In search of an evidence-based strategy for quality assessment of human tissue samples: report of the tissue Biospecimen Research Working Group of the Spanish Biobank Network.

The purpose of the present work is to underline the importance of obtaining a standardized procedure to ensure and evaluate both clinical and research usability of human tissue samples. The study, which was carried out by the Biospecimen Science Working Group of the Spanish Biobank Network, is based on a general overview of the current situation about quality assurance in human tissue biospecimens. It was conducted an exhaustive review of the analytical techniques used to evaluate the quality of human tissue samples over the past 30 years, as well as their reference values if they were published, and classified them according to the biomolecules evaluated: (i) DNA, (ii) RNA, and (iii) soluble or/and fixed proteins for immunochemistry. More than 130 publications released between 1989 and 2019 were analysed, most of them reporting results focused on the analysis of tumour and biopsy samples. A quality assessment proposal with an algorithm has been developed for both frozen tissue samples and formalin-fixed paraffin-embedded (FFPE) samples, according to the expected quality of sample based on the available pre-analytical information and the experience of the participants in the Working Group. The high heterogeneity of human tissue samples and the wide number of pre-analytic factors associated to quality of samples makes it very difficult to harmonize the quality criteria. However, the proposed method to assess human tissue sample integrity and antigenicity will not only help to evaluate whether stored human tissue samples fit for the purpose of biomarker development, but will also allow to perform further studies, such as assessing the impact of different pre-analytical factors on very well characterized samples or evaluating the readjustment of tissue sample collection, processing and storing procedures. By ensuring the quality of the samples used on research, the reproducibility of scientific results will be guaranteed.

Mitotic cell-cycle progression is regulated by CPEB1 and CPEB4-dependent translational control.

Meiotic and early-embryonic cell divisions in vertebrates take place in the absence of transcription and rely on the translational regulation of stored maternal messenger RNAs. Most of these mRNAs are regulated by the cytoplasmic-polyadenylation-element-binding protein (CPEB), which mediates translational activation and repression through cytoplasmic changes in their poly(A) tail length. It was unknown whether translational regulation by cytoplasmic polyadenylation and CPEB can also regulate mRNAs at specific points of mitotic cell-cycle divisions. Here we show that CPEB-mediated post-transcriptional regulation by phase-specific changes in poly(A) tail length is required for cell proliferation and specifically for entry into M phase in mitotically dividing cells. This translational control is mediated by two members of the CPEB family of proteins, CPEB1 and CPEB4. We conclude that regulation of poly(A) tail length is not only required to compensate for the lack of transcription in specialized cell divisions but also acts as a general mechanism to control mitosis.

CHOP induces death by promoting protein synthesis and oxidation in the stressed endoplasmic reticulum.

Unfolded and malfolded client proteins impose a stress on the endoplasmic reticulum (ER), which contributes to cell death in pathophysiological conditions. The transcription factor C/EBP homologous protein (CHOP) is activated by ER stress, and CHOP deletion protects against its lethal consequences. We find that CHOP directly activates GADD34, which promotes ER client protein biosynthesis by dephosphorylating phospho-Ser 51 of the alpha-subunit of translation initiation factor 2 (eIF2alpha) in stressed cells. Thus, impaired GADD34 expression reduces client protein load and ER stress in CHOP(-/-) cells exposed to perturbations that impair ER function. CHOP(-/-) and GADD34 mutant cells accumulate less high molecular weight protein complexes in their stressed ER than wild-type cells. Furthermore, mice lacking GADD34-directed eIF2alpha dephosphorylation, like CHOP(-/-) mice, are resistant to renal toxicity of the ER stress-inducing drug tunicamycin. CHOP also activates ERO1alpha, which encodes an ER oxidase. Consequently, the ER of stressed CHOP(-/-) cells is relatively hypo-oxidizing. Pharmacological and genetic manipulations that promote a hypo-oxidizing ER reduce abnormal high molecular weight protein complexes in the stressed ER and protect from the lethal consequences of ER stress. CHOP deletion thus protects cells from ER stress by decreasing ER client protein load and changing redox conditions within the organelle.

Cytoprotection by pre-emptive conditional phosphorylation of translation initiation factor 2.

Transient phosphorylation of the alpha-subunit of translation initiation factor 2 (eIF2alpha) represses translation and activates select gene expression under diverse stressful conditions. Defects in the eIF2alpha phosphorylation-dependent integrated stress response impair resistance to accumulation of malfolded proteins in the endoplasmic reticulum (ER stress), to oxidative stress and to nutrient deprivations. To study the hypothesized protective role of eIF2alpha phosphorylation in isolation of parallel stress signaling pathways, we fused the kinase domain of pancreatic endoplasmic reticulum kinase (PERK), an ER stress-inducible eIF2alpha kinase that is normally activated by dimerization, to a protein module that binds a small dimerizer molecule. The activity of this artificial eIF2alpha kinase, Fv2E-PERK, is subordinate to the dimerizer and is uncoupled from upstream stress signaling. Fv2E-PERK activation enhanced the expression of numerous stress-induced genes and protected cells from the lethal effects of oxidants, peroxynitrite donors and ER stress. Our findings indicate that eIF2alpha phosphorylation can initiate signaling in a cytoprotective gene expression pathway independently of other parallel stress-induced signals and that activation of this pathway can single-handedly promote a stress-resistant preconditioned state.

Inhibition of a constitutive translation initiation factor 2alpha phosphatase, CReP, promotes survival of stressed cells.

Phosphorylation of eukaryotic translation initiation factor 2alpha (eIF2alpha) on serine 51 is effected by specific stress-activated protein kinases. eIF2alpha phosphorylation inhibits translation initiation promoting a cytoprotective gene expression program known as the integrated stress response (ISR). Stress-induced activation of GADD34 feeds back negatively on this pathway by promoting eIF2alpha dephosphorylation, however, GADD34 mutant cells retain significant eIF2alpha-directed phosphatase activity. We used a somatic cell genetic approach to identify a gene encoding a novel regulatory subunit of a constitutively active holophosphatase complex that dephosphorylates eIF2alpha. RNAi of this gene, which we named constitutive repressor of eIF2alpha phosphorylation (CReP, or PPP1R15B), repressed the constitutive eIF2alpha-directed phosphatase activity and activated the ISR. CReP RNAi strongly protected mammalian cells against oxidative stress, peroxynitrite stress, and more modestly against accumulation of malfolded proteins in the endoplasmic reticulum. These findings suggest that therapeutic inhibition of eIF2alpha dephosphorylation by targeting the CReP-protein-phosphatase-1 complex may be used to access the salubrious qualities of the ISR.

An integrated stress response regulates amino acid metabolism and resistance to oxidative stress.

Eukaryotic cells respond to unfolded proteins in their endoplasmic reticulum (ER stress), amino acid starvation, or oxidants by phosphorylating the alpha subunit of translation initiation factor 2 (eIF2alpha). This adaptation inhibits general protein synthesis while promoting translation and expression of the transcription factor ATF4. Atf4(-/-) cells are impaired in expressing genes involved in amino acid import, glutathione biosynthesis, and resistance to oxidative stress. Perk(-/-) cells, lacking an upstream ER stress-activated eIF2alpha kinase that activates Atf4, accumulate endogenous peroxides during ER stress, whereas interference with the ER oxidase ERO1 abrogates such accumulation. A signaling pathway initiated by eIF2alpha phosphorylation protects cells against metabolic consequences of ER oxidation by promoting the linked processes of amino acid sufficiency and resistance to oxidative stress.

Stress-induced gene expression requires programmed recovery from translational repression.

Active repression of protein synthesis protects cells against protein malfolding during endoplasmic reticulum stress, nutrient deprivation and oxidative stress. However, long-term adaptation to these conditions requires synthesis of new stress-induced proteins. Phosphorylation of the alpha-subunit of translation initiation factor 2 (eIF2alpha) represses translation in diverse stressful conditions. GADD34 is a stress-inducible regulatory subunit of a holophosphatase complex that dephosphorylates eIF2alpha, and has been hypothesized to play a role in translational recovery. Here, we report that GADD34 expression correlated temporally with eIF2alpha dephosphorylation late in the stress response. Inactivation of both alleles of GADD34 prevented eIF2alpha dephosphorylation and blocked the recovery of protein synthesis, normally observed late in the stress response. Furthermore, defective recovery of protein synthesis markedly impaired translation of stress-induced proteins and interfered with programmed activation of stress-induced genes in the GADD34 mutant cells. These observations indicate that GADD34 controls a programmed shift from translational repression to stress-induced gene expression, and reconciles the apparent contradiction between the translational and transcriptional arms of cellular stress responses.

Control of PERK eIF2alpha kinase activity by the endoplasmic reticulum stress-induced molecular chaperone P58IPK.

P58(IPK) is an Hsp40 family member known to inhibit the interferon (IFN)-induced, double-stranded RNA-activated, eukaryotic initiation factor 2alpha (eIF2alpha) protein kinase R (PKR) by binding to its kinase domain. We find that the stress of unfolded proteins in the endoplasmic reticulum (ER) activates P58(IPK) gene transcription through an ER stress-response element in its promoter region. P58(IPK) interacts with and inhibits the PKR-like ER-localized eIF2alpha kinase PERK, which is normally activated during the ER-stress response to protect cells from ER stress by attenuating protein synthesis and reducing ER client protein load. Levels of phosphorylated eIF2alpha were lower in ER-stressed P58(IPK)-overexpressing cells and were enhanced in P58(IPK) mutant cells. In the ER-stress response, PKR-like ER kinase (PERK)-mediated translational repression is transient and is followed by translational recovery and enhanced expression of genes that increase the capacity of the ER to process client proteins. The absence of P58(IPK) resulted in increased expression levels of two ER stress-inducible genes, BiP and Chop, consistent with the enhanced eIF2alpha phosphorylation in the P58(IPK) deletion cells. Our studies suggest that P58(IPK) induction during the ER-stress response represses PERK activity and plays a functional role in the expression of downstream markers of PERK activity in the later phase of the ER-stress response.

Transcriptional and translational control in the Mammalian unfolded protein response.

Cells monitor the physiological load placed on their endoplasmic reticulum (ER) and respond to perturbations in ER function by a process known as the unfolded protein response (UPR). In metazoans the UPR has a transcriptional component that up-regulates expression of genes that enhance the capacity of the organelle to deal with the load of client proteins and a translational component that insures tight coupling between protein biosynthesis on the cytoplasmic side and folding in the ER lumen. Together, these two components adapt the secretory apparatus to physiological load and protect cells from the consequences of protein malfolding.