Proteomic discovery of chemical probes that perturb protein complexes in human cells.

Most human proteins lack chemical probes, and several large-scale and generalizable small-molecule binding assays have been introduced to address this problem. How compounds discovered in such "binding-first" assays affect protein function, nonetheless, often remains unclear. Here, we describe a "function-first" proteomic strategy that uses size exclusion chromatography (SEC) to assess the global impact of electrophilic compounds on protein complexes in human cells. Integrating the SEC data with cysteine-directed activity-based protein profiling identifies changes in protein-protein interactions that are caused by site-specific liganding events, including the stereoselective engagement of cysteines in PSME1 and SF3B1 that disrupt the PA28 proteasome regulatory complex and stabilize a dynamic state of the spliceosome, respectively. Our findings thus show how multidimensional proteomic analysis of focused libraries of electrophilic compounds can expedite the discovery of chemical probes with site-specific functional effects on protein complexes in human cells.

A class of anti-inflammatory lipids decrease with aging in the central nervous system.

Lipids contribute to the structure, development, and function of healthy brains. Dysregulated lipid metabolism is linked to aging and diseased brains. However, our understanding of lipid metabolism in aging brains remains limited. Here we examined the brain lipidome of mice across their lifespan using untargeted lipidomics. Co-expression network analysis highlighted a progressive decrease in 3-sulfogalactosyl diacylglycerols (SGDGs) and SGDG pathway members, including the potential degradation products lyso-SGDGs. SGDGs show an age-related decline specifically in the central nervous system and are associated with myelination. We also found that an SGDG dramatically suppresses LPS-induced gene expression and release of pro-inflammatory cytokines from macrophages and microglia by acting on the NF-κB pathway. The detection of SGDGs in human and macaque brains establishes their evolutionary conservation. This work enhances interest in SGDGs regarding their roles in aging and inflammatory diseases and highlights the complexity of the brain lipidome and potential biological functions in aging.

Mitochondrial DNA variation in Alzheimer's disease reveals a unique microprotein called SHMOOSE.

Mitochondrial DNA variants have previously associated with disease, but the underlying mechanisms have been largely elusive. Here, we report that mitochondrial SNP rs2853499 associated with Alzheimer's disease (AD), neuroimaging, and transcriptomics. We mapped rs2853499 to a novel mitochondrial small open reading frame called SHMOOSE with microprotein encoding potential. Indeed, we detected two unique SHMOOSE-derived peptide fragments in mitochondria by using mass spectrometry-the first unique mass spectrometry-based detection of a mitochondrial-encoded microprotein to date. Furthermore, cerebrospinal fluid (CSF) SHMOOSE levels in humans correlated with age, CSF tau, and brain white matter volume. We followed up on these genetic and biochemical findings by carrying out a series of functional experiments. SHMOOSE acted on the brain following intracerebroventricular administration, differentiated mitochondrial gene expression in multiple models, localized to mitochondria, bound the inner mitochondrial membrane protein mitofilin, and boosted mitochondrial oxygen consumption. Altogether, SHMOOSE has vast implications for the fields of neurobiology, Alzheimer's disease, and microproteins.

Biochemistry and Protein Interactions of the CYREN Microprotein.

Over the past decade, advances in genomics have identified thousands of additional protein-coding small open reading frames (smORFs) missed by traditional gene finding approaches. These smORFs encode peptides and small proteins, commonly termed micropeptides or microproteins. Several of these newly discovered microproteins have biological functions and operate through interactions with proteins and protein complexes within the cell. CYREN1 is a characterized microprotein that regulates double-strand break repair in mammalian cells through interaction with Ku70/80 heterodimer. Ku70/80 binds to and stabilizes double-strand breaks and recruits the machinery needed for nonhomologous end join repair. In this study, we examined the biochemical properties of CYREN1 to better understand and explain its cellular protein interactions. Our findings support that CYREN1 is an intrinsically disordered microprotein and this disordered structure allows it to enriches several proteins, including a newly discovered interaction with SF3B1 via a distinct short linear motif (SLiMs) on CYREN1. Since many microproteins are predicted to be disordered, CYREN1 is an exemplar of how microproteins interact with other proteins and reveals an unknown scaffolding function of this microprotein that may link NHEJ and splicing.

AIG1 and ADTRP are endogenous hydrolases of fatty acid esters of hydroxy fatty acids (FAHFAs) in mice.

Fatty acid esters of hydroxy fatty acids (FAHFAs) are a newly discovered class of signaling lipids with anti-inflammatory and anti-diabetic properties. However, the endogenous regulation of FAHFAs remains a pressing but unanswered question. Here, using MS-based FAHFA hydrolysis assays, LC-MS-based lipidomics analyses, and activity-based protein profiling, we found that androgen-induced gene 1 (AIG1) and androgen-dependent TFPI-regulating protein (ADTRP), two threonine hydrolases, control FAHFA levels in vivo in both genetic and pharmacologic mouse models. Tissues from mice lacking ADTRP (Adtrp-KO), or both AIG1 and ADTRP (DKO) had higher concentrations of FAHFAs particularly isomers with the ester bond at the 9th carbon due to decreased FAHFA hydrolysis activity. The levels of other lipid classes were unaltered indicating that AIG1 and ADTRP specifically hydrolyze FAHFAs. Complementing these genetic studies, we also identified a dual AIG1/ADTRP inhibitor, ABD-110207, which is active in vivo Acute treatment of WT mice with ABD-110207 resulted in elevated FAHFA levels, further supporting the notion that AIG1 and ADTRP activity control endogenous FAHFA levels. However, loss of AIG1/ADTRP did not mimic the changes associated with pharmacologically administered FAHFAs on extent of upregulation of FAHFA levels, glucose tolerance, or insulin sensitivity in mice, indicating that therapeutic strategies should weigh more on FAHFA administration. Together, these findings identify AIG1 and ADTRP as the first endogenous FAHFA hydrolases identified and provide critical genetic and chemical tools for further characterization of these enzymes and endogenous FAHFAs to unravel their physiological functions and roles in health and disease.

HtrA1 Proteolysis of ApoE In Vitro Is Allele Selective.

Apolipoprotein E (ApoE) belongs to a large class of proteins that solubilize lipids for physiological transport. Humans have three different APOE alleles, APOE ε2, APOE ε3, and APOE ε4, and genetic studies identified ApoE4 as the strongest genetic risk factor for Alzheimer's disease (AD). People who are homozygous for ApoE4 (i.e., ApoE4/E4) are an order of magnitude more likely to develop late-onset AD (LOAD) than ApoE3/E3 carriers. Several differences between ApoE3 and ApoE4 may contribute to AD including the observation that ApoE4 is degraded to a greater extent than ApoE3 in the human brain. Experiments with high-temperature requirement serine peptidase A1 (HtrA1), which is found in the nervous system, demonstrate that HtrA1 is an allele-selective ApoE-degrading enzyme that degrades ApoE4 more quickly than ApoE3. This activity is specific to HtrA1, as similar assays with HtrA2 showed minimal ApoE4 proteolysis and trypsin had no preference between ApoE4 and ApoE3. HtrA1 has also been reported to cleave the tau protein (Tau) and the amyloid protein precursor (APP) to hinder the formation of toxic amyloid deposits associated with AD. Competition assays with ApoE4, ApoE3, and Tau revealed that ApoE4 inhibits Tau degradation. Thus, the identification of ApoE4 as an in vitro HtrA1 substrate suggests a potential biochemical mechanism that links ApoE4 regulation of AD proteins such as Tau.

Improved Identification and Analysis of Small Open Reading Frame Encoded Polypeptides.

Computational, genomic, and proteomic approaches have been used to discover nonannotated protein-coding small open reading frames (smORFs). Some novel smORFs have crucial biological roles in cells and organisms, which motivates the search for additional smORFs. Proteomic smORF discovery methods are advantageous because they detect smORF-encoded polypeptides (SEPs) to validate smORF translation and SEP stability. Because SEPs are shorter and less abundant than average proteins, SEP detection using proteomics faces unique challenges. Here, we optimize several steps in the SEP discovery workflow to improve SEP isolation and identification. These changes have led to the detection of several new human SEPs (novel human genes), improved confidence in the SEP assignments, and enabled quantification of SEPs under different cellular conditions. These improvements will allow faster detection and characterization of new SEPs and smORFs.

Characterization of a Pachymedusa dacnicolor-Sauvagine analog as a new high-affinity radioligand for corticotropin-releasing factor receptor studies.

The corticotropin-releasing factor (CRF) peptide family comprises the mammalian peptides CRF and the urocortins as well as frog skin sauvagine and fish urophyseal urotensin. Advances in understanding the roles of the CRF ligand family and associated receptors have often relied on radioreceptor assays using labeled CRF ligands. These assays depend on stable, high-affinity CRF analogs that can be labeled, purified, and chemically characterized. Analogs of several of the native peptides have been used in this context, most prominently including sauvagine from the frog Phyllomedusa sauvageii (PS-Svg). Because each of these affords both advantages and disadvantages, new analogs with superior properties would be welcome. We find that a sauvagine-like peptide recently isolated from a different frog species, Pachymedusa dacnicolor (PD-Svg), is a high-affinity agonist whose radioiodinated analog, [(125)ITyr(0)-Glu(1), Nle(17)]-PD-Svg, exhibits improved biochemical properties over those of earlier iodinated agonists. Specifically, the PD-Svg radioligand binds both CRF receptors with comparably high affinity as its PS-Svg counterpart, but detects a greater number of sites on both type 1 and type 2 receptors. PD-Svg is also ∼10 times more potent at stimulating cAMP accumulation in cells expressing the native receptors. Autoradiographic localization using the PD-Svg radioligand shows robust specific binding to rodent brain and peripheral tissues that identifies consensus CRF receptor-expressing sites in a greater number and/or with greater sensitivity than its PS-Svg counterpart. We suggest that labeled analogs of PD-Svg may be useful tools for biochemical, structural, pharmacological, and anatomic studies of CRF receptors.

An Inner Mitochondrial Membrane Microprotein from the SLC35A4 Upstream ORF Regulates Cellular Metabolism.

Upstream open reading frames (uORFs) are cis-acting elements that can dynamically regulate the translation of downstream ORFs by suppressing downstream translation under basal conditions and, in some cases, increasing downstream translation under stress conditions. Computational and empirical methods have identified uORFs in the 5'-UTRs of approximately half of all mouse and human transcripts, making uORFs one of the largest regulatory elements known. Because the prevailing dogma was that eukaryotic mRNAs produce a single functional protein, the peptides and small proteins, or microproteins, encoded by uORFs were rarely studied. We hypothesized that a uORF in the SLC35A4 mRNA is producing a functional microprotein (SLC35A4-MP) because of its conserved amino acid sequence. Through a series of biochemical and cellular experiments, we find that the 103-amino acid SLC35A4-MP is a single-pass transmembrane inner mitochondrial membrane (IMM) microprotein. The IMM contains the protein machinery crucial for cellular respiration and ATP generation, and loss of function studies with SLC35A4-MP significantly diminish maximal cellular respiration, indicating a vital role for this microprotein in cellular metabolism. The findings add SLC35A4-MP to the growing list of functional microproteins and, more generally, indicate that uORFs that encode conserved microproteins are an untapped reservoir of functional microproteins.

CRFR1 is expressed on pancreatic beta cells, promotes beta cell proliferation, and potentiates insulin secretion in a glucose-dependent manner.

Corticotropin-releasing factor (CRF), originally characterized as the principal neuroregulator of the hypothalamus-pituitary-adrenal axis, has broad central and peripheral distribution and actions. We demonstrate the presence of CRF receptor type 1 (CRFR1) on primary beta cells and show that activation of pancreatic CRFR1 promotes insulin secretion, thus contributing to the restoration of normoglycemic equilibrium. Stimulation of pancreatic CRFR1 initiates a cAMP response that promotes insulin secretion in vitro and in vivo and leads to the phosphorylation of cAMP response element binding and the induction of the expression of several immediate-early genes. Thus, the insulinotropic actions of pancreatic CRFR1 oppose the activation of CRFR1 on anterior pituitary corticotropes, leading to the release of glucocorticoids that functionally antagonize the actions of insulin. Stimulation of the MIN6 insulinoma line and primary rat islets with CRF also activates the MAPK signaling cascade leading to rapid phosphorylation of Erk1/2 in response to CRFR1-selective ligands, which induce proliferation in primary rat neonatal beta cells. Importantly, CRFR1 stimulates insulin secretion only during conditions of intermediate to high ambient glucose, and the CRFR1-dependent phosphorylation of Erk1/2 is greater with elevated glucose concentrations. This response is reminiscent of the actions of the incretins, which potentiate insulin secretion only during elevated glucose conditions. The presence of CRFR1 on beta cells adds another layer of complexity to the intricate network of paracrine and autocrine factors and their cognate receptors whose coordinated efforts can dictate islet hormone output and regulate beta cell proliferation.

The TINCR ubiquitin-like microprotein is a tumor suppressor in squamous cell carcinoma.

The TINCR (Terminal differentiation-Induced Non-Coding RNA) gene is selectively expressed in epithelium tissues and is involved in the control of human epidermal differentiation and wound healing. Despite its initial report as a long non-coding RNA, the TINCR locus codes for a highly conserved ubiquitin-like microprotein associated with keratinocyte differentiation. Here we report the identification of TINCR as a tumor suppressor in squamous cell carcinoma (SCC). TINCR is upregulated by UV-induced DNA damage in a TP53-dependent manner in human keratinocytes. Decreased TINCR protein expression is prevalently found in skin and head and neck squamous cell tumors and TINCR expression suppresses the growth of SCC cells in vitro and in vivo. Consistently, Tincr knockout mice show accelerated tumor development following UVB skin carcinogenesis and increased penetrance of invasive SCCs. Finally, genetic analyses identify loss-of-function mutations and deletions encompassing the TINCR gene in SCC clinical samples supporting a tumor suppressor role in human cancer. Altogether, these results demonstrate a role for TINCR as protein coding tumor suppressor gene recurrently lost in squamous cell carcinomas.

Profiling mouse brown and white adipocytes to identify metabolically relevant small ORFs and functional microproteins.

Microproteins (MPs) are a potentially rich source of uncharacterized metabolic regulators. Here, we use ribosome profiling (Ribo-seq) to curate 3,877 unannotated MP-encoding small ORFs (smORFs) in primary brown, white, and beige mouse adipocytes. Of these, we validated 85 MPs by proteomics, including 33 circulating MPs in mouse plasma. Analyses of MP-encoding mRNAs under different physiological conditions (high-fat diet) revealed that numerous MPs are regulated in adipose tissue in vivo and are co-expressed with established metabolic genes. Furthermore, Ribo-seq provided evidence for the translation of Gm8773, which encodes a secreted MP that is homologous to human and chicken FAM237B. Gm8773 is highly expressed in the arcuate nucleus of the hypothalamus, and intracerebroventricular administration of recombinant mFAM237B showed orexigenic activity in obese mice. Together, these data highlight the value of this adipocyte MP database in identifying MPs with roles in fundamental metabolic and physiological processes such as feeding.

Transcriptional co-activator regulates melanocyte differentiation and oncogenesis by integrating cAMP and MAPK/ERK pathways.

The cyclic AMP pathway promotes melanocyte differentiation by activating CREB and the cAMP-regulated transcription co-activators 1-3 (CRTC1-3). Differentiation is dysregulated in melanomas, although the contributions of CRTC proteins is unclear. We report a selective differentiation impairment in CRTC3 KO melanocytes and melanoma cells, due to downregulation of oculo-cutaneous albinism II (OCA2) and block of melanosome maturation. CRTC3 stimulates OCA2 expression by binding to CREB on a conserved enhancer, a regulatory site for pigmentation and melanoma risk. CRTC3 is uniquely activated by ERK1/2-mediated phosphorylation at Ser391 and by low levels of cAMP. Phosphorylation at Ser391 is constitutively elevated in human melanoma cells with hyperactivated ERK1/2 signaling; knockout of CRTC3 in this setting impairs anchorage-independent growth, migration, and invasiveness, whereas CRTC3 overexpression supports cell survival in response to the mitogen-activated protein kinase (MAPK) inhibitor vemurafenib. As melanomas expressing gain-of-function mutations in CRTC3 are associated with reduced survival, our results suggest that CRTC3 inhibition may provide therapeutic benefit in this setting.

Injection of Urocortin 3 into the ventromedial hypothalamus modulates feeding, blood glucose levels, and hypothalamic POMC gene expression but not the HPA axis.

Urocortin 3 (Ucn 3) is a corticotropin-releasing factor (CRF)-related peptide with high affinity for the type 2 CRF receptor (CRFR2). Central administration of Ucn 3 stimulates the hypothalamic-pituitary-adrenal axis, suppresses feeding, and elevates blood glucose levels, suggesting that activation of brain CRFR2 promotes stress-like responses. Several CRFR2-expressing brain areas, including the ventromedial hypothalamus (VMH) and the posterior amygdala (PA), may be potential sites mediating the effects of Ucn 3. In the present study, Ucn 3 or vehicle was bilaterally injected into the VMH or PA, and food intake and plasma levels of ACTH, corticosterone, glucose, and insulin were determined. Food intake was greatly reduced in rats following Ucn 3 injection into the VMH. Ucn 3 injection into the VMH rapidly elevated plasma levels of glucose and insulin but did not affect ACTH and corticosterone secretion. Injection of Ucn 3 into the PA did not alter any of the parameters measured. We determined that the majority of CRFR2-positive neurons in the VMH were excitatory glutamatergic, and a subset of these neurons project to the arcuate nucleus of the hypothalamus (ARH). Importantly, stimulation of CRFR2 in the VMH increased proopiomelanocortin mRNA expression in the ARH. In conclusion, the present study demonstrates that CRFR2 in the VMH mediates some of the central effects of Ucn 3, and the ARH melanocortin system may be a downstream target of VMH CRFR2 neurons.

A novel corticotropin-releasing factor receptor splice variant exhibits dominant negative activity: a putative link to stress-induced heart disease.

A growing body of experimental and clinical studies supports a strong association between psychological stress and cardiovascular disease. An important endogenous cardioprotective role in heart physiology has been attributed to corticotropin-releasing factor receptor type 2beta (CRFR2beta). Here, we report the isolation of cDNA from mouse (m) heart encoding a novel CRFR2beta splice variant. Translation of this insertion variant (iv)-mCRFR2beta isoform produces a 421-aa protein that includes a unique C-terminal cytoplasmic tail. Our functional analysis and cellular localization studies demonstrated that when coexpressed with wild-type mCRFR2beta, iv-mCRFR2beta significantly inhibited the wild-type mCRFR2beta membrane expression and its functional signaling by ER-Golgi complex retention, suggesting a dose-dependent dominant negative effect. Interestingly, mice exposed to a 4-wk paradigm of chronic variable stress, a model of chronic psychological stress in humans, presented significantly lower levels of mCRFR2beta and higher levels of iv-mCRFR2beta mRNA expression in their hearts, compared to nonstressed control mice. The dominant-negative effect of iv-mCRFR2beta and its up-regulation by psychological stress suggest a new form of regulation of the mCRFR2beta cardioprotective effect and a potential role for this novel isoform in stress-induced heart disease.

The Activin Receptor, Activin-Like Kinase 4, Mediates Toxoplasma Gondii Activation of Hypoxia Inducible Factor-1.

To grow and cause disease, intracellular pathogens modulate host cell processes. Identifying these processes as well as the mechanisms used by the pathogens to manipulate them is important for the development of more effective therapeutics. As an example, the intracellular parasite Toxoplasma gondii induces a wide variety of changes to its host cell, including altered membrane trafficking, cytoskeletal reorganization, and differential gene expression. Although several parasite molecules and their host targets have been identified that mediate- these changes, few are known to be required for parasite replication. One exception is the host cell transcription factor, hypoxia-inducible factor-1 (HIF-1), which is required for parasite replication in an oxygen-dependent manner. Toxoplasma activates HIF-1 by stabilizing the HIF-1α subunit, and this is dependent on the signaling from the Activin-Like Kinase (ALK) receptor superfamily. Here, we demonstrate that specific overexpression of the ALK family member, ALK4, increased HIF-1 activity in Toxoplasma-infected cells, and this increase required ALK4 kinase activity. Moreover, Toxoplasma stimulated ALK4 to dimerize with its co-receptor, ActRII, and also increased ALK4 kinase activity, thereby demonstrating that Toxoplasma activates the ALK4 receptor. ALK4 activation of HIF-1 was independent of canonical SMAD signaling but rather was dependent on the non-canonical Rho GTPase and JNK MAP kinase signaling pathways. Finally, Toxoplasma increased rates of ALK4 ubiquitination and turnover. These data provide the first evidence indicating that ALK4 signaling is a target for a microbial pathogen to manipulate its host cell.

Mitogenic Signals Stimulate the CREB Coactivator CRTC3 through PP2A Recruitment.

The second messenger 3',5'-cyclic adenosine monophosphate (cAMP) stimulates gene expression via the cAMP-regulated transcriptional coactivator (CRTC) family of cAMP response element-binding protein coactivators. In the basal state, CRTCs are phosphorylated by salt-inducible kinases (SIKs) and sequestered in the cytoplasm by 14-3-3 proteins. cAMP signaling inhibits the SIKs, leading to CRTC dephosphorylation and nuclear translocation. Here we show that although all CRTCs are regulated by SIKs, their interactions with Ser/Thr-specific protein phosphatases are distinct. CRTC1 and CRTC2 associate selectively with the calcium-dependent phosphatase calcineurin, whereas CRTC3 interacts with B55 PP2A holoenzymes via a conserved PP2A-binding region (amino acids 380-401). CRTC3-PP2A complex formation was induced by phosphorylation of CRTC3 at S391, facilitating the subsequent activation of CRTC3 by dephosphorylation at 14-3-3 binding sites. As stimulation of mitogenic pathways promoted S391 phosphorylation via the activation of ERKs and CDKs, our results demonstrate how a ubiquitous phosphatase enables cross talk between growth factor and cAMP signaling pathways at the level of a transcriptional coactivator.

Regulation of the ER stress response by a mitochondrial microprotein.

Cellular homeostasis relies on having dedicated and coordinated responses to a variety of stresses. The accumulation of unfolded proteins in the endoplasmic reticulum (ER) is a common stress that triggers a conserved pathway called the unfolded protein response (UPR) that mitigates damage, and dysregulation of UPR underlies several debilitating diseases. Here, we discover that a previously uncharacterized 54-amino acid microprotein PIGBOS regulates UPR. PIGBOS localizes to the mitochondrial outer membrane where it interacts with the ER protein CLCC1 at ER-mitochondria contact sites. Functional studies reveal that the loss of PIGBOS leads to heightened UPR and increased cell death. The characterization of PIGBOS reveals an undiscovered role for a mitochondrial protein, in this case a microprotein, in the regulation of UPR originating in the ER. This study demonstrates microproteins to be an unappreciated class of genes that are critical for inter-organelle communication, homeostasis, and cell survival.