Kaminska, M. | bioRxiv Science

bioRxiv2024

Glycosaminoglycans act as activators of peptidylarginine deiminase 4

Peptidylarginine deiminase 4 (PAD4) is a citrullinating enzyme that is gathering increasing attention due to its possible involvement in physiological processes as well as in the pathogenesis of diseases like rheumatoid arthritis or thrombosis. PAD4 is activated by calcium ions, but the details of this mechanism are elusive, because in the human body, Ca2+ concentrations are too low for full activity. Given that glycosaminoglycans (GAGs) are also implicated in the development and progression of rheumatoid arthritis, we investigated the activation of PAD4 by GAGs using heparin as a model. We employed activity assays, chromatography techniques, molecular interaction measurements (MST and SPR), FACS, and immunocytochemistry to demonstrate the activation of PAD4 by GAGs. Our data show that PAD4 binds heparin with high affinity and forms high molecular weight complexes with heparin, consistent with heparin-bound tetramer formation. Heparin activates PAD4 by increasing the enzymes Ca2+ affinity threefold. We also show that the effectiveness of activation with heparin depends on the length of GAG used and its negative charge. Direct measurement of heparin binding to PAD4 confirmed tight interaction with nanomolar affinity. Mutagenesis of regions likely responsible for heparin binding showed that dimerization of PAD4 is necessary for efficient activation, but the distinct binding site was not determined as interaction with heparin likely occurs over larger surface of PAD4. Furthermore, we show that other GAG family members, including heparan and chondroitin sulphates, are also able to activate PAD4. We also found that disturbed production of GAGs by CHO cells results in reduced PAD4 binding efficiency. Finally, heparin induces NETosis in hPMNs in concentration-dependent manner, as measured by the release of DNA and citrullination of histone H3. In summary, we identify the first natural coactivator of PAD4, which is present in all individuals, potentially explaining the regulation of PAD4 activity in physiological conditions, and providing new insight into the development of rheumatoid arthritis and other PAD4-related diseases.

biochemistry↗

bioRxiv2023

Sleep spindle density and temporal clustering are associated with sleep-dependent memory consolidation in Parkinson's disease

Sleep is required for successful memory consolidation. Sleep spindles, bursts of oscillatory activity occurring during non-REM sleep, are known to be crucial for this process and, recently, it has been proposed that the temporal organization of spindles into clusters might additionally play a role in memory consolidation. In Parkinsons disease, spindle activity is reduced, and this reduction has been found to be predictive of cognitive decline. However, it remains unknown whether alterations in sleep spindles in Parkinsons disease are predictive of sleep-dependent cognitive processes like memory consolidation, leaving open questions about the possible mechanisms linking sleep and more general cognitive state in Parkinsons patients. The current study sought to fill this gap by recording overnight polysomnography and measuring overnight declarative memory consolidation in a sample of thirty-five Parkinsons patients. Memory consolidation was measured using a verbal paired-associates task administered before and after the night of recorded sleep. We found that lower sleep spindle density at frontal leads during non-REM stage 3 was associated with worse overnight declarative memory consolidation. We also found that patients who showed less temporal clustering of spindles exhibited worse declarative memory consolidation. These results suggest alterations to sleep spindles, which are known to be a consequence of Parkinsons disease, might represent a mechanism by which poor sleep leads to worse cognitive function in Parkinsons patients. Statement of significanceSleep -- particularly spindle activity -- is critical for memory consolidation, a core cognitive process. Changes to the architecture and oscillations of sleep are well documented in Parkinsons disease (PD) and have been associated with worse overall cognition. However, whether altered sleep plays a causal role in this relationship, by directly interfering with sleep-dependent cognitive processes, or whether it represents a mere epiphenomenon of advancing disease, remains unknown. Our study is the first to investigate a possible direct relationship between sleep and cognition in PD. We show that sleep spindles and their temporal clustering into trains relate to impairments in overnight declarative memory consolidation in patients. These findings are an important first step towards identifying modifiable sources of cognitive impairment in PD.

neuroscience↗