The immunomodulatory receptor Siglec-3/CD33 influences risk for late-onset Alzheimer's disease (LOAD), an apparently human-specific post-reproductive disease. 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 β-amyloid and have a lower LOAD risk. How the CD33m isoform increases β-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 β-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 allele that protects post-reproductive individuals from LOAD may have evolved by adaptive loss-of-function, an example of the less-is-more hypothesis.

Huang et al; DNMT3A variants confer protein instability 1 Systematic profiling of DNMT3A variants reveals protein instability mediated by the DCAF8 E3 ubiquitin ligase adaptor

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.

Mutations in three functional domains of DNA methyltransferase 3A (DNMT3A) have been found in individuals with clonal hematopoiesis of indeterminate potential (CHIP), hematological malignancies, and cohorts of patients with overgrowth syndrome (Tatton-Brown Rahman Syndrome, or TBRS). In CHIP, the majority of DNMT3A mutations (~85%) are not at the Arginine 882 residue that is most frequently seen in AML (around 58% of DNMT3A-mutant cases). While most studies to date have focused on DNMT3A mutations in the hotspot residue R882, frameshift or premature stop codon mutations resulting in haploinsufficiency of DNMT3A also have been shown to predispose toward myeloid malignancies. However, the functional impact of these non-R882 missense mutations, and the implications for prognosis, have not yet been examined. To address this question, we generated a murine model with a single amino acid deletion of tryptophan 293 (corresponding to aa 297 in human) in the PWWP domain of DNMT3A (Dnmt3aW293Del model). We found that, similar to Dnmt3a-null mice, Dnmt3aW293Del/W293Del mice do not survive beyond postnatal day 24, suggesting that Dnmt3aW293Del is a hypomorphic mutation. In addition, Dnmt3aW293Del/+ mice recapitulated many of the features of human TBRS syndrome including obesity and neurological defects. Using methylation-deficient mouse embryonic stem cells, we re-introduced doxycycline-inducible mutant DNMT3AW297Del and observed only a negligible increase in DNA methylation, while intact DNMT3AWT protein showed a global 60% increase of DNA methylation, as measured by whole genome bisulfite sequencing. To determine the molecular mechanisms through which DNMT3AW293Del acted as DNMT3A-null mutation, we first examined both RNA and protein expression in the Dnmt3aW293Del murine model. Quantitative PCR revealed that both Dnmt3aWT and Dnmt3aW293Del mRNA were expressed at normal levels in the Dnmt3aW293Del/+ mice. Unexpectedly, however, we found that DNMT3AW293Del mutant protein could not be detected. Using bicistronic vectors to measure protein stability in human embryonic kidney 293 cells (HEK293T), we demonstrated that loss of DNMT3AW297Del protein is not due to a translational defect but to impaired protein stability. Additionally, we found application of a proteasome inhibitor could rescue expression of mutant DNMT3AW297Del expression. Furthermore, using lymphoblastoid cell lines (LCLs) derived from patients, we found DNMT3A protein expression was lower in DNMT3AW297Del/+ LCLs and that exposure to a proteasome inhibitor could increase protein levels, reinforcing the concept that deletion of aa 297 reduced DNMT3A protein stability. To determine whether reduced protein stability was a common feature of DNMT3A mutations, we examined stability of 105 additional missense mutations across three functional domains frequently mutated in CHIP, hematological malignancies, and TBRS. Surprisingly, 45 out of 105 missense DNMT3A mutations (42.9%) examined had impaired protein stability, the majority of which were located in the PWWP and catalytic domains. Using variant allelic frequencies (VAFs) from a publicly available dataset of patients with non-hematological malignancies, we found that mutants with reduced protein stability were associated with significantly higher VAFs compared to mutants with unknown function. Notably, R882 mutants had significantly higher VAFs compared to the mutants with impaired protein stability. These data indicate that individuals with R882 mutations may have the highest likelihood of conversion to malignancies, with mutants with impaired stability having moderate likelihoods, and mutants in domains with unknown function may have lowest chance to convert to malignancies and best prognosis. In this study, we show that mice with a mutation in the PWWP domain of Dnmt3a recapitulate the phenotypes of human overgrowth patients. We further discovered that a large portion of missense DNMT3A mutations have impaired protein stability. This study highlights the importance of understanding how DNMT3A protein expression is regulated and suggests proteasome inhibition may be a potential treatment for patients with particular mutations in DNMT3A, found in overgrowth syndrome, CHIP, and hematological malignancies. Disclosures No relevant conflicts of interest to declare.

Blood pH is tightly regulated between 7.35-7.45, with values below 7.3 during sepsis being associated with lactic acidosis, low serum zinc, and release of proinflammatory HMGB1 from activated and/or necrotic cells. Using an ex vivo whole blood system to model lactic acidosis, we show that while HMGB1 does not engage leukocyte receptors at physiological pH, lowering pH with lactic acid facilitates binding. At normal pH, micromolar zinc supports plasma sialoglycoprotein binding by HMGB1, which is markedly reduced when pH is adjusted with lactic acid to sepsis levels. Glycan array studies confirmed zinc and pH-dependent HMGB1 binding to sialoglycans typical of plasma glycoproteins. Thus, proinflammatory effects of HMGB1 are suppressed via plasma sialoglycoproteins until drops in pH and zinc release HMGB1 to trigger downstream immune activation.Significance StatementHMGB1 sequestered by plasma sialoglycoproteins at physiological pH is released when pH and zinc concentrations fall in sepsis.