Prolyl isomerization of FAAP20 catalyzed by PIN1 regulates the Fanconi anemia pathway.

The Fanconi Anemia (FA) pathway is a multi-step DNA repair process at stalled replication forks in response to DNA interstrand cross-links (ICLs). Pathological mutation of key FA genes leads to the inherited disorder FA, characterized by progressive bone marrow failure and cancer predisposition. The study of FA is of great importance not only to children suffering from FA but also as a model to study cancer pathogenesis in light of genome instability among the general population. FANCD2 monoubiquitination by the FA core complex is an essential gateway that connects upstream DNA damage signaling to enzymatic steps of repair. FAAP20 is a key component of the FA core complex, and regulated proteolysis of FAAP20 mediated by the ubiquitin E3 ligase SCFFBW7 is critical for maintaining the integrity of the FA complex and FA pathway signaling. However, upstream regulatory mechanisms that govern this signaling remain unclear. Here, we show that PIN1, a phosphorylation-specific prolyl isomerase, regulates the integrity of the FA core complex, thus FA pathway activation. We demonstrate that PIN1 catalyzes cis-trans isomerization of the FAAP20 pSer48-Pro49 motif and promotes FAAP20 stability. Mechanistically, PIN1-induced conformational change of FAAP20 enhances its interaction with the PP2A phosphatase to counteract SCFFBW7-dependent proteolytic signaling at the phosphorylated degron motif. Accordingly, PIN1 deficiency impairs FANCD2 activation and the DNA ICL repair process. Together, our study establishes PIN1-dependent prolyl isomerization as a new regulator of the FA pathway and genomic integrity.

Dissecting Substrate Specificities of the Mitochondrial AFG3L2 Protease.

Human AFG3L2 is a compartmental AAA+ protease that performs ATP-fueled degradation at the matrix face of the inner mitochondrial membrane. Identifying how AFG3L2 selects substrates from the diverse complement of matrix-localized proteins is essential for understanding mitochondrial protein biogenesis and quality control. Here, we create solubilized forms of AFG3L2 to examine the enzyme's substrate specificity mechanisms. We show that conserved residues within the presequence of the mitochondrial ribosomal protein, MrpL32, target the subunit to the protease for processing into a mature form. Moreover, these residues can act as a degron, delivering diverse model proteins to AFG3L2 for degradation. By determining the sequence of degradation products from multiple substrates using mass spectrometry, we construct a peptidase specificity profile that displays constrained product lengths and is dominated by the identity of the residue at the P1' position, with a strong preference for hydrophobic and small polar residues. This specificity profile is validated by examining the cleavage of both fluorogenic reporter peptides and full polypeptide substrates bearing different P1' residues. Together, these results demonstrate that AFG3L2 contains multiple modes of specificity, discriminating between potential substrates by recognizing accessible degron sequences and performing peptide bond cleavage at preferred patterns of residues within the compartmental chamber.

The scaffold proteins of lignin biosynthetic cytochrome P450 enzymes.

Lignin is a complex and irregular biopolymer of crosslinked phenylpropanoid units in plant secondary cell walls. Its biosynthesis requires three endoplasmic reticulum (ER)-resident cytochrome P450 monooxygenases, C4H, C3'H and F5H, to establish the structural characteristics of its monomeric precursors. These P450 enzymes were reported to associate with each other or potentially with other soluble monolignol biosynthetic enzymes to form an enzyme complex or a metabolon. However, the molecular basis governing such enzyme or pathway organization remains elusive. Here, we show that Arabidopsis membrane steroid-binding proteins (MSBPs) serve as a scaffold to physically organize monolignol P450 monooxygenases, thereby regulating the lignin biosynthetic process. We find that although C4H, C3'H and F5H are in spatial proximity to each other on the ER membrane in vivo, they do not appear to directly interact with each other. Instead, two MSBP proteins physically interact with all three P450 enzymes and, moreover, MSBPs themselves associate as homomers and heteromers on the ER membrane, thereby organizing P450 clusters. Downregulation of MSBP genes does not affect the transcription levels of monolignol biosynthetic P450 genes but substantially impairs the stability and activity of the MSBP-interacting P450 enzymes and, consequently, lignin deposition, and the accumulation of soluble phenolics in the monolignol branch but not in the flavonoid pathway. Our study suggests that MSBP proteins are essential structural components in the ER membrane that physically organize and stabilize the monolignol biosynthetic P450 enzyme complex, thereby specifically controlling phenylpropanoid-monolignol branch biosynthesis.

Initial steps in RNA processing and ribosome assembly occur at mitochondrial DNA nucleoids.

Mammalian mitochondrial DNA (mtDNA) resides in compact nucleoids, where it is replicated and transcribed into long primary transcripts processed to generate rRNAs, tRNAs, and mRNAs encoding 13 proteins. This situation differs from bacteria and eukaryotic nucleoli, which have dedicated rRNA transcription units. The assembly of rRNAs into mitoribosomes has received little study. We show that mitochondrial RNA processing enzymes involved in tRNA excision, ribonuclease P (RNase P) and ELAC2, as well as a subset of nascent mitochondrial ribosomal proteins (MRPs) associate with nucleoids to initiate RNA processing and ribosome assembly. SILAC pulse-chase labeling experiments show that nascent MRPs recruited to the nucleoid fraction were highly labeled after the pulse in a transcription-dependent manner and decreased in labeling intensity during the chase. These results provide insight into the landscape of binding events required for mitochondrial ribosome assembly and firmly establish the mtDNA nucleoid as a control center for mitochondrial biogenesis.

Systematic analysis of microRNA fingerprints in thrombocythemic platelets using integrated platforms.

Posttranscriptional and translational controls mediated by microRNAs (miRNA) regulate diverse biologic processes. We dissected regulatory effects of miRNAs relevant to megakaryocytopoiesis and platelet biology by analyzing expression patterns in 79 subjects with thrombocytosis and controls, and integrated data with transcriptomic and proteomic platforms. We validated a unique 21-miRNA genetic fingerprint associated with thrombocytosis, and demonstrated that a 3-member subset defines essential thrombocythemia (ET). The genetic signature includes functional guide and passenger strands of the previously uncharacterized miR 490 (5p and 3p), which displayed restricted, low-level expression in megakaryocytes/platelets (compared with leukocytes), and aberrant expression during thrombocytosis, most profound in ET. Overexpression of miR 490 in a bilineage differentiation model of megakaryocyte/erythroid progenitor formation was insufficient for hematopoietic colony differentiation and/or lineage specification. Integration of transcriptomic and mass spectrometric datasets with functional reporter assays identified dishevelled associated activator of morphogenesis 1 (DAAM1) as a miR 490 5p protein target demonstrating decreased expression in ET platelets, putatively by translational control (and not by mRNA target degradation). Our data define a dysregulated miRNA fingerprint in thrombocytosis and support a developmentally restricted function of miR 490 (and its putative DAAM1 target) to conditions associated with exaggerated megakaryocytopoiesis and/or proplatelet formation.

Vasoactive Intestinal Peptide Knockout (VIP KO) mouse model of sulfite-sensitive asthma: up-regulation of novel lung carbonyl reductase.

BACKGROUND: We earlier reported spontaneous features of asthma in Vasoactive Intestinal Peptide knockout mice (VIP KO): 1) peribronchiolar airway inflammation, with accumulation of lymphocytes and eosinophils, 2) pro-inflammatory cytokine production of IL-5, IL-6, with IFN-γ, and 3) airway hyper-responsiveness to inhaled methacholine. In human asthma, a phenotype with sulfite sensitivity leads to airway inflammation and hyper-responsiveness to inhaled sulfites, and is associated with upregulation of anti-oxidant protein lung carbonyl reductase. For the present experiments, we examined the role of VIP in modulating anti-oxidant genes and their proteins, including lung carbonyl reductase. RESULTS: Four male VIP KO mice and four wild-type age- and gender matched mice had lungs examined for whole genome microarray and a proteomics approach using mass spectrometry. The proteomics analysis revealed that a novel variant of anti-oxidant protein lung carbonyl reductase (car3) was uniquely and markedly elevated in the VIP KO mice. RT-PCR indicated that carbonic anhydrase 3, which is an anti-oxidant protein, was elevated in the VIP KO mice. CONCLUSIONS: These data support the concept that VIP influences the endogenous oxidant/antioxidant balance. One potential implication is that VIP and its analogues may be used to treat inflammatory diseases, including asthma.

A transgenic mouse model of heart failure using inducible Galpha q.

Receptors coupled to Galpha q play a key role in the development of heart failure. Studies using genetically modified mice suggest that Galpha q mediates a hypertrophic response in cardiac myocytes. Galpha q signaling in these models is modified during early growth and development, whereas most heart failure in humans occurs after cardiac damage sustained during adulthood. To determine the phenotype of animals that express increased Galpha q signaling only as adults, we generated transgenic mice that express a silent Galpha q protein (Galpha qQ209L-hbER) in cardiac myocytes that can be activated by tamoxifen. Following drug treatment to activate Galpha q Q209L-hbER, these mice rapidly develop a dilated cardiomyopathy and heart failure. This phenotype does not appear to involve myocyte hypertrophy but is associated with dephosphorylation of phospholamban (PLB), decreased sarcoplasmic reticulum Ca2+-ATPase activity, and a decrease in L-type Ca2+ current density. Changes in Ca2+ handling and decreased cardiac contractility are apparent 1 week after Galpha qQ209L-hbER activation. In contrast, transgenic mice that express an inducible Galpha q mutant that cannot activate phospholipase Cbeta (PLCbeta) do not develop heart failure or changes in PLB phosphorylation, but do show decreased L-type Ca2+ current density. These results demonstrate that activation of Galpha q in cardiac myocytes of adult mice causes a dilated cardiomyopathy that requires the activation of PLCbeta. However, increased PLCbeta signaling is not required for all of the Galpha q-induced cardiac abnormalities.

Structure-function relationships in the NA+,K+-pump.

The Na,K-pump was discovered about 50 years ago. Since then there has been a methodic investigation of its structure and functional characteristics. The development of the Albers-Post model for the transport cycle was a milestone that provided the framework for detailed understanding of the transport process. The pump is composed of 2 subunits that exist in the membrane as an alphabeta heterodimer. All known enzymatic functions of the pump occur through the alpha subunit. Although necessary for activity, the complete role of the beta subunit is not understood fully. Numerous studies have established that the alphabeta protomer is the minimal functional unit needed to perform the Albers-Post reaction cycle. However, higher orders of aggregation [(alphabeta)n] are commonly detected. There is little evidence that oligomerization has functional consequence for ion transport. The Na+,K+-adenosine triphosphatase (ATPase) is a member of the P-type ATPase family of transporters. Proteins within this family have common amino acid sequence motifs that share functional characteristics and structure. Low-resolution 3-dimensional reconstruction of 2-dimensional crystal diffractions provide evidence for the similarity in tertiary structure of the alpha subunit and the Ca2+ATPase (a closely related P-type ATPase). The spatial location of the beta subunit also is obvious in these reconstructions. Recent high-resolution reconstructions from 3-dimensional crystals of the Ca2+ATPase provide structural details at the atomic level. It now is possible to interpret structurally some of the key steps in the Albers-Post reaction. Some of these high-resolution interpretations are translatable to the Na+,K+-ATPase, but a high-resolution structure of the Na,K-pump is needed for the necessary details of those aspects that are unique to this transporter.