Mechanisms of protein homeostasis (proteostasis) maintain stem cell identity in mammalian pluripotent stem cells

Protein homeostasis, or proteostasis, is essential for cell function, development, and organismal viability. The composition of the proteome is adjusted to the specific requirements of a particular cell type and status. Moreover, multiple metabolic and environmental conditions challenge the integrity of the proteome. To maintain the quality of the proteome, the proteostasis network monitors proteins from their synthesis through their degradation. Whereas somatic stem cells lose their ability to maintain proteostasis with age, immortal pluripotent stem cells exhibit a stringent proteostasis network associated with their biological function and intrinsic characteristics. Moreover, growing evidence indicates that enhanced proteostasis mechanisms play a central role in immortality and cell fate decisions of pluripotent stem cells. Here, we will review new insights into the melding fields of proteostasis and pluripotency and their implications for the understanding of organismal development and survival.     

The insulin-degrading enzyme is an allosteric modulator of the 20S proteasome and a potential competitor of the 19S

The interaction of insulin-degrading enzyme (IDE) with the main intracellular proteasome assemblies (i.e, 30S, 26S and 20S) was analyzed by enzymatic activity, mass spectrometry and native gel electrophoresis. IDE was mainly detected in association with assemblies with at least one free 20S end and biochemical investigations suggest that IDE competes with the 19S in vitro. IDE directly binds the 20S and affects its proteolytic activities in a bimodal fashion, very similar in human and yeast 20S, inhibiting at (IDE)?≤?30 nM and activating at (IDE)?≥?30 nM. Only an activating effect is observed in a yeast mutant locked in the “open” conformation (i.e., the α-3ΔN 20S), envisaging a possible role of IDE as modulator of the 20S “open”–”closed” allosteric equilibrium. Protein–protein docking in silico proposes that the interaction between IDE and the 20S could involve the C-term helix of the 20S α-3 subunit which regulates the gate opening of the 20S.     

Structural features underlying the selectivity of the kinase inhibitors NBC and dNBC: role of a nitro group that discriminates between CK2 and DYRK1A

8-hydroxy-4-methyl-9-nitrobenzo(g)chromen-2-one (NBC) has been found to be a fairly potent ATP site-directed inhibitor of protein kinase CK2 (Ki = 0.22 μM). Here, we show that NBC also inhibits PIM kinases, especially PIM1 and PIM3, the latter as potently as CK2. Upon removal of the nitro group, to give 8-hydroxy-4-methyl-benzo(g)chromen-2-one (here referred to as “denitro NBC”, dNBC), the inhibitory power toward CK2 is almost entirely lost (IC₅₀ > 30 μM) whereas that toward PIM1 and PIM3 is maintained; in addition, dNBC is a potent inhibitor of a number of other kinases that are weakly inhibited or unaffected by NBC, with special reference to DYRK1A whose IC₅₀ values with NBC and dNBC are 15 and 0.60 μM, respectively. Therefore, the observation that NBC, unlike dNBC, is a potent inducer of apoptosis is consistent with the notion that this effect is mediated by inhibition of endogenous CK2. The structural features underlying NBC selectivity have been revealed by inspecting its 3D structure in complex with the catalytic subunit of Z. mays CK2. The crucial role of the nitro group is exerted both through a direct electrostatic interaction with the side chain of Lys68 and, indirectly, by enhancing the acidic dissociation constant of the adjacent hydroxyl group which interacts with a conserved water molecule in the deepest part of the cavity. By contrast, the very same nitro group is deleterious for the binding to the active site of DYRK1A, as disclosed by molecular docking. This provides the rationale for preferential inhibition of DYRK1A by dNBC.     

SHARP1 suppresses breast cancer metastasis by promoting degradation of hypoxia-inducible factors

The molecular determinants of malignant cell behaviours in breast cancer remain only partially understood. Here we show that SHARP1 (also known as BHLHE41 or DEC2) is a crucial regulator of the invasive and metastatic phenotype in triple-negative breast cancer (TNBC), one of the most aggressive types of breast cancer. SHARP1 is regulated by the p63 metastasis suppressor and inhibits TNBC aggressiveness through inhibition of hypoxia-inducible factor 1α (HIF-1α) and HIF-2α (HIFs). SHARP1 opposes HIF-dependent TNBC cell migration in vitro, and invasive or metastatic behaviours in vivo. SHARP1 is required, and sufficient, to limit expression of HIF-target genes. In primary TNBC, endogenous SHARP1 levels are inversely correlated with those of HIF targets. Mechanistically, SHARP1 binds to HIFs and promotes HIF proteasomal degradation by serving as the HIF-presenting factor to the proteasome. This process is independent of pVHL (von Hippel-Lindau tumour suppressor), hypoxia and the ubiquitination machinery. SHARP1 therefore determines the intrinsic instability of HIF proteins to act in parallel to, and cooperate with, oxygen levels. This work sheds light on the mechanisms and pathways by which TNBC acquires invasiveness and metastatic propensity.     

Thymidine H3 incorporation in the nurse cells of amphigonic and parthenogenetic ovaries ofMegoura viciae (Horn. Aph.)

Conclusions In the amphigonic females ofM. viciae an active nuclear incorporation of thymidine H³ occurs during growth which may be attributed to continuous endomitotic divisions. However, even when the nurse cells are functioning fully, and when the nuclei appear to have achieved maximum development, incorporation of the thymidine H³ continues, and, as was seen in other insects, does not affect all the nuclei. Incorporation would therefore seem to be due not to the continuance of endomitotic divisions but rather to the synthesis of metabolic DNA. On the other hand, even if one supposes that in the nurse cells of the amphigonic ovary endomitotic processes continue right up to the end of vitellogenesis of the amphigonic winter egg, this is quite out of the question so far as the parthenogenetic ovary is concerned. Diploid nurse cells are functioning continuously, since in a parthenogenetic ovary a great many ovocytes reach maturity one after the other, passing through all stages of development to produce the embryos. The nurse cells always retain, in such cases, their characteristic appearance right from the beginning of their differentiation³ and therefore thymidine H³ incorporation cannot be ascribed to continuous endomitotic divisions. It can therefore be assumed that the active synthesis which occurs in the nuclei does not concern genetically stable DNA but a metabolic DNA. The above results thus add new weight to the assumption by former authors that metabolic DNA may be synthesized in the nurse cells of amphigonic insects as well.

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Riassunto Nell'afideMegoura viciae le cellule nutrici diploidi dell'ovario partenogenetico e quelle poliploidi dell'ovario anfigonico si comportano in maniera analoga incorporando timidina H³ durante l'accrescimento ovocitario. Tale incorporazione viene attribuita alla sintesi di DNA metabolico.

     

Non-redundant role of the long pentraxin PTX3 in anti-fungal innate immune response

Pentraxins are a superfamily of conserved proteins that are characterized by a cyclic multimeric structure. The classical short pentraxins, C-reactive protein (CRP) and serum amyloid P component (SAP), are acute-phase proteins produced in the liver in response to inflammatory mediators. Short pentraxins regulate innate resistance to microbes and the scavenging of cellular debris and extracellular matrix components. In contrast, long pentraxins have an unrelated, long amino-terminal domain coupled to the carboxy-terminal pentraxin domain, and differ, with respect to short pentraxins, in their gene organization, chromosomal localization, cellular source, and in their stimuli-inducing and ligand-recognition ability. To investigate the in vivo function of the long pentraxin PTX3, we generated mice deficient in Ptx3 by homologous recombination. Ptx3-null mice were susceptible to invasive pulmonary aspergillosis. Ptx3 binds selected microbial agents, including conidia of Aspergillus fumigatus, and we found that susceptibility of Ptx3-null mice was associated with defective recognition of conidia by alveolar macrophages and dendritic cells, as well as inappropriate induction of an adaptive type 2 response. Thus, the long pentraxin Ptx3 is a secreted pattern-recognition receptor that has a non-redundant role in resistance to selected microbial agents, in particular to the opportunistic fungal pathogen Aspergillus fumigatus.