The Escherichia coli Lon protease degrades the E. coli DNA-binding protein HUbeta, but not the related protein HUalpha. Here we show that the Lon protease binds to both HUbeta and HUalpha, but selectively degrades only HUbeta in the presence of ATP. Mass spectrometry of HUbeta peptide fragments revealed that region K18-G22 is the preferred cleavage site, followed in preference by L36-K37. The preferred cleavage site was further refined to A20-A21 by constructing and testing mutant proteins; Lon degraded HUbeta-A20Q and HUbeta-A20D more slowly than HUbeta. We used optical tweezers to measure the rupture force between HU proteins and Lon; HUalpha, HUbeta, and HUbeta-A20D can bind to Lon, and in the presence of ATP, the rupture force between each of these proteins and Lon became weaker. Our results support a mechanism of Lon protease cleavage of HU proteins in at least three stages: binding of Lon with the HU protein (HUbeta, HUalpha, or HUbeta-A20D); hydrolysis of ATP by Lon to provide energy to loosen the binding to the HU protein and to allow an induced-fit conformational change; and specific cleavage of only HUbeta.

On agar plates, daughter cells of Escherichia coli mutually slide and align side-by-side in parallel during the first round of binary fission. This phenomenon has been previously attributed to an elastic material that restricts apparently separated bacteria from being in string. We hypothesize that the interaction between bacteria and the underneath substratum may affect the arrangement of the daughter bacteria. To test this hypothesis, bacterial division on hyaluronic acid (HA) gel, as an alternative substratum, was examined. Consistent with our proposition, the HA gel differs from agar by suppressing the typical side-by-side alignments to a rare population. Examination of bacterial surface molecules that may contribute to the daughter cells' arrangement yielded an observation that, with disrupted lpp, the E. coli daughter cells increasingly formed non-typical patterns, i.e. neither sliding side-by-side in parallel nor forming elongated strings. Therefore, our results suggest strongly that the early cell patterning is affected by multiple interaction factors. With oscillatory optical tweezers, we further demonstrated that the interaction force decreased in bacteria without Lpp, a result substantiating our notion that the side-by-side sliding phenomenon directly reflects the strength of in-situ interaction between bacteria and substratum.

Following vascular injury, blood coagulation and platelet activation trigger the wound healing process. The migration of blood cells through the leaky vessels, formation of new blood vessels, and the synthesis of extracellular matrix (ECM) are essential for tissue repair. Tissue transglutaminase (TTG) is a unique extracellular and intracellular enzyme that stabilizes tissues, binds and releases nitric oxide (NO), and hydrolyzes GTP and ATP. TTG’s crosslinking (TGase) activity makes ECM resistant to protease digestion and aids wound healing. Recent studies demonstrate TTG binds and releases NO inhibiting platelet aggregation and neutrophil migration which inhibiting the inflammatory response. TTG is an enzyme with 687 amino acid residues. The active site involved in protein crosslinking is located at Cys277, while GTP/ATP binding domain is located in the N- and C-terminus and there are 18 free Cys-SH groups distributed throughout the molecule to bind NO. When vascular tissues derived from different phases of wound healing were analyzed on immunoblotting, we detected full-length and truncated forms of TTG antigens. To determine whether the truncated forms of TTG were formed by protein proteolysis or alternative splicing, we screened a human smooth muscle cell lambda cDNA library using a full-length human TTG cDNA as a probe. DNA sequencing analysis of the positive clones revealed that, in additional to wild type, two C-terminal truncated forms, TTG3 and TTG4, were also present. TTG3 and TTG4 were produced by a rare alternate splicing event utilizing alternate 5′ and 3′ splice site, located within exons XII and XIII, respectively. TTG3 and TTG4 were composed of 674 and 646 amino acid residues that shared identical N-terminal 622 amino acids with TTG with distinct 52 and 23 amino acids at the C-terminus that translated into proteins with the predicted Mr of 75 and 70 KDa, respectively. Structure-function studies using purified enzymes demonstrated that TTG3 and TTG4 showed the same calcium requirement as TTG, but had only 9 and 8% of residual TGase activity, respectively. TGase activity of TTG was inhibited by GTP with an IC50 of 6 microM, while both isoforms were not inhibited by up to 400 microM of GTP. GTP also failed to induce a conformational change in the molecule and both isoforms were proteolyzed by tyrpsin while full-length TTG remained intact. Both isoforms retained GTPase and ATPase activities. RT-PCR and immunoblotting demonstrated that TTG3 and TTG4 were expressed at less than 10 and 5% of TTG and were localized in the nucleus in human umbilical vascular endothelial (HUVEC) and vascular smooth muscle (VSMC) cells. In contrast, human leukocytes and platelets contained ~7-fold higher levels of both isoforms than TTG. In conclusion, we identified two novel C-terminal truncated forms of TTG that are expressed by HUVEC, VSMC, human leukocytes and platelets. This is the first report of the expression of two novel TTG isoforms in human blood cells. The different affinity for GTP and TGase activities, distinct intracellular localization and high expression levels in human leukocytes and platelets suggest a unique physiological function of these isoforms during hemostasis. In addition, TTG3 has two additional Cys-SH groups which could bind NO. The physiological significance of both isoforms of TTG in regulating wound repair is currently under investigation.

in the original publication. The affiliations have been corrected here and in the article online. In addition, ''Republic of China'' or ''ROC'' has been replaced with ''Taiwan'' in the affiliations and the Acknowledgments.