP)-dependent reduction in the density of core histones in chromatin containing UV-induced DNA lesions, which strictly required functional DDB2 and involved the activity of poly(adenosine diphosphate [ADP]-ribose) polymerase 1. Finally, we show that lesion recognition by XPC, but not DDB2, was strongly reduced in ATP-depleted cells and was regulated by the steady-state levels of poly(ADP-ribose) chains.

C-terminal UBA domains protect ubiquitin receptors by preventing initiation of protein degradation.

The ubiquitin receptors Rad23 and Dsk2 deliver polyubiquitylated substrates to the proteasome for destruction. The C-terminal ubiquitin-associated (UBA) domain of Rad23 functions as a cis-acting stabilization signal that protects this protein from proteasomal degradation. Here, we provide evidence that the C-terminal UBA domains guard ubiquitin receptors from destruction by preventing initiation of degradation at the proteasome. We show that introduction of unstructured polypeptides that are sufficiently long to function as initiation sites for degradation abrogates the protective effect of UBA domains. Vice versa, degradation of substrates that contain an unstructured extension can be attenuated by the introduction of C-terminal UBA domains. Our study gains insight into the molecular mechanism responsible for the protective effect of UBA domains and explains how ubiquitin receptors can shuttle substrates to the proteasome without themselves becoming subject to proteasomal degradation.

The AAA-ATPase VCP/p97 promotes 53BP1 recruitment by removing L3MBTL1 from DNA double-strand breaks.

The accumulation of the human tumor suppressor 53BP1 at DNA damage sites requires the ubiquitin ligases RNF8 and RNF168. As 53BP1 recognizes dimethylated Lys20 in histone H4 (H4K20me2), the requirement for RNF8- and RNF168-mediated ubiquitylation has been unclear. Here we show that RNF8-mediated ubiquitylation facilitates the recruitment of the AAA-ATPase valosin-containing protein (VCP, also known as p97) and its cofactor NPL4 to sites of double-strand breaks. RIDDLE cells, which lack functional RNF168, also show impaired recruitment of VCP to DNA damage. The ATPase activity of VCP promotes the release of the Polycomb protein L3MBTL1 from chromatin, which also binds the H4K20me2 histone mark, thereby facilitating 53BP1 recruitment. Consistent with this, nematodes lacking the VCP orthologs CDC-48.1 or CDC-48.2, or cofactors UFD-1 or NPL-4, are highly sensitive to ionizing radiation. Our data suggest that human RNF8 and RNF168 promote VCP-mediated displacement of L3MBTL1 to unmask 53BP1 chromatin binding sites.

Genetic variability in the species Rhizopus stolonifer, assessed by random amplified polymorphic DNA analysis.

Rhizopus stolonifer is an important post-harvest pathogenic fungus. Recent taxonomic findings based on morphological and growth characteristics led to a dramatic reduction in the number of accepted species within the genus. The aim of this study was to examine this situation with molecular markers. Twenty-nine R. stolonifer strains isolated from various locations and substrates were characterized by random amplified polymorphic DNA (RAPD) analysis. The numerical analysis of the RAPD data revealed four main clusters with extremely high dissimilarity values, but only low or moderate variability was observed within these groups. These results suggest a high genetic heterogeneity in the case of R. stolonifer: isolates of R. stolonifer var. stolonifer, R. stolonifer var. reflexus and R. niveus displayed species-level genetic distances, which gives rise to considerations that they might be separate species.

Cloning and sequence analysis of the glyceraldehyde-3-phosphate dehydrogenase gene from the zygomycetes fungus Rhizomucor miehei.

Rhizomucor miehei is important from a biotechnological aspect in consequence of its content of aspartic proteinase, which has high milk-clotting activity. A genomic library of R. miehei NRRL 5901 has been constructed in a phage (Lambda Fix II) vector. The glyceraldehyde-3-phosphate dehydrogenase (gpd) gene was isolated from this library by hybridization of the recombinant phage clones with a gpd-specific gene probe generated by polymerase chain reaction. The complete nucleotide sequence encodes a putative polypeptide chain of 336 amino acids interrupted by 5 introns. The predicted amino acid sequence of this gene shows a high degree of sequence similarity to the glyceraldehyde-3-phosphate dehydrogenase proteins from yeast and filamentous fungi. The promoter region, containing a consensus TATA box, and 246-bp downstream from the putative stop codon were also determined. The possibility of using the gpd promoter in the construction of new transformation vectors is discussed.

Cloning and sequence analysis of Mucor circinelloides glyceraldehyde-3-phosphate dehydrogenase gene.

A genomic library of Mucor circinelloides ATCC 1216b has been constructed in Lambda Fix II vector. The library has an average insert site of 10 kb and covers the genome 12 times. The M. circinelloides gene encoding glyceraldehyde-3-phosphate dehydrogenase (gpd) was isolated from this library by hybridization of the recombinant phage clones with a gpd-specific gene probe generated by PCR reaction. The complete nucleotide sequence encodes a putative polypeptide chain of 339 amino acids interrupted by 3 introns. The predicted amino acid sequence of this gene shows a high degree of sequence similarity to the GPD proteins from other filamentous fungi. The promoter region, containing a consensus TATA and CAAT box and a 298 nucleotid long termination region were also determined.