Kinases leave their mark on caspase substrates

Apoptosis is a cell death program that is executed by the caspases, a family of cysteine proteases that typically cleave after aspartate residues during a proteolytic cascade that systematically dismantles the dying cell. Extensive signaling crosstalk occurs between caspase-mediated proteolysis and kinase-mediated phosphorylation, enabling integration of signals from multiple pathways into the decision to commit to apoptosis. A new study from Maluch et al. examines how phosphorylation within caspase cleavage sites impacts the efficiency of substrate cleavage. The results demonstrate that while phosphorylation in close proximity to the scissile bond is generally inhibitory, it does not necessarily abrogate substrate cleavage, but instead attenuates the rate. In some cases, this inhibition can be overcome by additional favorable substrate features. These findings suggest potential nuanced physiological roles for phosphorylation of caspase substrates with exciting implications for targeting caspases with chemical probes and therapeutics.

C1q binding to surface-bound IgG is stabilized by C1r2s2 proteases

Complement is an important effector mechanism for antibody-mediated clearance of infections and tumor cells. Upon binding to target cells, the antibody’s constant (Fc) domain recruits complement component C1 to initiate a proteolytic cascade that generates lytic pores and stimulates phagocytosis. The C1 complex (C1qr2s2) consists of the large recognition protein C1q and a heterotetramer of proteases C1r and C1s (C1r2s2). While interactions between C1 and IgG-Fc are believed to be mediated by the globular heads of C1q, we here find that C1r2s2 proteases affect the capacity of C1q to form an avid complex with surface-bound IgG molecules (on various 2,4-dinitrophenol [DNP]-coated surfaces and pathogenic Staphylococcus aureus). The extent to which C1r2s2 contributes to C1q–IgG stability strongly differs between human IgG subclasses. Using antibody engineering of monoclonal IgG, we reveal that hexamer-enhancing mutations improve C1q–IgG stability, both in the absence and presence of C1r2s2. In addition, hexamer-enhanced IgGs targeting S. aureus mediate improved complement-dependent phagocytosis by human neutrophils. Altogether, these molecular insights into complement binding to surface-bound IgGs could be important for optimal design of antibody therapies.

SDC mediates DNA methylation-controlled clock pace by interacting with ZTL in Arabidopsis

Molecular bases of eukaryotic circadian clocks mainly rely on transcriptional-translational feedback loops (TTFLs), while epigenetic codes also play critical roles in fine-tuning circadian rhythms. However, unlike histone modification codes that play extensive and well-known roles in the regulation of circadian clocks, whether DNA methylation (5mC) can affect the circadian clock, and the associated underlying molecular mechanisms, remains largely unexplored in many organisms. Here we demonstrate that global genome DNA hypomethylation can significantly lengthen the circadian period of Arabidopsis. Transcriptomic and genetic evidence demonstrate that SUPPRESSOR OF drm1 drm2 cmt3 (SDC), encoding an F-box containing protein, is required for the DNA hypomethylation-tuned circadian clock. Moreover, SDC can physically interact with another F-box containing protein ZEITLUPE (ZTL) to diminish its accumulation. Genetic analysis further revealed that ZTL and its substrate TIMING OF CAB EXPRESSION 1 (TOC1) likely act downstream of DNA methyltransferases to control circadian rhythm. Together, our findings support the notion that DNA methylation is important to maintain proper circadian pace in Arabidopsis, and further established that SDC links DNA hypomethylation with a proteolytic cascade to assist in tuning the circadian clock.

Nanobodies Provide Insight into the Molecular Mechanisms of the Complement Cascade and Offer New Therapeutic Strategies

The complement system is part of the innate immune response, where it provides immediate protection from infectious agents and plays a fundamental role in homeostasis. Complement dysregulation occurs in several diseases, where the tightly regulated proteolytic cascade turns offensive. Prominent examples are atypical hemolytic uremic syndrome, paroxysmal nocturnal hemoglobinuria and Alzheimer’s disease. Therapeutic intervention targeting complement activation may allow treatment of such debilitating diseases. In this review, we describe a panel of complement targeting nanobodies that allow modulation at different steps of the proteolytic cascade, from the activation of the C1 complex in the classical pathway to formation of the C5 convertase in the terminal pathway. Thorough structural and functional characterization has provided a deep mechanistic understanding of the mode of inhibition for each of the nanobodies. These complement specific nanobodies are novel powerful probes for basic research and offer new opportunities for in vivo complement modulation.

C1q binding to surface-bound IgG is stabilized by C1r2s2 proteases

Complement is an important effector mechanism for antibody-mediated clearance of infections and tumor cells. Upon binding to target cells, the antibody’s constant (Fc) domain recruits complement component C1 to initiate a proteolytic cascade that generates lytic pores and stimulates phagocytosis. The C1 complex (C1qr2s2) consists of the large recognition protein C1q and a heterotetramer of proteases C1r and C1s (C1r2s2). While interactions between C1 and IgG-Fc’s are believed to be mediated by the globular heads of C1q, we here find that C1r2s2 proteases affect the capacity of C1q to form an avid complex with surface-bound IgG molecules (on various DNP-coated surfaces and pathogenic Staphylococcus aureus). The extent to which C1r2s2 contribute to C1q-IgG stability strongly differs between human IgG subclasses. Using antibody engineering of monoclonal IgG we reveal that hexamer-enhancing mutations improve C1q-IgG stability, both in absence and presence of C1r2s2. In addition, hexamer-enhanced IgGs targeting S. aureus mediate improved complement-dependent phagocytosis by human neutrophils. Altogether, these molecular insights into complement binding to surface-bound IgGs could be important for optimal design of antibody therapies.

Nanobodies Provide Insight Into the Molecular Mechanisms of the Complement Cascade and Offer New Therapeutic Strategies

The complement system is part of the innate immune response, where it provides immediate protection from infectious agents and plays a fundamental role in homeostasis. Complement dysregulation occurs in several diseases, where the tightly regulated proteolytic cascade turns offensive. Prominent examples are atypical hemolytic uremic syndrome, paroxysmal nocturnal hemoglobinuria and Alzheimer’s disease. Therapeutic intervention targeting complement activation may allow treatment of such debilitating diseases. In this review, we describe a panel of complement targeting nanobodies that allow modulation at different steps of the proteolytic cascade, from the activation of the C1 complex in the classical pathway to formation of the C5 convertase in the terminal pathway. Thorough structural and functional characterization has provided a deep mechanistic understanding of the mode of inhibition for each of the nanobodies. These complement specific nanobodies are novel powerful probes for basic research and offer new opportunities for in vivo complement modulation.

Cathepsin B Localizes in the Caveolae and Participates in the Proteolytic Cascade in Trabecular Meshwork Cells. Potential New Drug Target for the Treatment of Glaucoma

Extracellular matrix (ECM) deposition in the trabecular meshwork (TM) is one of the hallmarks of glaucoma, a group of human diseases and leading cause of permanent blindness. The molecular mechanisms underlying ECM deposition in the glaucomatous TM are not known, but it is presumed to be a consequence of excessive synthesis of ECM components, decreased proteolytic degradation, or both. Targeting ECM deposition might represent a therapeutic approach to restore outflow facility in glaucoma. Previous work conducted in our laboratory identified the lysosomal enzyme cathepsin B (CTSB) to be expressed on the cellular surface and to be secreted into the culture media in trabecular meshwork (TM) cells. Here, we further investigated the role of CTSB on ECM remodeling and outflow physiology in vitro and in CSTBko mice. Our results indicate that CTSB localizes in the caveolae and participates in the pericellular degradation of ECM in TM cells. We also report here a novel role of CTSB in regulating the expression of PAI-1 and TGFβ/Smad signaling in TM cells vitro and in vivo in CTSBko mice. We propose enhancing CTSB activity as a novel therapeutic target to attenuate fibrosis and ECM deposition in the glaucomatous outflow pathway.