Quantitative flow cytometric selection of tau conformational nanobodies specific for pathological aggregates.

Single-domain antibodies, also known as nanobodies, are broadly important for studying the structure and conformational states of several classes of proteins, including membrane proteins, enzymes, and amyloidogenic proteins. Conformational nanobodies specific for aggregated conformations of amyloidogenic proteins are particularly needed to better target and study aggregates associated with a growing class of associated diseases, especially neurodegenerative disorders such as Alzheimer's and Parkinson's diseases. However, there are few reported nanobodies with both conformational and sequence specificity for amyloid aggregates, especially for large and complex proteins such as the tau protein associated with Alzheimer's disease, due to difficulties in selecting nanobodies that bind to complex aggregated proteins. Here, we report the selection of conformational nanobodies that selectively recognize aggregated (fibrillar) tau relative to soluble (monomeric) tau. Notably, we demonstrate that these nanobodies can be directly isolated from immune libraries using quantitative flow cytometric sorting of yeast-displayed libraries against tau aggregates conjugated to quantum dots, and this process eliminates the need for secondary nanobody screening. The isolated nanobodies demonstrate conformational specificity for tau aggregates in brain samples from both a transgenic mouse model and human tauopathies. We expect that our facile approach will be broadly useful for isolating conformational nanobodies against diverse amyloid aggregates and other complex antigens.

PTEN is both an activator and a substrate of chaperone-mediated autophagy.

PTEN is a crucial negative regulator of the INS/PI3K/AKT pathway and is one of the most commonly mutated tumor suppressors in cancer. Global overexpression (OE) of PTEN in mice shifts metabolism to favor oxidative phosphorylation over glycolysis, reduces fat mass, and extends the lifespan of both sexes. We demonstrate that PTEN regulates chaperone-mediated autophagy (CMA). Using cultured cells and mouse models, we show that PTEN OE enhances CMA, dependent upon PTEN's lipid phosphatase activity and AKT inactivation. Reciprocally, PTEN knockdown reduces CMA, which can be rescued by inhibiting class I PI3K or AKT. Both PTEN and CMA are negative regulators of glycolysis and lipid droplet formation. We show that suppression of glycolysis and lipid droplet formation downstream of PTEN OE depends on CMA activity. Finally, we show that PTEN protein levels are sensitive to CMA and that PTEN accumulates in lysosomes with elevated CMA. Collectively, these data suggest that CMA is both an effector and a regulator of PTEN.

Flow cytometric isolation of drug-like conformational antibodies specific for amyloid fibrils.

Antibodies that recognize specific protein conformational states are broadly important for research, diagnostic and therapeutic applications, yet they are difficult to generate in a predictable and systematic manner using either immunization or in vitro antibody display methods. This problem is particularly severe for conformational antibodies that recognize insoluble antigens such as amyloid fibrils associated with many neurodegenerative disorders. Here we report a quantitative fluorescence-activated cell sorting (FACS) method for directly selecting high-quality conformational antibodies against different types of insoluble (amyloid fibril) antigens using a single, off-the-shelf human library. Our approach uses quantum dots functionalized with antibodies to capture insoluble antigens, and the resulting quantum dot conjugates are used in a similar manner as conventional soluble antigens for multi-parameter FACS selections. Notably, we find that this approach is robust for isolating high-quality conformational antibodies against tau and α-synuclein fibrils from the same human library with combinations of high affinity, high conformational specificity and, in some cases, low off-target binding that rival or exceed those of clinical-stage antibodies specific for tau (zagotenemab) and α-synuclein (cinpanemab). This approach is expected to enable conformational antibody selection and engineering against diverse types of protein aggregates and other insoluble antigens (e.g., membrane proteins) that are compatible with presentation on the surface of antibody-functionalized quantum dots.

Ubiquilin-2 regulates pathological alpha-synuclein.

The key protein implicated in Parkinson's disease and other synucleinopathies is α-synuclein, and a post-translationally modified form of the protein, phosphorylated at serine 129 (pS129), is a principal component in Lewy bodies, a pathological hallmark of PD. While altered proteostasis has been implicated in the etiology of Parkinson's disease, we still have a limited understanding of how α-synuclein is regulated in the nervous system. The protein quality control protein Ubiquilin-2 (UBQLN2) is known to accumulate in synucleinopathies, but whether it directly regulates α-synuclein is unknown. Using cellular and mouse models, we find that UBQLN2 decreases levels of α-synuclein, including the pS129 phosphorylated isoform. Pharmacological inhibition of the proteasome revealed that, while α-synuclein may be cleared by parallel and redundant quality control pathways, UBQLN2 preferentially targets pS129 for proteasomal degradation. Moreover, in brain tissue from human PD and transgenic mice expressing pathogenic α-synuclein (A53T), native UBQLN2 becomes more insoluble. Collectively, our studies support a role for UBQLN2 in directly regulating pathological forms of α-synuclein and indicate that UBQLN2 dysregulation in disease may contribute to α-synuclein-mediated toxicity.

Recapitulation of anti-aging phenotypes by global, but not by muscle-specific, deletion of PAPP-A in mice.

Deletion of pregnancy-associated plasma protein-A (PAPP-A), a protease that cleaves some but not all IGF1 binding proteins, postpones late-life diseases and extends lifespan in mice, but the mechanism of this effect is unknown. Here we show that PAPP-A knockout (PKO) mice display a set of changes, in multiple tissues, that are characteristic of other varieties of slow-aging mice with alterations in GH production or GH responsiveness, including Ames dwarf, Snell dwarf, and GHRKO mice. PKO mice have elevated UCP1 in brown and white adipose tissues (WAT), and a change in fat-associated macrophage subsets that leads to diminished production of inflammatory cytokines. PKO mice also show increased levels of muscle FNDC5 and its cleavage product, the myokine irisin, thought to cause changes in fat cell differentiation. PKO mice have elevated production of hepatic GPLD1 and plasma GPLD1, consistent with their elevation of hippocampal BDNF and DCX, used as indices of neurogenesis. In contrast, disruption of PAPP-A limited to muscle ("muPKO" mice) produces an unexpectedly complex set of changes, in most cases opposite in direction from those seen in PKO mice. These include declines in WAT UCP1, increases in inflammatory macrophages and cytokines in WAT, and a decline in muscle FNDC5 and plasma irisin. muPKO mice do, however, resemble global PKO mice in their elevation of hippocampal BDNF and DCX. The data for the PKO mice support the idea that these changes in fat, macrophages, liver, muscle, plasma, and brain are consistent and biologically significant features of the slow-aging phenotype in mice. The results on the muPKO mice provide a foundation for further investigation of the complex, local, and global circuits by which PAPP-A modulates signals ordinarily controlled by GH and/or IGF1.

Sirtuin 5 levels are limiting in preserving cardiac function and suppressing fibrosis in response to pressure overload.

Heart failure (HF) is the inability of the heart to pump blood sufficiently to meet the metabolic demands of the body. HF with reduced systolic function is characterized by cardiac hypertrophy, ventricular fibrosis and remodeling, and decreased cardiac contractility, leading to cardiac functional impairment and death. Transverse aortic constriction (TAC) is a well-established model for inducing hypertrophy and HF in rodents. Mice globally deficient in sirtuin 5 (SIRT5), a NAD+-dependent deacylase, are hypersensitive to cardiac stress and display increased mortality after TAC. Prior studies assessing SIRT5 functions in the heart have all employed loss-of-function approaches. In this study, we generated SIRT5 overexpressing (SIRT5OE) mice, and evaluated their response to chronic pressure overload using TAC. Compared to littermate controls, SIRT5OE mice were protected against adverse functional consequences of TAC, left ventricular dilation and impaired ejection fraction. Transcriptomic analysis revealed that SIRT5 suppresses key HF sequelae, including the metabolic switch from fatty acid oxidation to glycolysis, immune activation, and fibrotic signaling pathways. We conclude that SIRT5 is a limiting factor in the preservation of cardiac function in response to experimental pressure overload.

Canagliflozin Increases Intestinal Adenoma Burden in Female ApcMin/+ Mice.

The diabetes drug canagliflozin extends life span in male mice. Since malignant neoplasms are the major cause of death in most mouse strains, this observation suggests that canagliflozin might exert anti-neoplastic effects in male mice. Here, we treated a mouse neoplasia model, the adenoma-prone ApcMin/+ strain, with canagliflozin, to test the effects of this drug on intestinal tumor burden. Surprisingly, canagliflozin increased the total area of intestine involved by adenomas, an effect most marked in the distal intestine and in female mice. Immunohistochemical analysis suggested that canagliflozin may not influence adenoma growth via direct SGLT1/2 inhibition in neoplastic cells. Our results are most consistent with a model where canagliflozin aggravates adenoma development by altering the anatomic distribution of intestinal glucose absorption, as evidenced by increases in postprandial GLP-1 levels driven by delayed glucose absorption. We hypothesize that canagliflozin exacerbates adenomatosis in the ApcMin/+ model via complex, cell-non-autonomous mechanisms, and that sex differences in GLP-1 responses may in part underlie sexually dimorphic effects of this drug on life span.

The deacylase SIRT5 supports melanoma viability by influencing chromatin dynamics.

Cutaneous melanoma remains the most lethal skin cancer, and ranks third among all malignancies in terms of years of life lost. Despite the advent of immune checkpoint and targeted therapies, only roughly half of patients with advanced melanoma achieve a durable remission. Sirtuin 5 (SIRT5) is a member of the sirtuin family of protein deacylases that regulates metabolism and other biological processes. Germline Sirt5 deficiency is associated with mild phenotypes in mice. Here we showed that SIRT5 was required for proliferation and survival across all cutaneous melanoma genotypes tested, as well as uveal melanoma, a genetically distinct melanoma subtype that arises in the eye and is incurable once metastatic. Likewise, SIRT5 was required for efficient tumor formation by melanoma xenografts and in an autochthonous mouse Braf Pten-driven melanoma model. Via metabolite and transcriptomic analyses, we found that SIRT5 was required to maintain histone acetylation and methylation levels in melanoma cells, thereby promoting proper gene expression. SIRT5-dependent genes notably included MITF, a key lineage-specific survival oncogene in melanoma, and the c-MYC proto-oncogene. SIRT5 may represent a druggable genotype-independent addiction in melanoma.

SIRT3 Regulates Macrophage-Mediated Inflammation in Diabetic Wound Repair.

Control of inflammation is critical for the treatment of nonhealing wounds, but a delicate balance exists between early inflammation that is essential for normal tissue repair and the pathologic inflammation that can occur later in the repair process. This necessitates the development of novel therapies that can target inflammation at the appropriate time during repair. Here, we found that SIRT3 is essential for normal healing and regulates inflammation in wound macrophages after injury. Under prediabetic conditions, SIRT3 was decreased in wound macrophages and resulted in dysregulated inflammation. In addition, we found that FABP4 regulates SIRT3 in human blood monocytes, and inhibition of FABP4 in wound macrophages decreases inflammatory cytokine expression, making FABP4 a viable target for the regulation of excess inflammation and wound repair in diabetes. Using a series of ex vivo and in vivo studies with genetically engineered mouse models and diabetic human monocytes, we showed that FABP4 expression is epigenetically upregulated in diabetic wound macrophages and, in turn, diminishes SIRT3 expression, thereby promoting inflammation. These findings have significant implications for controlling inflammation and promoting tissue repair in diabetic wounds.

Sirtuins: guardians of mammalian healthspan.

The first link between sirtuins and longevity was made 15 years ago in yeast. These initial studies sparked efforts by many laboratories working in diverse model organisms to elucidate the relations between sirtuins, lifespan, and age-associated dysfunction. Here, we discuss the current understanding of how sirtuins relate to aging. We focus primarily on mammalian sirtuins SIRT1, SIRT3, and SIRT6, the three sirtuins for which the most relevant data are available. Strikingly, a large body of evidence now indicates that these and other mammalian sirtuins suppress a variety of age-related pathologies and promote healthspan. Moreover, increased expression of SIRT1 or SIRT6 extends mouse lifespan. Overall, these data point to important roles for sirtuins in promoting mammalian health, and perhaps in modulating the aging process.

Mass spectrometry-based detection of protein acetylation.

Improved sample preparation techniques and increasingly sensitive mass spectrometry (MS) analysis have revolutionized the study of protein post-translational modifications (PTMs) (Rush et al., Nat Biotechnol 23:94-101, 2005). Here, we describe a general approach for immunopurification and MS-based identification of acetylated proteins in biological samples. This approach is useful to characterize changes in the acetylome in response to biological interventions (Schwer et al., Aging Cell 8:604-606, 2009).

SIRT5-mediated lysine desuccinylation impacts diverse metabolic pathways.

Protein function is regulated by diverse posttranslational modifications. The mitochondrial sirtuin SIRT5 removes malonyl and succinyl moieties from target lysines. The spectrum of protein substrates subject to these modifications is unknown. We report systematic profiling of the mammalian succinylome, identifying 2,565 succinylation sites on 779 proteins. Most of these do not overlap with acetylation sites, suggesting differential regulation of succinylation and acetylation. Our analysis reveals potential impacts of lysine succinylation on enzymes involved in mitochondrial metabolism; e.g., amino acid degradation, the tricarboxylic acid cycle (TCA) cycle, and fatty acid metabolism. Lysine succinylation is also present on cytosolic and nuclear proteins; indeed, we show that a substantial fraction of SIRT5 is extramitochondrial. SIRT5 represses biochemical activity of, and cellular respiration through, two protein complexes identified in our analysis, pyruvate dehydrogenase complex and succinate dehydrogenase. Our data reveal widespread roles for lysine succinylation in regulating metabolism and potentially other cellular functions.

Deep sequencing of voodoo lily (Amorphophallus konjac): an approach to identify relevant genes involved in the synthesis of the hemicellulose glucomannan.

A Roche 454 cDNA deep sequencing experiment was performed on a developing corm of Amorphophallus konjac--also known as voodoo lily. The dominant storage polymer in the corm of this plant is the polysaccharide glucomannan, a hemicellulose known to exist in the cell walls of higher plants and a major component of plant biomass derived from softwoods. A total of 246 mega base pairs of sequence data was obtained from which 4,513 distinct contigs were assembled. Within this voodoo lily expressed sequence tag collection genes representing the carbohydrate related pathway of glucomannan biosynthesis were identified, including sucrose metabolism, nucleotide sugar conversion pathways for the formation of activated precursors as well as a putative glucomannan synthase. In vivo expression of the putative glucomannan synthase and subsequent in vitro activity assays unambiguously demonstrate that the enzyme has indeed glucomannan mannosyl- and glucosyl transferase activities. Based on the expressed sequence tag analysis hitherto unknown pathways for the synthesis of GDP-glucose, a necessary precursor for glucomannan biosynthesis, could be proposed. Moreover, the results highlight transcriptional bottlenecks for the synthesis of this hemicellulose.