The C-terminal domain of p53 orchestrates the interplay between non-covalent and covalent poly(ADP-ribosyl)ation of p53 by PARP1.

The post-translational modification poly(ADP-ribosyl)ation (PARylation) plays key roles in genome maintenance and transcription. Both non-covalent poly(ADP-ribose) binding and covalent PARylation control protein functions, however, it is unknown how the two modes of modification crosstalk mechanistically. Employing the tumor suppressor p53 as a model substrate, this study provides detailed insights into the interplay between non-covalent and covalent PARylation and unravels its functional significance in the regulation of p53. We reveal that the multifunctional C-terminal domain (CTD) of p53 acts as the central hub in the PARylation-dependent regulation of p53. Specifically, p53 bound to auto-PARylated PARP1 via highly specific non-covalent PAR-CTD interaction, which conveyed target specificity for its covalent PARylation by PARP1. Strikingly, fusing the p53-CTD to a protein that is normally not PARylated, renders this a target for covalent PARylation as well. Functional studies revealed that the p53-PAR interaction had substantial implications on molecular and cellular levels. Thus, PAR significantly influenced the complex p53-DNA binding properties and controlled p53 functions, with major implications on the p53-dependent interactome, transcription, and replication-associated recombination. Remarkably, this mechanism potentially also applies to other PARylation targets, since a bioinformatics analysis revealed that CTD-like regions are highly enriched in the PARylated proteome.

Analyzing structure-function relationships of artificial and cancer-associated PARP1 variants by reconstituting TALEN-generated HeLa PARP1 knock-out cells.

Genotoxic stress activates PARP1, resulting in the post-translational modification of proteins with poly(ADP-ribose) (PAR). We genetically deleted PARP1 in one of the most widely used human cell systems, i.e. HeLa cells, via TALEN-mediated gene targeting. After comprehensive characterization of these cells during genotoxic stress, we analyzed structure-function relationships of PARP1 by reconstituting PARP1 KO cells with a series of PARP1 variants. Firstly, we verified that the PARP1\E988K mutant exhibits mono-ADP-ribosylation activity and we demonstrate that the PARP1\L713F mutant is constitutively active in cells. Secondly, both mutants exhibit distinct recruitment kinetics to sites of laser-induced DNA damage, which can potentially be attributed to non-covalent PARP1-PAR interaction via several PAR binding motifs. Thirdly, both mutants had distinct functional consequences in cellular patho-physiology, i.e. PARP1\L713F expression triggered apoptosis, whereas PARP1\E988K reconstitution caused a DNA-damage-induced G2 arrest. Importantly, both effects could be rescued by PARP inhibitor treatment, indicating distinct cellular consequences of constitutive PARylation and mono(ADP-ribosyl)ation. Finally, we demonstrate that the cancer-associated PARP1 SNP variant (V762A) as well as a newly identified inherited PARP1 mutation (F304L\V762A) present in a patient with pediatric colorectal carcinoma exhibit altered biochemical and cellular properties, thereby potentially supporting human carcinogenesis. Together, we establish a novel cellular model for PARylation research, by revealing strong structure-function relationships of natural and artificial PARP1 variants.

The oncoprotein DEK affects the outcome of PARP1/2 inhibition during mild replication stress.

DNA replication stress is a major source of genomic instability and is closely linked to tumor formation and progression. Poly(ADP-ribose)polymerases1/2 (PARP1/2) enzymes are activated in response to replication stress resulting in poly(ADP-ribose) (PAR) synthesis. PARylation plays an important role in the remodelling and repair of impaired replication forks, providing a rationale for targeting highly replicative cancer cells with PARP1/2 inhibitors. The human oncoprotein DEK is a unique, non-histone chromatin architectural protein whose deregulated expression is associated with the development of a wide variety of human cancers. Recently, we showed that DEK is a high-affinity target of PARylation and that it promotes the progression of impaired replication forks. Here, we investigated a potential functional link between PAR and DEK in the context of replication stress. Under conditions of mild replication stress induced either by topoisomerase1 inhibition with camptothecin or nucleotide depletion by hydroxyurea, we found that the effect of acute PARP1/2 inhibition on replication fork progression is dependent on DEK expression. Reducing DEK protein levels also overcomes the restart impairment of stalled forks provoked by blocking PARylation. Non-covalent DEK-PAR interaction via the central PAR-binding domain of DEK is crucial for counteracting PARP1/2 inhibition as shown for the formation of RPA positive foci in hydroxyurea treated cells. Finally, we show by iPOND and super resolved microscopy that DEK is not directly associated with the replisome since it binds to DNA at the stage of chromatin formation. Our report sheds new light on the still enigmatic molecular functions of DEK and suggests that DEK expression levels may influence the sensitivity of cancer cells to PARP1/2 inhibitors.

Effect of Calstabin1 depletion on calcium transients and energy utilization in muscle fibers and treatment opportunities with RyR1 stabilizers.

Depletion of calstabin1 (FKBP12) from the RyR1 channel and consequential calcium leakage from the sarcoplasmic reticulum (SR) is found in certain disease conditions such as dystrophy, aging or muscle overuse. Here, we first assessed the effect of calstabin1 depletion on resting Ca(2+) levels and transients. We found that depletion of calstabin1 with the calstabin1-dissociation compound FK506 increased the release of calcium from the SR by 14 % during tetanic stimulation (50 Hz, 300 ms) and delayed cytosolic calcium removal. However, we did not find a significant increase in resting cytosolic Ca(2+) levels. Therefore, we tested if increased SERCA activity could counterbalance calcium leakage. By measuring the energy utilization of muscle fibers with and without FK506 treatment, we observed that FK506-treatment increased oxygen consumption by 125% compared to baseline levels. Finally, we found that pretreatment of muscle fibers with the RyR1 stabilizer JTV-519 led to an almost complete normalization of calcium flux dynamics and energy utilization. We conclude that cytosolic calcium levels are mostly preserved in conditions with leaky RyR1 channels due to increased SERCA activity. Therefore, we suggest that RyR1 leakiness might lead to chronic metabolic stress, followed by cellular damage, and RyR1 stabilizers could potentially protect diseased muscle tissue.

Regulation of nuclear envelope permeability in cell death and survival.

The nuclear pore complex (NPC) mediates macromolecular exchange between nucleus and cytoplasm. It is a regulated channel whose functional properties are modulated in response to the physiological status of the cell. Identifying the factors responsible for regulating NPC activity is crucial to understand how intracellular signaling cues are integrated at the level of this channel to control nucleocytoplasmic trafficking. For proteins lacking active translocation signals the NPC acts as a molecular sieve limiting passage across the nuclear envelope (NE) to proteins with a MW below ~40 kD. Here, we investigate how this permeability barrier is altered in paradigms of cell death and cell survival, i.e., apoptosis induction via staurosporine, and enhanced viability via overexpression of Bcl-2. We monitor dynamic changes of the NPC's size-exclusion limit for passive diffusion by confocal time-lapse microscopy of cells undergoing apoptosis, and use different diffusion markers to determine how Bcl-2 expression affects steady-state NE permeability. We show that staurosporine triggers an immediate and gradual leakiness of the NE preceding the appearance of apoptotic hallmarks. Bcl-2 expression leads to a constitutive increase in NE permeability, and its localization at the NE is sufficient for the effect, evincing a functional role for Bcl-2 at the nuclear membrane. In both settings, NPC leakiness correlates with reduced Ca²âº in internal stores, as demonstrated by fluorometric measurements of ER/NE Ca²âº levels. By comparing two cellular models with opposite outcome these data pinpoint ER/NE Ca²âº as a general and physiologically relevant regulator of the permeability barrier function of the NPC.