Sialoglycan recognition is a common connection linking acidosis, zinc, and HMGB1 in sepsis.

Blood pH is tightly maintained between 7.35 and 7.45, and acidosis (pH <7.3) indicates poor prognosis in sepsis, wherein lactic acid from anoxic tissues overwhelms the buffering capacity of blood. Poor sepsis prognosis is also associated with low zinc levels and the release of High mobility group box 1 (HMGB1) from activated and/or necrotic cells. HMGB1 added to whole blood at physiological pH did not bind leukocyte receptors, but lowering pH with lactic acid to mimic sepsis conditions allowed binding, implying the presence of natural inhibitor(s) preventing binding at normal pH. Testing micromolar concentrations of divalent cations showed that zinc supported the robust binding of sialylated glycoproteins with HMGB1. Further characterizing HMGB1 as a sialic acid-binding lectin, we found that optimal binding takes place at normal blood pH and is markedly reduced when pH is adjusted with lactic acid to levels found in sepsis. Glycan array studies confirmed the binding of HMGB1 to sialylated glycan sequences typically found on plasma glycoproteins, with binding again being dependent on zinc and normal blood pH. Thus, HMGB1-mediated hyperactivation of innate immunity in sepsis requires acidosis, and micromolar zinc concentrations are protective. We suggest that the potent inflammatory effects of HMGB1 are kept in check via sequestration by plasma sialoglycoproteins at physiological pH and triggered when pH and zinc levels fall in late stages of sepsis. Current clinical trials independently studying zinc supplementation, HMGB1 inhibition, or pH normalization may be more successful if these approaches are combined and perhaps supplemented by infusions of heavily sialylated molecules.

The Alzheimer's disease-protective CD33 splice variant mediates adaptive loss of function via diversion to an intracellular pool.

The immunomodulatory receptor Siglec-3/CD33 influences risk for late-onset Alzheimer's disease (LOAD), an apparently human-specific post-reproductive disease. CD33 generates two splice variants: a full-length CD33M transcript produced primarily by the "LOAD-risk" allele and a shorter CD33m isoform lacking the sialic acid-binding domain produced primarily from the "LOAD-protective" allele. An SNP that modulates CD33 splicing to favor CD33m is associated with enhanced microglial activity. Individuals expressing more protective isoform accumulate less brain ß-amyloid and have a lower LOAD risk. How the CD33m isoform increases ß-amyloid clearance remains unknown. We report that the protection by the CD33m isoform may not be conferred by what it does but, rather, from what it cannot do. Analysis of blood neutrophils and monocytes and a microglial cell line revealed that unlike CD33M, the CD33m isoform does not localize to cell surfaces; instead, it accumulates in peroxisomes. Cell stimulation and activation did not mobilize CD33m to the surface. Thus, the CD33m isoform may neither interact directly with amyloid plaques nor engage in cell-surface signaling. Rather, production and localization of CD33m in peroxisomes is a way of diminishing the amount of CD33M and enhancing ß-amyloid clearance. We confirmed intracellular localization by generating a CD33m-specific monoclonal antibody. Of note, CD33 is the only Siglec with a peroxisome-targeting sequence, and this motif emerged by convergent evolution in toothed whales, the only other mammals with a prolonged post-reproductive lifespan. The CD33 allele that protects post-reproductive individuals from LOAD may have evolved by adaptive loss-of-function, an example of the less-is-more hypothesis.

Cyclic-AMP response element binding protein (CREB) and microRNA miR-29b regulate renalase gene expression under catecholamine excess conditions.

AIMS: Renalase, a key mediator of cross-talk between kidneys and sympathetic nervous system, exerts protective roles in various cardiovascular/renal disease states. However, molecular mechanisms underpinning renalase gene expression remain incompletely understood. Here, we sought to identify the key molecular regulators of renalase under basal/catecholamine-excess conditions. MATERIALS AND METHODS: Identification of the core promoter domain of renalase was carried out by promoter-reporter assays in N2a/HEK-293/H9c2 cells. Computational analysis of the renalase core promoter domain, over-expression of cyclic-AMP-response-element-binding-protein (CREB)/dominant negative mutant of CREB, ChIP assays were performed to determine the role of CREB in transcription regulation. Role of the miR-29b-mediated-suppression of renalase was validated in-vivo by using locked-nucleic-acid-inhibitors of miR-29. qRT-PCR and Western-blot analyses measured the expression of renalase, CREB, miR-29b and normalization controls in cell lysates/ tissue samples under basal/epinephrine-treated conditions. KEY FINDINGS: CREB, a downstream effector in epinephrine signaling, activated renalase expression via its binding to the renalase-promoter. Physiological doses of epinephrine and isoproterenol enhanced renalase-promoter activity and endogenous renalase protein level while propranolol diminished the promoter activity and endogenous renalase protein level indicating a potential role of beta-adrenergic receptor in renalase gene regulation. Multiple animal models (acute exercise, genetically hypertensive/stroke-prone mice/rat) displayed directionally-concordant expression of CREB and renalase. Administration of miR-29b inhibitor in mice upregulated endogenous renalase expression. Moreover, epinephrine treatment down-regulated miR-29b promoter-activity/transcript levels. SIGNIFICANCE: This study provides evidence for renalase gene regulation by concomitant transcriptional activation via CREB and post-transcriptional attenuation via miR-29b under excess epinephrine conditions. These findings have implications for disease states with dysregulated catecholamines.

Systematic Profiling of DNMT3A Variants Reveals Protein Instability Mediated by the DCAF8 E3 Ubiquitin Ligase Adaptor.

Clonal hematopoiesis is a prevalent age-related condition associated with a greatly increased risk of hematologic disease; mutations in DNA methyltransferase 3A (DNMT3A) are the most common driver of this state. DNMT3A variants occur across the gene with some particularly associated with malignancy, but the functional relevance and mechanisms of pathogenesis of the majority of mutations are unknown. Here, we systematically investigated the methyltransferase activity and protein stability of 253 disease-associated DNMT3A mutations, and found that 74% were loss-of-function mutations. Half of these variants exhibited reduced protein stability and, as a class, correlated with greater clonal expansion and acute myeloid leukemia development. We investigated the mechanisms underlying the instability using a CRISPR screen and uncovered regulated destruction of DNMT3A mediated by the DCAF8 E3 ubiquitin ligase adaptor. We establish a new paradigm to classify novel variants that has prognostic and potential therapeutic significance for patients with hematologic disease. SIGNIFICANCE: DNMT3A has emerged as the most important epigenetic regulator and tumor suppressor in the hematopoietic system. Our study represents a systematic and high-throughput method to characterize the molecular impact of DNMT3A missense mutations and the discovery of a regulated destruction mechanism of DNMT3A offering new prognostic and future therapeutic avenues.See related commentary by Ma and Will, p. 23.This article is highlighted in the In This Issue feature, p. 1.