Investigation of the Cellular Response to Environmental Stresses

Abstract

Cells have a repertoire of responses to stress, spanning from triggering survival mechanisms to initiating cell death, which ultimately clears out compromised cells. The decision between activating protective or detrimental stress reactions hinges greatly on the stress's characteristics, duration, and the specific type of cell involved. Microbial infections, spanning bacterial, viral, fungal, and parasitic realms, frequently result in diverse cellular stresses, constituting a prevalent affliction in humans. Naturally occurring in the Earth's crust, metal compositions vary across locales, 10 of which are essential elements for human body to function correctly. While essential for biochemical and physiological functions in organisms at low concentrations, metals become harmful when exceeding certain thresholds. Copper (Cu) is a required trace element for the growth and development of mammalian cells. However, excess Cu triggers cell death by apoptosis, necrosis, cuproptosis, or other types of cell death. Kaposi's sarcoma-associated herpesvirus (KSHV; human herpesvirus 8) is a member of the Gammaherpesvirinae subfamily and is responsible for causing Kaposi's sarcoma as well as two lymphoproliferative disorders: primary effusion lymphoma and multicentric Castleman disease. KSHV PAN RNA, a 1.1 kb long non-coding RNA (lncRNA), plays a crucial role in regulating both cellular and viral gene expression by interacting with chromatin-modifying complexes and binding to cellular and viral DNA. Our initial investigations, employing RNA Antisense Purification (RAP) combined with deep sequencing (pseudo-seq, Ψ-seq) and CLAP, suggest that up to 50% of PAN RNA exhibited a prominent pseudouridine (Ψ) modification at position 444 in latent BCBL1 cells. PUS7 knockdown (KD) diminished Ψ modification at position 444 and leads to reduced levels of PAN RNA compared to wild-type cells. Confocal microscopy analysis verified the temporal and spatial subcellular availability of PUS7 for PAN modification. Furthermore, the KSHV copy number and viral ORFs expression were greatly inhibited in the PUS7 KD cells. This study contributes to the understanding of PAN RNA plasticity during viral replication revealing the sophisticated relationship that exists between the viral transcript and cellular epitranscriptomic machinery and creates a paradigm for future studies in the field of host-pathogen interactions. Under normal conditions, the mitochondrial carrier family protein SLC25A3 is required for Cu transport to the mitochondrial matrix. Previous experiments, as well as our own, showed that the mouse heart developed hypertrophy in conditional SLC25A3 knockout (KO) mice. Mouse cardiac tissues and HEK293 cells lacking SLC25A3 exhibited increased phosphorylation of MAPK/ERK1/2 and elevated cellular Cu levels. The increased phosphorylation of MAPK/ERK1/2 in HEK293 cells can be inhibited by Cu chelators. Additionally, the introduction of Cu not only triggered MAPK/ERK1/2 activation but also induced the cleavage of caspase 8 and PARP, both recognized as signatures of apoptotic cell death. The Cu-dependent activation of apoptosis was enhanced by U0126, a small molecule inhibitor of MAPKK/MEK, upstream of ERK1/2. Furthermore, Cu-induced apoptosis was decreased by knockdown (KD) of both CTR1 and CTR2, which were also downregulated by SLC25A3 KD. These findings provide valuable insights into the molecular interplay among Cu transport, MAPK/ERK1/2 activation, and cellular apoptosis. In summary, this research uncovered two mechanisms against environmental stresses for human cells. One mechanism involves the PAN lncRNA pseudouridine (Ψ) modification at position 444 by PUS7, which regulates viral replication and activation. The other mechanism is the regulation of Cu transport by SLC25A3, which affects MAPK/ERK1/2 activation and cellular apoptosis in response to environmental stresses.