Extracellular proteolysis in cancer: Proteases, substrates, and mechanisms in tumor progression and metastasis.

A vast ensemble of extracellular proteins influences the development and progression of cancer, shaped and reshaped by a complex network of extracellular proteases. These proteases, belonging to the distinct classes of metalloproteases, serine proteases, cysteine proteases, and aspartic proteases, play a critical role in cancer. They often become dysregulated in cancer, with increases in pathological protease activity frequently driven by the loss of normal latency controls, diminished regulation by endogenous protease inhibitors, and changes in localization. Dysregulated proteases accelerate tumor progression and metastasis by degrading protein barriers within the extracellular matrix (ECM), stimulating tumor growth, reactivating dormant tumor cells, facilitating tumor cell escape from immune surveillance, and shifting stromal cells toward cancer-promoting behaviors through the precise proteolysis of specific substrates to alter their functions. These crucial substrates include ECM proteins and proteoglycans, soluble proteins secreted by tumor and stromal cells, and extracellular domains of cell surface proteins, including membrane receptors and adhesion proteins. The complexity of the extracellular protease web presents a significant challenge to untangle. Nevertheless, technological strides in proteomics, chemical biology, and the development of new probes and reagents are enabling progress and advancing our understanding of the pivotal importance of extracellular proteolysis in cancer.

Assessing proteolytic events in bioinformatic reanalysis of public secretome data from melanoma cell lines.

Autocrine and paracrine signals are of paramount importance in both normal and oncogenic events and the composition of such secreted molecular signals (i.e the secretome) designate the communication status of cells. In this context, the analysis of post-translational modifications in secreted proteins may unravel biological circuits regulated by irreversible modifications such as proteolytic processing. In the present study, we have performed a bioinformatic reanalysis of public proteomics data on melanoma cell line secretomes, changing database searching parameters to allow for the identification of proteolytic events generated by active proteases. Such approach enabled the identification of proteolytic signatures which suggested active proteases and whose expression profiles might be targeted in patient tissues or liquid biopsies, as well as their cleaved substrates. Although N-terminomics approaches continue to be the method of choice for the evaluation of proteolytic signaling events in complex samples, the simple approach performed in this work resulted in the gain of biological insights derived from shotgun proteomics data.

Cullin 3, a cellular scripter of the non-proteolytic ubiquitin code.

Cullin-RING ubiquitin ligases (CRLs) represent the largest family of E3 ubiquitin ligases that control most if not all cellular processes. In CUL3-based CRLs, the substrate specificity is conferred by the interaction with one of around 183 existing BTB proteins, implying a broad spectrum of possible ubiquitylation signals and possible direct ubiquitylation substrates. Indeed, CUL3-based E3-ligases can catalyze various proteolytic and non-proteolytic ubiquitin signals regulating many physiological and pathophysiological states. Here, we discuss the recent studies focusing on the non-proteolytic CUL3-based signaling in mammalian cells, which emerge as important pathways during cell division, embryonic development as well as other biological processes. Mechanistically, non-proteolytic ubiquitin signals generated by CUL3 E3-ligases often regulate substrates' interactions with other downstream factors or their subcellular localization. Existing data also demonstrate an interplay with the proteolytic ubiquitylation catalyzed on the same substrates by different E3-ligases or by the same CUL3-BTB CRL3s on different substrates. In future, a deeper understanding of the upstream spatiotemporal regulatory mechanisms will help to dissect this fascinating CUL3 ubiquitin code.

The effect of different acute muscle contraction regimens on the expression of muscle proteolytic signaling proteins and genes.

Previous studies have reported that different modes of muscle contraction (i.e., eccentric or concentric contraction) with similar contraction times can affect muscle proteolytic responses. However, the effect of different contraction modes on muscle proteolytic response under the same force-time integral (FTI: contraction force × time) has not been investigated. The purpose of this study was to investigate the effect of different contraction modes, with the same FTI, on acute proteolytic signaling responses. Eleven-week-old male Sprague-Dawley rats were randomly assigned to eccentric (EC), concentric (CC), or isometric contraction (IC) groups. Different modes of muscle contraction were performed on the right gastrocnemius muscle using electrical stimulation, with the left muscle acting as a control. In order to apply an equivalent FTI, the number of stimulation sets was modified between the groups. Muscle samples were taken immediately and three hours after exercise. Phosphorylation of FoxO3a at Ser253 was significantly increased immediately after exercise compared to controls irrespective of contraction mode. The mRNA levels of the ubiquitin ligases, MuRF1, and MAFbx mRNA were unchanged by contraction mode or time. Phosphorylation of ULK1 at Ser317 (positive regulatory site) and Ser757 (negative regulatory site) was significantly increased compared to controls, immediately or 3 h after exercise, in all contraction modes. The autophagy markers (LC3B-II/I ratio and p62 expression) were unchanged, regardless of contraction mode. These data suggest that differences in contraction mode during resistance exercise with a constant FTI, are not factors in regulating proteolytic signaling in the early phase of skeletal muscle contraction.