Microtubule-Associated Serine/Threonine (MAST) Kinases in Development and Disease.

Microtubule-Associated Serine/Threonine (MAST) kinases represent an evolutionary conserved branch of the AGC protein kinase superfamily in the kinome. Since the discovery of the founding member, MAST2, in 1993, three additional family members have been identified in mammals and found to be broadly expressed across various tissues, including the brain, heart, lung, liver, intestine and kidney. The study of MAST kinases is highly relevant for unraveling the molecular basis of a wide range of different human diseases, including breast and liver cancer, myeloma, inflammatory bowel disease, cystic fibrosis and various neuronal disorders. Despite several reports on potential substrates and binding partners of MAST kinases, the molecular mechanisms that would explain their involvement in human diseases remain rather obscure. This review will summarize data on the structure, biochemistry and cell and molecular biology of MAST kinases in the context of biomedical research as well as organismal model systems in order to provide a current profile of this field.

Mutant Phosphodiesterase 3A Protects From Hypertension-Induced Cardiac Damage.

BACKGROUND: Phosphodiesterase 3A (PDE3A) gain-of-function mutations cause hypertension with brachydactyly (HTNB) and lead to stroke. Increased peripheral vascular resistance, rather than salt retention, is responsible. It is surprising that the few patients with HTNB examined so far did not develop cardiac hypertrophy or heart failure. We hypothesized that, in the heart, PDE3A mutations could be protective. METHODS: We studied new patients. CRISPR-Cas9-engineered rat HTNB models were phenotyped by telemetric blood pressure measurements, echocardiography, microcomputed tomography, RNA-sequencing, and single nuclei RNA-sequencing. Human induced pluripotent stem cells carrying PDE3A mutations were established, differentiated to cardiomyocytes, and analyzed by Ca2+ imaging. We used Förster resonance energy transfer and biochemical assays. RESULTS: We identified a new PDE3A mutation in a family with HTNB. It maps to exon 13 encoding the enzyme's catalytic domain. All hitherto identified HTNB PDE3A mutations cluster in exon 4 encoding a region N-terminally from the catalytic domain of the enzyme. The mutations were recapitulated in rat models. Both exon 4 and 13 mutations led to aberrant phosphorylation, hyperactivity, and increased PDE3A enzyme self-assembly. The left ventricles of our patients with HTNB and the rat models were normal despite preexisting hypertension. A catecholamine challenge elicited cardiac hypertrophy in HTNB rats only to the level of wild-type rats and improved the contractility of the mutant hearts, compared with wild-type rats. The ß-adrenergic system, phosphodiesterase activity, and cAMP levels in the mutant hearts resembled wild-type hearts, whereas phospholamban phosphorylation was decreased in the mutants. In our induced pluripotent stem cell cardiomyocyte models, the PDE3A mutations caused adaptive changes of Ca2+ cycling. RNA-sequencing and single nuclei RNA-sequencing identified differences in mRNA expression between wild-type and mutants, affecting, among others, metabolism and protein folding. CONCLUSIONS: Although in vascular smooth muscle, PDE3A mutations cause hypertension, they confer protection against hypertension-induced cardiac damage in hearts. Nonselective PDE3A inhibition is a final, short-term option in heart failure treatment to increase cardiac cAMP and improve contractility. Our data argue that mimicking the effect of PDE3A mutations in the heart rather than nonselective PDE3 inhibition is cardioprotective in the long term. Our findings could facilitate the search for new treatments to prevent hypertension-induced cardiac damage.

The Tails of Protein Kinase A.

Protein kinase A (PKA) is a holoenzyme consisting of a regulatory (R)-subunit dimer and two catalytic (C)-subunits. There are two major families of C-subunits, Cα and Cß, and four functionally nonredundant R-subunits (RIα, RIß, RIIα, RIIß). In addition to binding to and being regulated by the R-subunits, the C-subunits are regulated by two tail regions that each wrap around the N- and C-lobes of the kinase core. Although the C-terminal (Ct-) tail is classified as an intrinsically disordered region (IDR), the N-terminal (Nt-) tail is dominated by a strong helix that is flanked by short IDRs. In contrast to the Ct-tail, which is a conserved and highly regulated feature of all PKA, PKG, and protein kinase C protein kinase group (AGC) kinases, the Nt-tail has evolved more recently and is highly variable in vertebrates. Surprisingly and in contrast to the kinase core and the Ct-tail, the entire Nt-tail is not conserved in nonmammalian PKAs. In particular, in humans, Cß actually represents a large family of C-subunits that are highly variable in their Nt-tail and also expressed in a highly tissue-specific manner. Although we know so much about the Cα1-subunit, we know almost nothing about these Cß isoforms wherein Cß2 is highly expressed in lymphocytes, and Cß3 and Cß4 isoforms account for ∼50% of PKA signaling in brain. Based on recent disease mutations, the Cß proteins appear to be functionally important and nonredundant with the Cα isoforms. Imaging in retina also supports nonredundant roles for Cß as well as isoform-specific localization to mitochondria. This represents a new frontier in PKA signaling. SIGNIFICANCE STATEMENT: How tails and adjacent domains regulate each protein kinase is a fundamental challenge for the biological community. Here we highlight how the N- and C-terminal tails of PKA (Nt-tails/Ct-tails) affect the structure and regulate the function of the kinase core and show the combinatorial variations that are introduced into the Nt-tail of the Cα- and Cß-subunits in contrast to the Ct-tail, which is conserved across the entire AGC subfamily of protein kinases.

Binding of the Human 14-3-3 Isoforms to Distinct Sites in the Leucine-Rich Repeat Kinase 2.

Proteins of the 14-3-3 family are well known modulators of the leucine-rich repeat kinase 2 (LRRK2) regulating kinase activity, cellular localization, and ubiquitylation. Although binding between those proteins has been investigated, a comparative study of all human 14-3-3 isoforms interacting with LRRK2 is lacking so far. In a comprehensive approach, we quantitatively analyzed the interaction between the seven human 14-3-3 isoforms and LRRK2-derived peptides covering both, reported and putative 14-3-3 binding sites. We observed that phosphorylation is an absolute prerequisite for 14-3-3 binding and generated binding patterns of 14-3-3 isoforms to interact with peptides derived from the N-terminal phosphorylation cluster (S910 and S935), the Roc domain (S1444) and the C-terminus. The tested 14-3-3 binding sites in LRRK2 preferentially were recognized by the isoforms γ and η, whereas the isoforms ϵ and especially σ showed the weakest or no binding. Interestingly, the possible pathogenic mutation Q930R in LRRK2 drastically increases binding affinity to a peptide encompassing pS935. We then identified the autophosphorylation site T2524 as a so far not described 14-3-3 binding site at the very C-terminus of LRRK2. Binding affinities of all seven 14-3-3 isoforms were quantified for all three binding regions with pS1444 displaying the highest affinity of all measured singly phosphorylated peptides. The strongest binding was detected for the combined phosphosites S910 and S935, suggesting that avidity effects are important for high affinity interaction between 14-3-3 proteins and LRRK2.

The short-chain fatty acid pentanoate suppresses autoimmunity by modulating the metabolic-epigenetic crosstalk in lymphocytes.

Short-chain fatty acids (SCFAs) have immunomodulatory effects, but the underlying mechanisms are not well understood. Here we show that pentanoate, a physiologically abundant SCFA, is a potent regulator of immunometabolism. Pentanoate induces IL-10 production in lymphocytes by reprogramming their metabolic activity towards elevated glucose oxidation. Mechanistically, this reprogramming is mediated by supplying additional pentanoate-originated acetyl-CoA for histone acetyltransferases, and by pentanoate-triggered enhancement of mTOR activity. In experimental mouse models of colitis and multiple sclerosis, pentanoate-induced regulatory B cells mediate protection from autoimmune pathology. Additionally, pentanoate shows a potent histone deacetylase-inhibitory activity in CD4+ T cells, thereby reducing their IL-17A production. In germ-free mice mono-colonized with segmented filamentous bacteria (SFB), pentanoate inhibits the generation of small-intestinal Th17 cells and ameliorates SFB-promoted inflammation in the central nervous system. Taken together, by enhancing IL-10 production and suppressing Th17 cells, the SCFA pentanoate might be of therapeutic relevance for inflammatory and autoimmune diseases.

Regulation of the effector function of CD8+ T cells by gut microbiota-derived metabolite butyrate.

The gut microbiota produces metabolites such as short-chain fatty acids (SCFAs) that regulate the energy homeostasis and impact on immune cell function of the host. Recently, innovative approaches based on the oral administration of SCFAs have been discussed for therapeutic modification of inflammatory immune responses in autoimmune diseases. So far, most studies have investigated the SCFA-mediated effects on CD4+ T cells and antigen presenting cells. Here we show that butyrate and, to a lesser degree, propionate directly modulate the gene expression of CD8+ cytotoxic T lymphocytes (CTLs) and Tc17 cells. Increased IFN-γ and granzyme B expression by CTLs as well as the molecular switch of Tc17 cells towards the CTL phenotype was mediated by butyrate independently of its interaction with specific SCFA-receptors GPR41 and GPR43. Our results indicate that butyrate strongly inhibited histone-deacetylases (HDACs) in CD8+ T cells thereby affecting the gene expression of effector molecules. Accordingly, the pan-HDAC inhibitors trichostatin A (TSA) and sodium valproate exerted similar influence on CD8+ T cells. Furthermore, higher acetate concentrations were also able to increase IFN-γ production in CD8+ T lymphocytes by modulating cellular metabolism and mTOR activity. These findings might have significant implications in adoptive immunotherapy of cancers and in anti-viral immunity.

Prevention of colitis-associated cancer by selective targeting of immunoproteasome subunit LMP7.

Chronic inflammation is a well-known risk factor in development of intestinal tumorigenesis, although the exact mechanisms underlying development of colitis-associated cancer (CAC) still remain obscure. The activity and function of immunoproteasome has been extensively analyzed in the context of inflammation and infectious diseases. Here, we show that the proteasomal immunosubunit LMP7 plays an essential role in development of CAC. Mice devoid of LMP7 were resistant to chronic inflammation and formation of neoplasia, and developed virtually no tumors after AOM/DSS treatment. Our data reveal that LMP7 deficiency resulted in reduced expression of pro-tumorigenic chemokines CXCL1, CXCL2 and CXCL3 as well as adhesion molecule VCAM-1. As a consequence, an impaired recruitment and activity of tumor-infiltrating leukocytes resulting in decreased secretion of cytokines IL-6 and TNF-α was observed. Further, the deletion or pharmacological inhibition of LMP7 and consequent blockade of NF-κB abrogated the production of IL-17A, which possesses a strong carcinogenic activity in the gut. Moreover, in vivo administration of the selective LMP7 inhibitor ONX-0914 led to a marked reduction of tumor numbers in wild-type (WT) mice. Collectively, we identified the immunoproteasome as a crucial mediator of inflammation-driven neoplasia highlighting a novel potential therapeutic approach to limit colonic tumorigenesis.

The Microbial Metabolite Butyrate Induces Expression of Th1-Associated Factors in CD4+ T Cells.

Short-chain fatty acids (SCFAs), which are generated by the bacterial fermentation of dietary fibers, promote expansion of regulatory T cells (Tregs). Potential therapeutic value of SCFAs has been recently highlighted in the experimental models of T cell-mediated autoimmunity and allergic inflammation. These studies suggest that physiological intestinal concentrations of SCFAs within the millimolar range are crucial for dampening inflammation-mediated processes. Here, we describe opposing effects of SCFAs on T cell-mediated immune responses. In accordance with published data, lower butyrate concentrations facilitated differentiation of Tregs in vitro and in vivo under steady-state conditions. In contrast, higher concentrations of butyrate induced expression of the transcription factor T-bet in all investigated T cell subsets resulting in IFN-γ-producing Tregs or conventional T cells. This effect was mediated by the inhibition of histone deacetylase activity and was independent of SCFA-receptors FFA2 and FFA3 as well as of Na+-coupled SCFA transporter Slc5a8. Importantly, while butyrate was not able to induce the generation of Tregs in the absence of TGF-ß1, the expression of T-bet and IFN-γ was triggered upon stimulation of CD4+ T cells with this SCFA alone. Moreover, the treatment of germ-free mice with butyrate enhanced the expression of T-bet and IFN-γ during acute colitis. Our data reveal that, depending on its concentration and immunological milieu, butyrate may exert either beneficial or detrimental effects on the mucosal immune system.

Protein Traffic to the Plasmodium falciparum apicoplast: evidence for a sorting branch point at the Golgi.

Plasmodium falciparum, similar to many other apicomplexan parasites, contains an apicoplast, a plastid organelle of secondary endosymbiotic origin. Nuclear-encoded proteins are targeted to the apicoplast by a bipartite topogenic signal consisting of (i) an endoplasmic reticulum (ER)-type N-terminal secretory signal peptide, followed by (ii) a plant-like transit peptide. Although the signals responsible for transport of most proteins to the apicoplast are well described, the route of trafficking from the ER to the outermost apicoplast membrane is still a matter of debate. Current models of trafficking to the apicoplast suggest that proteins destined for this organelle are, on entry into the lumen of the ER, diverted from the default secretory pathway to a specialized vesicular system which carries proteins directly from the ER to the outer apicoplast membrane. Here, we have re-examined this trafficking pathway. By titrating wild-type and mutant apicoplast transit peptides against different ER retrieval sequences and studying protein transport in a brefeldin A-resistant parasite line, we generated data which suggest a direct involvement of the Golgi in traffic of soluble proteins to the P. falciparum apicoplast.