The phylogenetically conserved eukaryotic translation initiation factor 5A (eIF5A) is the only known cellular protein to contain the post-translationally derived amino acid hypusine [N e -(4-amino-2-hydroxybutyl)lysine]. Both eIF5A and its hypusine modification are essential for sustained cell proliferation. Normally only one eIF5A protein is expressed in human cells. Recently, we identified a second human EIF5A gene that would encode an isoform (eIF5A-2) of 84% sequence identity. Overexpression of eIF5A-2 mRNA in certain human cancer cells, in contrast to weak normal expression limited to human testis and brain, suggests EIF5A2 as a potential oncogene. However, eIF5A-2 protein has not been described in human or mammalian cells heretofore. Here, we describe the identification of eIF5A-2 protein in human colorectal and ovarian cancer lines, SW-480 and UACC-1598, that overexpress eIF5A-2 mRNAs. Functional characterization of the human isoforms revealed that either human EIF5A gene can complement growth of a yeast strain in which the yeast EIF5A genes were disrupted. This indicates functional similarity of the human isoforms in yeast and suggests that eIF5A-2 has an important role in eukaryotic cell survival similar to that of the ubiquitous eIF5A-1. Detectable structural differences were also noted, including lack of immunological cross-reactivity, formation of different complexes with deoxyhypusine synthase, and K m values (1.5 ± 0.2 vs. 8.3 ± 1.4 lM for eIF5A-1 and -2, respectively) as substrates for deoxyhypusine synthase in vitro. These physical characteristics and distinct amino acid sequences in the C-terminal domain together with differences in gene expression patterns imply differentiated, tissue-specific functions of the eIF5A-2 isoform in the mammalian organism and in cancer.

The Togavirus (Alphavirus) Mayaro virus (MAYV) was initially described in 1954 from Mayaro County (Trinidad) and has been responsible for outbreaks in South America and the Caribbean. Imported MAYV cases are on the rise, leading to invasion concerns similar to Chikungunya and Zika viruses. Little is known about the range of mosquito species that are competent MAYV vectors. We tested vector competence of 2 MAYV genotypes in laboratory strains of six mosquito species (Aedes aegypti, Anopheles freeborni, An. gambiae, An. quadrimaculatus, An. stephensi, Culex quinquefasciatus). Ae. aegypti and Cx. quinquefasciatus were poor MAYV vectors, and had either poor or null infection and transmission rates at the tested viral challenge titers. In contrast, all Anopheles species were able to transmit MAYV, and 3 of the 4 species transmitted both genotypes. The Anopheles species tested are divergent and native to widely separated geographic regions (Africa, Asia, North America), suggesting that Anopheles may be important in the invasion and spread of MAYV across diverse regions of the world.

Eukaryotic initiation factor 5A (eIF5A) is a putative translation initiation factor and is the only cellular protein that contains the unique modified Lys, hypusine [N e -(4-amino-2-hydroxybutyl)lysine] [1]. Hypusine is formed post-translationally at one specific Lys residue of the eIF5A precursor in two consecutive enzymatic reactions [2,3]. The first enzyme, deoxyhypusine synthase (DHS) [4,5], catalyzes the transfer of the

Protein degradation by eukaryotic proteasomes is a multistep process involving substrate recognition, ATP-dependent unfolding, translocation into the proteolytic core particle, and finally proteolysis. To date, most investigations of proteasome function have focused on the first and the last steps in this process. Here we examine the relationship between the stability of a folded protein domain and its degradation rate. Test proteins were targeted to the proteasome independently of ubiquitination by directly tethering them to the protease. Degradation kinetics were compared for test protein pairs whose stability was altered by either point mutation or ligand binding, but were otherwise identical. In both intact cells and in reactions using purified proteasomes and substrates, increased substrate stability led to an increase in substrate turnover time. The steadystate time for degradation ranged from ϳ5 min (dihydrofolate reductase) to 40 min (I27 domain of titin). ATP turnover was 110/min./proteasome, and was not markedly changed by substrate. Proteasomes engage tightly folded substrates in multiple iterative rounds of ATP hydrolysis, a process that can be ratelimiting for degradation.To degrade folded proteins, chambered protease complexes unfold substrates in an energy-dependent manner that requires two components (1). The first, a regulatory complex, recognizes substrates and initiates their processing. The second, an associated proteolytic complex, contains sites at which peptide bonds are hydrolyzed. These sites are present in a closed chamber sequestered from the general cellular environment. The proteolytic chamber is accessed through a pore that excludes folded protein domains but accommodates an unfolded or unstructured polypeptide (2, 3). Regulatory and catalytic complexes must thus collaborate to degrade native proteins: the regulatory complex actively unfolds substrates containing structured domains and translocates them into the catalytic complex. Substrate unfolding and translocation by bacterial ATP-dependent proteases has been extensively studied. It was found that substrate unfolding can be rate-limiting for degradation and that increased mechanical stability of substrates prolongs degradation (4). Is that also true of eukaryotic proteasomes? Studies from our laboratory (5, 6) and by others (7-10) are consistent with this conclusion, but studies using purified proteasomes and substrates of well-defined structure have been limited and have not determined the kinetic parameters associated with proteasome action.There are several requirements for rigorously performing such an analysis. Homogeneous substrates must be available that differ in their resistance to unfolding, but are otherwise identical in structure and proteasomal interaction. Most substrates are designated for destruction by the conjugation of ubiquitin chains, which provide a high affinity tag for proteasome association, but some substrates utilize alternate tags to designate degradation (11,12). Capture of ubiquitin chains and their...

Innate immune cells shape the host response to microbial pathogens. Here we elucidate critical differences in the molecular response of macrophages vs. dendritic cells (DCs) to Histoplasma capsulatum, an intracellular fungal pathogen of humans. It has long been known that macrophages are permissive for Histoplasma growth and succumb to infection, whereas DCs restrict fungal growth and survive infection. We used murine macrophages and DCs to identify host pathways that influence fungal proliferation and host-cell viability. Transcriptional profiling experiments revealed that DCs produced a strong Type I interferon (IFN-I) response to infection with Histoplasma yeasts. Toll-like receptors 7 and 9 (TLR7/9), which recognize nucleic acids, were required for IFN-I production and restriction of fungal growth in DCs, but mutation of TLR7/9 had no effect on the outcome of macrophage infection. Moreover, TLR7/9 were essential for the ability of infected DCs to elicit production of the critical cytokine IFNγ from primed CD4+ T cells in vitro, indicating the role of this pathway in T cell activation. In a mouse model of infection, TLR7/9 were required for optimal production of IFN-I and IFNγ, host survival, and restriction of cerebral fungal burden. These data demonstrate the critical role of this pathway in eliciting an appropriate adaptive immune response in the host. Finally, although other fungal pathogens have been shown to elicit IFN-I in mouse models, the specific host cell responsible for producing IFN-I has not been elucidated. We found that CD103+ conventional DCs were the major producer of IFN-I in the lungs of wild-type mice infected with Histoplasma. Mice deficient in this DC subtype displayed reduced IFN-I production in vivo. These data reveal a previously unknown role for CD103+ conventional DCs and uncover the pivotal function of these cells in modulating the host immune response to endemic fungi.