Rab1A is an mTORC1 activator and a colorectal oncogene.

Amino acid (AA) is a potent mitogen that controls growth and metabolism. Here we describe the identification of Rab1 as a conserved regulator of AA signaling to mTORC1. AA stimulates Rab1A GTP binding and interaction with mTORC1 and Rheb-mTORC1 interaction in the Golgi. Rab1A overexpression promotes mTORC1 signaling and oncogenic growth in an AA- and mTORC1-dependent manner. Conversely, Rab1A knockdown selectively attenuates oncogenic growth of Rab1-overexpressing cancer cells. Moreover, Rab1A is overexpressed in colorectal cancer (CRC), which is correlated with elevated mTORC1 signaling, tumor invasion, progression, and poor prognosis. Our results demonstrate that Rab1 is an mTORC1 activator and an oncogene and that hyperactive AA signaling through Rab1A overexpression drives oncogenesis and renders cancer cells prone to mTORC1-targeted therapy.

Vaccinia virus leads to ATG12­ATG3 conjugation and deficiency in autophagosome formation.

The interactions between viruses and cellular autophagy have been widely reported. On the one hand, autophagy is an important innate immune response against viral infection. On the other hand, some viruses exploit the autophagy pathway for their survival and proliferation in host cells. Vaccinia virus is a member of the family of Poxviridae which includes the smallpox virus. The biogenesis of vaccinia envelopes, including the core envelope of the immature virus (IV), is not fully understood. In this study we investigated the possible interaction between vaccinia virus and the autophagy membrane biogenesis machinery. Massive LC3 lipidation was observed in mouse fibroblast cells upon vaccinia virus infection. Surprisingly, the vaccinia virus induced LC3 lipidation was shown to be independent of ATG5 and ATG7, as the atg5 and atg7 null mouse embryonic fibroblasts (MEFs) exhibited the same high levels of LC3 lipidation as compared with the wild-type MEFs. Mass spectrometry and immunoblotting analyses revealed that the viral infection led to the direct conjugation of ATG3, which is the E2-like enzyme required for LC3-phosphoethanonamine conjugation, to ATG12, which is a component of the E3-like ATG12­ATG5-ATG16 complex for LC3 lipidation. Consistently, ATG3 was shown to be required for the vaccinia virus induced LC3 lipidation. Strikingly, despite the high levels of LC3 lipidation, subsequent electron microscopy showed that vaccinia virus-infected cells were devoid of autophagosomes, either in normal growth medium or upon serum and amino acid deprivation. In addition, no autophagy flux was observed in virus-infected cells. We further demonstrated that neither ATG3 nor LC3 lipidation is crucial for viral membrane biogenesis or viral proliferation and infection. Together, these results indicated that vaccinia virus does not exploit the cellular autophagic membrane biogenesis machinery for their viral membrane production. Moreover, this study demonstrated that vaccinia virus instead actively disrupts the cellular autophagy through a novel molecular mechanism that is associated with aberrant LC3 lipidation and a direct conjugation between ATG12 and ATG3.

A sensitive coupled HPLC/electrospray mass spectrometry assay for SPM-1 metallo-beta-lactamase inhibitors.

Antibiotic-resistant bacteria continue to threaten human health through multiple mechanisms, including hydrolytic inactivation of beta-lactam antibiotics by metallo-beta-lactamases (MBLs). The SPM-1 enzyme, originally identified from a Pseudomonas aeruginosa clinical isolate, is a Class B beta-lactamase responsible for resistance in bacteria against antibiotics such as penicillins, cephalosporins, and carbapenems. Unlike Class A, C, and D beta-lactamases, which employ a serine residue in their active site, Class B enzymes possess one or two Zn atoms in the active site that play both a structural and catalytic role. A beta-lactamase inhibitor with co-administration of a beta-lactam antibiotic has proven to be an effective treatment against antibiotic-resistant bacteria whose resistance is due to serine-based beta-lactamases (e.g., amoxicillin/clavulanic acid). A similar clinical approach has not yet been developed for resistant bacteria possessing MBLs. The identification and development of specific and effective MBL inhibitors to combat this resistance could extend the utility of currently prescribed antibiotics such as cephalosporins and carbapenems. To discover MBL inhibitors, compound libraries are screened typically by enzymatic hydrolysis of a chromogenic substrate such as nitrocefin monitored by absorbance. Spectrophotometric assays, while valuable, lack the sensitivity and selectivity to screen natural product extract libraries because of the strongly absorbing nature of some extracts and the dilute concentrations of active components. An assay is described herein that monitors the SPM-1-catalyzed hydrolysis of penicillin G by high-performance (high-pressure) liquid chromatography-electrospray mass spectroscopy, which permits investigations with greater sensitivity and selectivity allowing the screening of natural product extracts for inhibitors of MBLs.