GAPDH Overexpression in the T Cell Lineage Promotes Angioimmunoblastic T Cell Lymphoma through an NF-κB-Dependent Mechanism.

GAPDH is emerging as a key player in T cell development and function. To investigate the role of GAPDH in T cells, we generated a transgenic mouse model overexpressing GAPDH in the T cell lineage. Aged mice developed a peripheral Tfh-like lymphoma that recapitulated key molecular, pathological, and immunophenotypic features of human angioimmunoblastic T cell lymphoma (AITL). GAPDH induced non-canonical NF-κB pathway activation in mouse T cells, which was strongly activated in human AITL. We developed a NIK inhibitor to reveal that targeting the NF-κB pathway prolonged AITL-bearing mouse survival alone and in combination with anti-PD-1. These findings suggest the therapeutic potential of targeting NF-κB signaling in AITL and provide a model for future AITL therapeutic investigations.

Low-Protein Diet Induces IRE1α-Dependent Anticancer Immunosurveillance.

Dietary restriction (DR) was shown to impact on tumor growth with very variable effects depending on the cancer type. However, how DR limits cancer progression remains largely unknown. Here, we demonstrate that feeding mice a low-protein (Low PROT) isocaloric diet but not a low-carbohydrate (Low CHO) diet reduced tumor growth in three independent mouse cancer models. Surprisingly, this effect relies on anticancer immunosurveillance, as depleting CD8+ T cells, antigen-presenting cells (APCs), or using immunodeficient mice prevented the beneficial effect of the diet. Mechanistically, we established that a Low PROT diet induces the unfolded protein response (UPR) in tumor cells through the activation of IRE1α and RIG1 signaling, thereby resulting in cytokine production and mounting an efficient anticancer immune response. Collectively, our data suggest that a Low PROT diet induces an IRE1α-dependent UPR in cancer cells, enhancing a CD8-mediated T cell response against tumors.

Melanocytes Sense Blue Light and Regulate Pigmentation through Opsin-3.

The shorter wavelengths of the visible light spectrum have been recently reported to induce a long-lasting hyperpigmentation but only in melano-competent individuals. Here, we provide evidence showing that OPN3 is the key sensor in melanocytes responsible for hyperpigmentation induced by the shorter wavelengths of visible light. The melanogenesis induced through OPN3 is calcium dependent and further activates CAMKII followed by CREB, extracellular signal-regulated kinase, and p38, leading to the phosphorylation of MITF and ultimately to the increase of the melanogenesis enzymes: tyrosinase and dopachrome tautomerase. Furthermore, blue light induces the formation of a protein complex that we showed to be formed by tyrosinase and dopachrome tautomerase. This multimeric tyrosinase/tyrosinase-related protein complex is mainly formed in dark-skinned melanocytes and induces a sustained tyrosinase activity, thus explaining the long-lasting hyperpigmentation that is observed only in skin type III and higher after blue light irradiation. OPN3 thus functions as the sensor for visible light pigmentation. OPN3 and the multimeric tyrosinase/tyrosinase-related protein complex induced after its activation appear as new potential targets for regulating melanogenesis but also to protect dark skins against blue light in physiological conditions and in pigmentary disorders.

An Arginine Finger Regulates the Sequential Action of Asymmetrical Hexameric ATPase in the Double-Stranded DNA Translocation Motor.

Biological motors are ubiquitous in living systems. Currently, how the motor components coordinate the unidirectional motion is elusive in most cases. Here, we report that the sequential action of the ATPase ring in the DNA packaging motor of bacteriophage ϕ29 is regulated by an arginine finger that extends from one ATPase subunit to the adjacent unit to promote noncovalent dimer formation. Mutation of the arginine finger resulted in the interruption of ATPase oligomerization, ATP binding/hydrolysis, and DNA translocation. Dimer formation reappeared when arginine mutants were mixed with other ATPase subunits that can offer the arginine to promote their interaction. Ultracentrifugation and virion assembly assays indicated that the ATPase was presenting as monomers and dimer mixtures. The isolated dimer alone was inactive in DNA translocation, but the addition of monomer could restore the activity, suggesting that the hexameric ATPase ring contained both dimer and monomers. Moreover, ATP binding or hydrolysis resulted in conformation and entropy changes of the ATPase with high or low DNA affinity. Taking these observations together, we concluded that the arginine finger regulates sequential action of the motor ATPase subunit by promoting the formation of the dimer inside the hexamer. The finding of asymmetrical hexameric organization is supported by structural evidence of many other ATPase systems showing the presence of one noncovalent dimer and four monomer subunits. All of these provide clues for why the asymmetrical hexameric ATPase gp16 of ϕ29 was previously reported as a pentameric configuration by cryo-electron microscopy (cryo-EM) since the contact by the arginine finger renders two adjacent ATPase subunits closer than other subunits. Thus, the asymmetrical hexamer would appear as a pentamer by cryo-EM, a technology that acquires the average of many images.

CD271 is an imperfect marker for melanoma initiating cells.

Understanding the molecular and cellular processes underlying melanoma plasticity and heterogeneity is of paramount importance to improve the efficiency of current treatment and to overcome resistance to chemotherapy drugs. The notion of plasticity and heterogeneity implies the existence of melanoma cell populations with different phenotypic and tumorigenic properties. Using melanoma cell lines and melanoma cells freshly isolated from patient biopsies, we investigated the relationship between ABCB5+, CD271+ and low-MITF, expressing populations that were reported to display melanoma initiating cell properties. Here, we showed that ABCB5+ and CD271+ populations poorly overlap. However, we found that the CD271+ population is enriched in low-MITF cells and expresses a higher level of stemness genes, such as OCT4, NANOG and NES. These features could explain the increased tumorigenicity of the CD271+ cells. The rapid conversion of CD271+ to CD271- cells in vitro demonstrates the plasticity ability of melanoma cells. Finally, we observed that the transient slow-growing population contains only CD271+ cells that are highly tumorigenic. However, the fast growing/CD271+ population exhibits a poor tumorigenic ability. Taking together, our data show that CD271 is an imperfect marker for melanoma initiating cells, but may be useful to identify melanoma cells with an increased stemness and tumorigenic potential.

The N-degradome of Escherichia coli: limited proteolysis in vivo generates a large pool of proteins bearing N-degrons.

The N-end rule is a conserved mechanism found in Gram-negative bacteria and eukaryotes for marking proteins to be degraded by ATP-dependent proteases. Specific N-terminal amino acids (N-degrons) are sufficient to target a protein to the degradation machinery. In Escherichia coli, the adaptor ClpS binds an N-degron and delivers the protein to ClpAP for degradation. As ClpS recognizes N-terminal Phe, Trp, Tyr, and Leu, which are not found at the N terminus of proteins translated and processed by the canonical pathway, proteins must be post-translationally modified to expose an N-degron. One modification is catalyzed by Aat, an enzyme that adds leucine or phenylalanine to proteins with N-terminal lysine or arginine; however, such proteins are also not generated by the canonical protein synthesis pathway. Thus, the mechanisms producing N-degrons in proteins and the frequency of their occurrence largely remain a mystery. To address these issues, we used a ClpS affinity column to isolate interacting proteins from E. coli cell lysates under non-denaturing conditions. We identified more than 100 proteins that differentially bound to a column charged with wild-type ClpS and eluted with a peptide bearing an N-degron. Thirty-two of 37 determined N-terminal peptides had N-degrons. Most of the proteins were N-terminally truncated by endoproteases or exopeptidases, and many were further modified by Aat. The identities of the proteins point to possible physiological roles for the N-end rule in cell division, translation, transcription, and DNA replication and reveal widespread proteolytic processing of cellular proteins to generate N-end rule substrates.

Revolution rather than rotation of AAA+ hexameric phi29 nanomotor for viral dsDNA packaging without coiling.

It has long been believed that the DNA-packaging motor of dsDNA viruses utilizes a rotation mechanism. Here we report a revolution rather than rotation mechanism for the bacteriophage phi29 DNA packaging motor. The phi29 motor contains six copies of the ATPase (Schwartz et al., this issue); ATP binding to one ATPase subunit stimulates the ATPase to adopt a conformation with a high affinity for dsDNA. ATP hydrolysis induces a new conformation with a lower affinity, thus transferring the dsDNA to an adjacent subunit by a power stroke. DNA revolves unidirectionally along the hexameric channel wall of the ATPase, but neither the dsDNA nor the ATPase itself rotates along its own axis. One ATP is hydrolyzed in each transitional step, and six ATPs are consumed for one helical turn of 360°. Transition of the same dsDNA chain along the channel wall, but at a location 60° different from the last contact, urges dsDNA to move forward 1.75 base pairs each step (10.5bp per turn/6ATP=1.75bp per ATP). Each connector subunit tilts with a left-handed orientation at a 30° angle in relation to its vertical axis that runs anti-parallel to the right-handed dsDNA helix, facilitating the one-way traffic of dsDNA. The connector channel has been shown to cause four steps of transition due to four positively charged lysine rings that make direct contact with the negatively charged DNA phosphate backbone. Translocation of dsDNA into the procapsid by revolution avoids the difficulties during rotation that are associated with DNA supercoiling. Since the revolution mechanism can apply to any stoichiometry, this motor mechanism might reconcile the stoichiometry discrepancy in many phage systems where the ATPase has been found as a tetramer, hexamer, or nonamer.

The ATPase of the phi29 DNA packaging motor is a member of the hexameric AAA+ superfamily.

The AAA+ superfamily of proteins is a class of motor ATPases performing a wide range of functions that typically exist as hexamers. The ATPase of phi29 DNA packaging motor has long been a subject of debate in terms of stoichiometry and mechanism of action. Here, we confirmed the stoichiometry of phi29 motor ATPase to be a hexamer and provide data suggesting that the phi29 motor ATPase is a member of the classical hexameric AAA+ superfamily. Native PAGE, EMSA, capillary electrophoresis, ATP titration, and binomial distribution assay show that the ATPase is a hexamer. Mutations in the known Walker motifs of the ATPase validated our previous assumptions that the protein exists as another member of this AAA+ superfamily. Our data also supports the finding that the phi29 DNA packaging motor uses a revolution mechanism without rotation or coiling (Schwartz et al., this issue).

"Push through one-way valve" mechanism of viral DNA packaging.

Double-stranded (ds)DNA viruses package their genomic DNA into a procapsid using a force-generating nanomotor powered by ATP hydrolysis. Viral DNA packaging motors are mainly composed of the connector channel and two DNA packaging enzymes. In 1998, it was proposed that viral DNA packaging motors exercise a mechanism similar to the action of AAA+ ATPases that assemble into ring-shaped oligomers, often hexamers, with a central channel (Guo et al. Molecular Cell, 2:149). This chapter focuses on the most recent findings in the bacteriophage ϕ29 DNA packaging nanomotor to address this intriguing notion. Almost all dsDNA viruses are composed entirely of protein, but in the unique case of ϕ29, packaging RNA (pRNA) plays an intermediate role in the packaging process. Evidence revealed that DNA packaging is accomplished via a "push through one-way valve" mechanism. The ATPase gp16 pushes dsDNA through the connector channel section by section into the procapsid. The dodecameric connector channel functions as a one-way valve that only allows dsDNA to enter but not exit the procapsid during DNA packaging. Although the roles of the ATPase gp16 and the motor connector channel are separate and independent, pRNA bridges these two components to ensure the coordination of an integrated motor. ATP induces a conformational change in gp16, leading to its stronger binding to dsDNA. Furthermore, ATP hydrolysis led to the departure of dsDNA from the ATPase/dsDNA complex, an action used to push dsDNA through the connector channel. It was found unexpectedly that by mutating the basic lysine rings of the connector channel or by changing the pH did not measurably impair DNA translocation or affect the one-way traffic property of the channel, suggesting that the positive charges in the lysine ring are not essential in gearing the dsDNA. The motor channel exercises three discrete, reversible, and controllable steps of gating, with each step altering the channel size by 31% to control the direction of translocation of dsDNA. Many DNA packaging models have been contingent upon the number of base pairs packaged per ATP relative to helical turns for B-type DNA. Both 2 and 2.5 bp per ATP have been used to argue for four, five, or six discrete steps of DNA translocation. The "push through one-way valve" mechanism renews the perception of dsDNA packaging energy calculations and provides insight into the discrepancy between 2 and 2.5 bp per ATP. Application of the DNA packaging motor in nanotechnology and nanomedicine is also addressed. Comparison with nine other DNA packaging models revealed that the "push through one-way valve" is the most agreeable mechanism to interpret most of the findings that led to historical models. The application of viral DNA packaging motors is also discussed.

Cysteinyl-glycine in the control of glutathione homeostasis in bovine lenses.

PURPOSE: To define a possible metabolic and/or signaling role for Cys-Gly in glutathione homeostasis in bovine eye lenses. METHODS: Bovine lenses were cultured up to 24 h in a medium containing 0.5 mM reduced glutathione (GSH) under different conditions. The intracellular and the extracellular contents of thiol compounds were evaluated using a free zone capillary electrophoresis method. RESULTS: Culture of lenses in the presence of GSH and the gamma-glutamyl transferase inhibitor serine-borate demonstrated a 1.5 fold increase in the level of extra-lenticular glutathione with respect to the initial value. Cys-Gly exogenously added impaired the extra-lenticular accumulation of glutathione. Both cysteine and gamma-Glu-Cys were ineffective in reducing extra-lenticular glutathione accumulation. In all conditions no differences in reduced and total intra-lenticular glutathione levels were observed. CONCLUSIONS: The impairment of Cys-Gly generation correlated with inhibition of gamma-glutamyl transferase by serine/borate, resulting in high extra-lenticular concentration of glutathione effluxed from the bovine lens. The possibility that Cys-Gly may intervene either in the replenishment processes for cysteine in the GSH biosynthetic step or in the function of the efflux GSH-transporters is considered.

Local and global mobility in the ClpA AAA+ chaperone detected by cryo-electron microscopy: functional connotations.

The ClpA chaperone combines with the ClpP peptidase to perform targeted proteolysis in the bacterial cytoplasm. ClpA monomer has an N-terminal substrate-binding domain and two AAA+ ATPase domains (D1 and D2). ClpA hexamers stack axially on ClpP heptamers to form the symmetry-mismatched protease. We used cryo-electron microscopy to visualize the ClpA-ATPgammaS hexamer, in the context of ClpAP complexes. Two segments lining the axial channel show anomalously low density, indicating that these motifs, which have been implicated in substrate translocation, are mobile. We infer that ATP hydrolysis is accompanied by substantial structural changes in the D2 but not the D1 tier. The entire N domain is rendered invisible by large-scale fluctuations. When deletions of 10 and 15 residues were introduced into the linker, N domain mobility was reduced but not eliminated and changes were observed in enzymatic activities. Based on these observations, we present a pseudo-atomic model of ClpAP holoenzyme, a dynamic proteolytic nanomachine.