NEDD8 modification of proteins is extensively studied in the recent years, and the ubiquitin-like molecule has been shown to be involved in numerous signalling pathways. In addition to its well-established roles, we showed that NEDD8 responds to various stress conditions, such as inhibition of the 26S proteasome, heat shock and oxidative stress. Modification of proteins with ubiquitin and ubiquitin-like molecules is involved in the regulation of almost every biological process. Historically, each conjugation pathway has its unique set of E1, E2 and E3 enzymes that lead to activation and conjugation of their cognate molecules. We also showed the unexpected finding that the ubiquitin E1 enzyme Ube1 activates the ubiquitin-like molecule NEDD8. The above-mentioned stress conditions cause a global increase in NEDDylation. Surprisingly, this does not depend on the NEDD8 E1 activating enzyme but rather on Ube1. A common event in the tested stress conditions is the depletion of “free” ubiquitin. A decrease in “free” ubiquitin levels in the absence of additional stress is sufficient to stimulate NEDDylation through Ube1. We also performed mass spectrometric analyses to investigate NEDD8 chain formation under stress. We found that NEDD8 forms chains with itself and with ubiquitin, and these chains are recognized by proteasome receptors and shuttle factors. Our studies revealed an unprecedented interplay between NEDD8 and ubiquitin pathways, operating in diverse cellular stress conditions. In a parallel project, we characterized the role of the deNEDDylating enzyme NEDP1 in response to DNA damage. The NEDP1 ortholog in C. elegans, Ulp-3 has been previously investigated in collaboration with Anton Gartner’s group. The enzyme has been found to be required for DNA damage-induced apoptosis in the worm germ line. Our results in human cell lines showed that the role of NEDP1 is conserved, since NEDP1 knockdown resulted in impaired effector caspase activation. Moreover, we showed that the nedp1 gene is induced upon ionizing irradiation. In the absence of the enzyme, we observed increased NEDDylation that was dependent on the NEDD8 E1 enzyme.