The proteasome: not just degrading anymore

The proteasome is a large multiprotein complex that has a critical role in the degradation of ubiquitylated proteins. A fascinating paper in this issue of Cell (Lee et al., 2005) now reveals that the proteasome recruits the SAGA histone acetyltransferase complex to a target promoter during gene activation. This finding adds to the growing body of evidence indicating that the proteasome has nonproteolytic functions.     

Ubiquitin: not just for proteasomes anymore

Ubiquitin is a small protein that can be covalently linked to itself or other proteins, either as single ubiquitin molecules or as chains of polyubiquitin. Addition of ubiquitin to a target protein requires a series of enzymatic activities (by ubiquitin-activating, -conjugating and -ligating enzymes). The first function attributed to ubiquitin was the covalent modification of misfolded cytoplasmic proteins, thereby directing proteasome-dependent proteolysis. More recently, additional functions have been ascribed to ubiquitin and ubiquitin-related proteins. Ubiquitin directs specific proteins through the endocytic pathway by modifying cargo proteins, and possibly also components of the cytoplasmic protein trafficking machinery.     

Not just for Eukarya anymore: protein glycosylation in Bacteria and Archaea

Of the many post-translational modifications proteins can undergo, glycosylation is the most prevalent and the most diverse. Today, it is clear that both N-glycosylation and O-glycosylation, once believed to be restricted to eukaryotes, also transpire in Bacteria and Archaea. Indeed, prokaryotic glycoproteins rely on a wider variety of monosaccharide constituents than do those of eukaryotes. In recent years, substantial progress in describing the enzymes involved in bacterial and archaeal glycosylation pathways has been made. It is becoming clear that enhanced knowledge of bacterial glycosylation enzymes may be of therapeutic value, while the demonstrated ability to introduce bacterial glycosylation genes into Escherichia coli represents a major step forward in glyco-engineering. A better understanding of archaeal protein glycosylation provides insight into this post-translational modification across evolution as well as protein processing under extreme conditions. Here, we discuss new structural and biosynthetic findings related to prokaryotic protein glycosylation, until recently a neglected topic.     

Metalloproteinases and cell fate: Notch just ADAMs anymore

Notch signaling is involved both in development as well as in multiple cancers, including pancreatic cancer. Its activity has been implicated early in pancreatic disease, shown to be essential for a pre-cancerous transdifferentiation event known as acinar-to-ductal metaplasia (ADM). Recently, we have shown that matrix metalloproteinase-7 (MMP-7) is essential for ADM by activating the Notch pathway, challenging the notion that ADAM metalloproteinases are the sole enzymes responsible for initiating Notch activity. In ADM, ADAMs do not compensate for the absence of MMP-7 activity. We propose that during development and stem cell maintenance, Notch activation is highly regulated by the binding of Notch ligand to receptor and employs the ubiquitously-expressed ADAMs, whereas in a disease state, high levels of induced MMP-7 activity can lead to aberrant ligand-independent Notch activation. Therefore, if ADM or PDA is to be blocked by inhibiting Notch, treatment with ADAM-specific inhibitors alone will be inadequate. Other approaches for Notch inhibition, including by γ-secretase and broad-spectrum MMP inhibitors, will be discussed.     

Calreticulin: not just another calcium-binding protein

In this paper we review some of the rapidly expanding information about calreticulin, a Ca²⁺-binding/storage protein of the endoplasmic reticulum. The emphasis is placed on the structure and function of calreticulin. We believe that calreticulin is a multifunctional Ca²⁺-binding protein and that distinct functional properties of the protein may be localized to each of the three structural domains of calreticulin. Most evidence indicates that calreticulin is a resident endoplasmic reticulum protein. However, it can also be found outside of the endoplasmic reticulum compartment, i.e. in the nuclear envelope, in the nucleus, in the cytotoxic granules in T-lymphocytes and in acrosomal vesicles of sperm cells. The evidence reviewed here clearly suggests that calreticulin has other functions in addition to its role as a Ca²⁺ storage protein in the endoplasmic reticulum.Abbreviations SR sarcoplasmic reticulum - ER endoplasmic reticulum     

Identification of two functional regions in Fis: the N-terminus is required to promote Hin-mediated DNA inversion but not lambda excision.

The Fis protein of E. coli binds to a recombinational enhancer sequence that is required to stimulate Hin-mediated DNA inversion. Fis is also required for efficient lambda prophase excision in vivo. The properties of mutant Fis proteins were examined in vivo and in vitro with respect to their stimulatory effects on these two different site-specific DNA recombination reactions. Both recombination reactions are dramatically affected by mutations altering a helix-turn-helix DNA binding motif located near the Fis C-terminus (residues 74-93). These mutations invariably decrease DNA binding affinity and some cause reduced DNA bending. Mutations in the Fis N-terminal region reduce or abolish the stimulation of Hin-mediated DNA recombination by Fis, but have little or no effect on DNA binding or lambda excision. We conclude that there are at least two functionally distinct domains in Fis: a C-terminal DNA binding region that is required for promoting both DNA recombination reactions and an N-terminal region that is uniquely required for Hin-mediated inversion.     

The strong-inference protocol: not just for grant proposals

The strong-inference protocol puts into action the important concepts in Platt's often-assigned, classic paper on the strong-inference method (10). Yet, perhaps because students are frequently performing experiments with known outcomes, the protocols they write as undergraduates are usually little more than step-by-step instructions for performing the experiment. The strong-inference protocol, however, includes an explicit statement of possible experimental outcomes and the interpretation that would follow from each. This approach encourages thorough planning, enhances the efficiency of experimental designs, and increases the power of statistical analysis by explicitly stating a priori predictions as well as the statistical methods that will be used to test them. A sample protocol for an experiment investigating temperature-metabolism relations in chicken embryos is provided to illustrate the important components of the strong-inference protocol and to encourage instructors to incorporate this powerful research tool into undergraduate laboratory courses.     

Xis and Fis proteins prevent site-specific DNA inversion in lysogens of phage HK022.

HK022, a temperate coliphage related to lambda, forms lysogens by inserting its DNA into the bacterial chromosome through site-specific recombination. The Escherichia coli Fis and phage Xis proteins promote excision of HK022 DNA from the bacterial chromosome. These two proteins also act during lysogenization to prevent a prophage rearrangement: lysogens formed in the absence of either Fis or Xis frequently carried a prophage that had suffered a site-specific internal DNA inversion. The inversion is a product of recombination between the phage attachment site and a secondary attachment site located within the HK022 left operon. In the absence of both Fis and Xis, the majority of lysogens carried a prophage with an inversion. Inversion occurs during lysogenization at about the same time as prophage insertion but is rare during lytic phage growth. Phages carrying the inverted segment are viable but have a defect in lysogenization, and we therefore suggest that prevention of this rearrangement is an important biological role of Xis and Fis for HK022. Although Fis and Xis are known to promote excision of lambda prophage, they had no detectable effect on lambda recombination at secondary attachment sites. HK022 cIts lysogens that were blocked in excisive recombination because of mutation in fis or xis typically produced high yields of phage after thermal induction, regardless of whether they carried an inverted prophage. The usual requirement for prophage excision was bypassed in these lysogens because they carried two or more prophages inserted in tandem at the bacterial attachment site; in such lysogens, viable phage particles can be formed by in situ packaging of unexcised chromosomes.     

Senescence: not just for tumor suppression

Cellular senescence provides an intrinsic barrier to tumor development by preventing the proliferation of cells that are at risk for malignant transformation. In this issue, Krizhanovsky et al. (2008) report that senescence is an important player not only in tumor suppression but also in the response of liver tissue to injury.