Response to immune checkpoint blockade improved in pre-clinical model of breast cancer after bariatric surgery.

Bariatric surgery is a sustainable weight loss approach, including vertical sleeve gastrectomy (VSG). Obesity exacerbates tumor growth, while diet-induced weight loss impairs progression. It remains unknown how bariatric surgery-induced weight loss impacts cancer progression or alters response to therapy. Using a pre-clinical model of obesity followed by VSG or diet-induced weight loss, breast cancer progression and immune checkpoint blockade therapy were investigated. Weight loss by VSG or weight-matched dietary intervention before tumor engraftment protected against obesity-exacerbated tumor progression. However, VSG was not as effective as diet in reducing tumor burden despite achieving similar weight and adiposity loss. Leptin did not associate with changes in tumor burden; however, circulating IL-6 was elevated in VSG mice. Uniquely, VSG tumors displayed elevated inflammation and immune checkpoint ligand PD-L1+ myeloid and non-immune cells. VSG tumors also had reduced T lymphocytes and markers of cytolysis, suggesting an ineffective anti-tumor microenvironment which prompted investigation of immune checkpoint blockade. While obese mice were resistant to immune checkpoint blockade, anti-PD-L1 potently impaired tumor progression after VSG through improved anti-tumor immunity. Thus, in formerly obese mice, surgical weight loss followed by immunotherapy reduced breast cancer burden. Finally, we compared transcriptomic changes in adipose tissue after bariatric surgery from patients and mouse models. A conserved bariatric surgery-associated weight loss signature (BSAS) was identified which significantly associated with decreased tumor volume. Findings demonstrate conserved impacts of obesity and bariatric surgery-induced weight loss pathways associated with breast cancer progression.

As the number of people classified as obese rises globally, so do obesity-related health risks. Studies show that people diagnosed with obesity have inflammation that contributes to tumor growth and their immune system is worse at detecting cancer cells. But weight loss is not currently used as a strategy for preventing or treating cancer. Surgical procedures for weight loss, also known as 'bariatric surgeries', are becoming increasingly popular. Recent studies have shown that individuals who lose weight after these treatments have a reduced risk of developing tumors. But how bariatric surgery directly impacts cancer progression has not been well studied: does it slow tumor growth or boost the anti-tumor immune response? To answer these questions, Sipe et al. compared breast tumor growth in groups of laboratory mice that were obese due to being fed a high fat diet. The first group of mice lost weight after undergoing a bariatric surgery in which part of their stomach was removed. The second lost the same amount of weight but after receiving a restricted diet, and the third underwent a fake surgery and did not lose any weight. The experiments found that surgical weight loss cuts breast cancer tumor growth in half compared with obese mice. But mice who lost the same amount of weight through dietary restrictions had even less tumor growth than surgically treated mice. The surgically treated mice who lost weight had more inflammation than mice in the two other groups, and had increased amounts of proteins and cells that block the immune response to tumors. Giving the surgically treated mice a drug that enhances the immune system's ability to detect and destroy cancer cells reduced inflammation and helped shrink the mice's tumors. Finally, Sipe et al. identified 54 genes which were turned on or off after bariatric surgery in both mice and humans, 11 of which were linked with tumor size. These findings provide crucial new information about how bariatric surgery can impact cancer progression. Future studies could potentially use the conserved genes identified by Sipe et al. to develop new ways to stimulate the anti-cancer benefits of weight loss without surgery.

Functionathon: a manual data mining workflow to generate functional hypotheses for uncharacterized human proteins and its application by undergraduate students.

About 10% of human proteins have no annotated function in protein knowledge bases. A workflow to generate hypotheses for the function of these uncharacterized proteins has been developed, based on predicted and experimental information on protein properties, interactions, tissular expression, subcellular localization, conservation in other organisms, as well as phenotypic data in mutant model organisms. This workflow has been applied to seven uncharacterized human proteins (C6orf118, C7orf25, CXorf58, RSRP1, SMLR1, TMEM53 and TMEM232) in the frame of a course-based undergraduate research experience named Functionathon organized at the University of Geneva to teach undergraduate students how to use biological databases and bioinformatics tools and interpret the results. C6orf118, CXorf58 and TMEM232 were proposed to be involved in cilia-related functions; TMEM53 and SMLR1 were proposed to be involved in lipid metabolism and C7orf25 and RSRP1 were proposed to be involved in RNA metabolism and gene expression. Experimental strategies to test these hypotheses were also discussed. The results of this manual data mining study may contribute to the project recently launched by the Human Proteome Organization (HUPO) Human Proteome Project aiming to fill gaps in the functional annotation of human proteins. Database URL: http://www.nextprot.org.

Mitochondrial peptide BRAWNIN is essential for vertebrate respiratory complex III assembly.

The emergence of small open reading frame (sORF)-encoded peptides (SEPs) is rapidly expanding the known proteome at the lower end of the size distribution. Here, we show that the mitochondrial proteome, particularly the respiratory chain, is enriched for small proteins. Using a prediction and validation pipeline for SEPs, we report the discovery of 16 endogenous nuclear encoded, mitochondrial-localized SEPs (mito-SEPs). Through functional prediction, proteomics, metabolomics and metabolic flux modeling, we demonstrate that BRAWNIN, a 71 a.a. peptide encoded by C12orf73, is essential for respiratory chain complex III (CIII) assembly. In human cells, BRAWNIN is induced by the energy-sensing AMPK pathway, and its depletion impairs mitochondrial ATP production. In zebrafish, Brawnin deletion causes complete CIII loss, resulting in severe growth retardation, lactic acidosis and early death. Our findings demonstrate that BRAWNIN is essential for vertebrate oxidative phosphorylation. We propose that mito-SEPs are an untapped resource for essential regulators of oxidative metabolism.

Crystal structure of a signal recognition particle Alu domain in the elongation arrest conformation.

The signal recognition particle (SRP) is a conserved ribonucleoprotein particle that targets membrane and secreted proteins to translocation channels in membranes. In eukaryotes, the Alu domain, which comprises the 5' and 3' extremities of the SRP RNA bound to the SRP9/14 heterodimer, is thought to interact with the ribosome to pause translation elongation during membrane docking. We present the 3.2 Å resolution crystal structure of a chimeric Alu domain, comprising Alu RNA from the archaeon Pyrococcus horikoshii bound to the human Alu binding proteins SRP9/14. The structure reveals how intricate tertiary interactions stabilize the RNA 5' domain structure and how an extra, archaeal-specific, terminal stem helps constrain the Alu RNA into the active closed conformation. In this conformation, highly conserved noncanonical base pairs allow unusually tight side-by-side packing of 5' and 3' RNA stems within the SRP9/14 RNA binding surface. The biological relevance of this structure is confirmed by showing that a reconstituted full-length chimeric archaeal-human SRP is competent to elicit elongation arrest in vitro. The structure will be useful in refining our understanding of how the SRP Alu domain interacts with the ribosome.

DERA is the human deoxyribose phosphate aldolase and is involved in stress response.

Deoxyribose-phosphate aldolase (EC 4.1.2.4), which converts 2-deoxy-d-ribose-5-phosphate into glyceraldehyde-3-phosphate and acetaldehyde, belongs to the core metabolism of living organisms. It was previously shown that human cells harbor deoxyribose phosphate aldolase activity but the protein responsible of this activity has never been formally identified. This study provides the first experimental evidence that DERA, which is mainly expressed in lung, liver and colon, is the human deoxyribose phosphate aldolase. Among human cell lines, the highest DERA mRNA level and deoxyribose phosphate aldolase activity were observed in liver-derived Huh-7 cells. DERA was shown to interact with the known stress granule component YBX1 and to be recruited to stress granules after oxidative or mitochondrial stress. In addition, cells in which DERA expression was down-regulated using shRNA formed fewer stress granules and were more prone to apoptosis after clotrimazole stress, suggesting the importance of DERA for stress granule formation. Furthermore, the expression of DERA was shown to permit cells in which mitochondrial ATP production was abolished to make use of extracellular deoxyinosine to maintain ATP levels. This study unraveled a previously undescribed pathway which may allow cells with high deoxyribose-phosphate aldolase activity, such as liver cells, to minimize or delay stress-induced damage by producing energy through deoxynucleoside degradation.

Efficient secretion of small proteins in mammalian cells relies on Sec62-dependent posttranslational translocation.

Mammalian cells secrete a large number of small proteins, but their mode of translocation into the endoplasmic reticulum is not fully understood. Cotranslational translocation was expected to be inefficient due to the small time window for signal sequence recognition by the signal recognition particle (SRP). Impairing the SRP pathway and reducing cellular levels of the translocon component Sec62 by RNA interference, we found an alternate, Sec62-dependent translocation path in mammalian cells required for the efficient translocation of small proteins with N-terminal signal sequences. The Sec62-dependent translocation occurs posttranslationally via the Sec61 translocon and requires ATP. We classified preproteins into three groups: 1) those that comprise ≤100 amino acids are strongly dependent on Sec62 for efficient translocation; 2) those in the size range of 120-160 amino acids use the SRP pathway, albeit inefficiently, and therefore rely on Sec62 for efficient translocation; and 3) those larger than 160 amino acids depend on the SRP pathway to preserve a transient translocation competence independent of Sec62. Thus, unlike in yeast, the Sec62-dependent translocation pathway in mammalian cells serves mainly as a fail-safe mechanism to ensure efficient secretion of small proteins and provides cells with an opportunity to regulate secretion of small proteins independent of the SRP pathway.

Functional identification of APIP as human mtnB, a key enzyme in the methionine salvage pathway.

The methionine salvage pathway is widely distributed among some eubacteria, yeast, plants and animals and recycles the sulfur-containing metabolite 5-methylthioadenosine (MTA) to methionine. In eukaryotic cells, the methionine salvage pathway takes place in the cytosol and usually involves six enzymatic activities: MTA phosphorylase (MTAP, EC 2.4.2.28), 5'-methylthioribose-1-phosphate isomerase (mtnA, EC 5.3.1.23), 5'-methylthioribulose-1-phosphate dehydratase (mtnB, EC: 4.2.1.109), 2,3-dioxomethiopentane-1-phosphate enolase/phosphatase (mtnC, EC 3.1.3.77), aci-reductone dioxygenase (mtnD, EC 1.13.11.54) and 4-methylthio-2-oxo-butanoate (MTOB) transaminase (EC 2.6.1.-). The aim of this study was to complete the available information on the methionine salvage pathway in human by identifying the enzyme responsible for the dehydratase step. Using a bioinformatics approach, we propose that a protein called APIP could perform this role. The involvement of this protein in the methionine salvage pathway was investigated directly in HeLa cells by transient and stable short hairpin RNA interference. We show that APIP depletion specifically impaired the capacity of cells to grow in media where methionine is replaced by MTA. Using a Shigella mutant auxotroph for methionine, we confirm that the knockdown of APIP specifically affects the recycling of methionine. We also show that mutation of three potential phosphorylation sites does not affect APIP activity whereas mutation of the potential zinc binding site completely abrogates it. Finally, we show that the N-terminal region of APIP that is missing in the short isoform is required for activity. Together, these results confirm the involvement of APIP in the methionine salvage pathway, which plays a key role in many biological functions like cancer, apoptosis, microbial proliferation and inflammation.

Residues in SRP9/14 essential for elongation arrest activity of the signal recognition particle define a positively charged functional domain on one side of the protein.

The signal recognition particle (SRP) is a ubiquitous cytoplasmic ribonucleoprotein complex required for the cotranslational targeting of proteins to the endoplasmic reticulum (ER). In eukaryotes, SRP has to arrest the elongation of the nascent chains during targeting to ensure efficient translocation of the preprotein, and this function of SRP is dependent on SRP9/14. Here we present the results of a mutational study on the human protein h9/14 that identified and characterized regions and single residues essential for elongation arrest activity. Effects of the mutations were assessed both in cell-free translation/translocation assays and in cultured mammalian cells. We identified two patches of basic amino acid residues that are essential for activity, whereas the internal loop of SRP14 was found to be dispensable. One patch of important basic residues comprises the previously identified basic pentapetide KRDKK, which can be substituted by four lysines without loss of function. The other patch includes three lysines in the solvent-accessible alpha2 of h9. All essential residues are located in proximity in SRP9/14 and their basic character suggests that they serve as a positively charged platform for interactions with ribosomal RNA. In addition, they can all be lysines consistent with the hypothesis that they recognize their target(s) via electrostatic contacts, most likely with the phosphate backbone, as opposed to contacts with specific bases.

SRP keeps polypeptides translocation-competent by slowing translation to match limiting ER-targeting sites.

SRP is essential for targeting nascent chains to the endoplasmic reticulum, and it delays nascent chain elongation in cell-free translation systems. However, the significance of this function has remained unclear. We show that efficient protein translocation into the ER is incompatible with normal cellular translation rates due to rate-limiting concentrations of SRP receptor (SR). We complemented mammalian cells depleted of SRP14 by expressing mutant versions of the protein lacking the elongation arrest function. The absence of a delay caused inefficient targeting of preproteins leading to defects in secretion, depletion of proteins in the endogenous membranes, and reduced cell growth. The detrimental effects were reversed by either reducing the cellular protein synthesis rate or increasing SR expression. SRP therefore ensures that nascent chains remain translocation competent during the targeting time window dictated by SR. Since SRP-signal sequence affinities vary, the delay may also regulate which proteins are preferentially targeted.

Natural synchronisation for the study of cell division in the green unicellular alga Ostreococcus tauri.

Ostreococcus tauri (Prasinophyceae) is a marine unicellular green alga which diverged early in the green lineage. The interest of O. tauri as a potential model to study plant cell division is based on its key phylogenetic position, its simple binary division, a very simple cellular organisation and now the availability of the full genome sequence. In addition O. tauri has a minimal yet complete set of cell cycle control genes. Here we show that division can be naturally synchronised by light/dark cycles and that organelles divide before the nucleus. This natural synchronisation, although being only partial, enables the study of the expression of CDKs throughout the cell cycle. The expression patterns of OtCDKA and OtCDKB were determined both at the mRNA and protein levels. The single OtCDKA gene is constantly expressed throughout the cell cycle, whereas OtCDKB is highly regulated and expressed only in S/G2/M phases. More surprisingly, OtCDKA is not phosphorylated at the tyrosine residue, in contrast to OtCDKB which is strongly phosphorylated during cell division. OtCDKA kinase activity appears before the S phase, indicating a possible role of this protein in the G1/S transition. OtCDKB kinase activity occurs later than OtCDKA, and its tyrosine phosphorylation is correlated to G2/M, suggesting a possible control of the mitotic activity. To our knowledge this is the first organism in the green lineage which showed CDKB tyrosine phosphorylation during cell cycle progression.