A multiubiquitin chain is confined to specific lysine in a targeted short-lived protein.

The ubiquitin-dependent degradation of a test protein beta-galactosidase (beta gal) is preceded by ubiquitination of beta gal. The many (from 1 to more than 20) ubiquitin moieties attached to a molecule of beta gal occur as an ordered chain of branched ubiquitin-ubiquitin conjugates in which the carboxyl-terminal Gly76 of one ubiquitin is jointed to the internal Lys48 of an adjacent ubiquitin. This multiubiquitin chain is linked to one of two specific Lys residues in beta gal. These same Lys residues have been identified by molecular genetic analysis as components of the aminoterminal degradation signal in beta gal. The experiments with ubiquitin mutated at its Lys48 residue indicate that the multiubiquitin chain in a targeted protein is essential for the degradation of the protein.

Minimally invasive thoracic corpectomy and anterior fusion in a patient with metastatic disease: case report and review of the literature.

For patients with metastatic disease to the spine there are numerous surgical approaches for decompression of neural elements and maintenance of mechanical stability. The challenge is to accomplish this while minimizing patient morbidity. Here we report on the feasibility and utility of a minimally invasive extreme lateral approach to the mid to high thoracic spine for anterior decompression and fusion.

Neural correlates of unstructured motor behaviors.

OBJECTIVE: We studied the relationship between uninstructed, unstructured movements and neural activity in three epilepsy patients with intracranial electroencephalographic (iEEG) recordings. APPROACH: We used a custom system to continuously record high definition video precisely time-aligned to clinical iEEG data. From these video recordings, movement periods were annotated via semi-automatic tracking based on dense optical flow. MAIN RESULTS: We found that neural signal features (8-32 Hz and 76-100 Hz power) previously identified from task-based experiments are also modulated before and during a variety of movement behaviors. These movement behaviors are coarsely labeled by time period and movement side (e.g. 'Idle' and 'Move', 'Right' and 'Left'); movements within a label can include a wide variety of uninstructed behaviors. A rigorous nested cross-validation framework was used to classify both movement onset and lateralization with statistical significance for all subjects. SIGNIFICANCE: We demonstrate an evaluation framework to study neural activity related to natural movements not evoked by a task, annotated over hours of video. This work further establishes the feasibility to study neural correlates of unstructured behavior through continuous recording in the epilepsy monitoring unit. The insights gained from such studies may advance our understanding of how the brain naturally controls movement, which may inform the development of more robust and generalizable brain-computer interfaces.

Novel CDC34 (UBC3) ubiquitin-conjugating enzyme mutants obtained by charge-to-alanine scanning mutagenesis.

CDC34 (UBC3) encodes a ubiquitin-conjugating (E2) enzyme required for transition from the G1 phase to the S phase of the budding yeast cell cycle. CDC34 consists of a 170-residue catalytic N-terminal domain onto which is appended an acidic C-terminal domain. A portable determinant of cell cycle function resides in the C-terminal domain, but determinants for specific function must reside in the N-terminal domain as well. We have explored the utility of "charge-to-alanine" scanning mutagenesis to identify novel N-terminal domain mutants of CDC34 that are enzymatically competent with respect to unfacilitated (E3-independent) ubiquitination but that nevertheless are defective with respect to its cell cycle function. Such mutants may reveal determinants of specific in vivo function, such as those required for interaction with substrates or trans-acting regulators of activity and substrate selectivity. Three of 18 "single-scan" mutants (in which small clusters of charged residues were mutated to alanine) were compromised with respect to in vivo function. One mutant (cdc34-109, 111, 113A) targeted a 12-residue segment of the Cdc34 protein not found in most other E2s and was unable to complement a cdc34 null mutant at low copy numbers but could complement a null mutant when overexpressed from an induced GAL1 promoter. Combining adjacent pairs of single-scan mutants to produce "double-scan" mutants yielded four additional mutants, two of which showed heat and cold sensitivity conditional defects. Most of the mutant proteins expressed in Escheria coli displayed unfacilitated (E3-independent) ubiquitin-conjugating activity, but two mutants differed from wild-type and other mutant Cdc34 proteins in the extent of multiubiquitination they catalyzed during an autoubiquitination reation-conjugating enzyme function and have identified additional mutant alleles of CDC34 that will be valuable in further genetic and biochemical studies of Cdc34-dependent ubiquitination.

Intermediates in homologous pairing promoted by recA protein. Isolation and characterization of active presynaptic complexes.

recA protein promotes homologous pairing and strand exchange by an ordered reaction in which the protein first polymerizes on single-stranded DNA. This presynaptic intermediate, which can be formed either in the presence or absence of Escherichia coli single-stranded binding protein (SSB), has been isolated by gel filtration and characterized. At saturation, purified complexes contained one molecule of recA protein per 3.6 nucleotide residues of single-stranded DNA. Complexes that had been formed in the presence of SSB contained up to one molecule of SSB per 15 nucleotide residues, but the content of SSB in different preparations of isolated complexes appeared to be inversely related to the content of recA protein. Even when they have lost as much as a third of their recA protein, presynaptic complexes can retain activity, because the formation of stable joint molecules depends principally on the binding of recA protein to the single-stranded DNA in the localized region that corresponds to the end of the duplex substrate.

Prognostic factors for stereotactic radiosurgery-treated patients with cerebral metastasis: implications on randomised control trial design and inter-institutional collaboration.

INTRODUCTION: Defining key prognostic factors for patients with cerebral metastases who underwent stereotactic radiosurgery (SRS) treatment will greatly facilitate future clinical trial designs. METHODS: We adopted a two-phase study design where results from one cohort were validated in a second independent cohort. The exploratory analysis reviewed the survival outcomes of 1017 consecutive patients (with 3610 metastases) who underwent Gamma radiosurgery at the University of California, San Diego (UCSD)/San Diego Gamma Knife Center (SDGKC). Multivariate analysis was performed to identify prognostic factors. Results were validated using data derived from 2519 consecutive patients (with 17,498 metastases) treated with SRS at the Katsuta Hospital. RESULTS: For the SDGKC cohort, the median overall survival of patients following SRS was 7 months. Two year follow-up data were available for 85% of the patients. Multivariate analysis found that patient age, Karnofsky Performance Status, systemic cancer status, tumour histology, number of metastasis and cumulative tumour volume independently associated with overall survival (p<0.001). All statistical associations were validated by multivariate analysis of data derived from the Katsuta Hospital cohort. CONCLUSIONS: This is the first integrated study that defined prognostic factors for SRS-treated patients with cerebral metastases using an inter-institutional validation study design. The work establishes a model for collaborative interactions between large volume centers and provides prognostic variables that should be incorporated into future clinical trial design.

The mechanism of the search for homology promoted by recA protein. Facilitated diffusion within nucleoprotein networks.

recA protein promotes the homologous pairing of single strands with duplex DNA by polymerizing on the single strands to make presynaptic nucleoprotein filaments which are polyvalent with respect to duplex DNA and which consequently form large networks or coaggregates when duplex DNA is added. Previous work has shown that efficient homologous pairing occurs within these networks. In the experiments described here, we observed that the length of the duplex DNA determined the stability of coaggregates, their steady state level, and the yield of joint molecules. Correspondingly, heterologous duplex DNA when preincubated with presynaptic filaments excluded subsequently added homologous duplex DNA from coaggregates and inhibited homologous pairing; the extents of exclusion and inhibition were determined by the length of the heterologous duplex DNA. On the other hand, long heterologous duplex DNA when added together with short homologous duplex DNA was capable of stimulating the absorption of the homologous molecules into coaggregates and increasing the rate of homologous pairing. In reactions involving short duplex molecules, polyamines exerted comparable effects on coaggregation and homologous pairing. We conclude that coaggregates are instrumental in homologous pairing, that they constitute distinct domains that are responsible for the processive or first order character of the pairing reaction, and that they act by concentrating DNA and facilitating diffusion.

By searching processively RecA protein pairs DNA molecules that share a limited stretch of homology.

RecA protein promotes homologous pairing by a reaction in which the protein first binds stoichiometrically to single-stranded DNA in a slow presynaptic step, and then conjoins single-stranded and duplex DNA, thereby forming a ternary complex. RecA protein did not pair molecules that shared only 30 bp homology, but, with full efficiency, it paired circular single-stranded and linear duplex molecules in which homology was limited to 151 bp at one end of the duplex DNA. The initial rate of the pairing reaction was directly related to the length of the heterologous part of the duplex DNA, which we varied from 0 to 3060 base pairs. Since interactions involving the heterologous part of a molecule speed the location of a small homologous region, we conclude that RecA protein promotes homologous alignment by a processive mechanism involving relative motion of conjoined molecules within the ternary complex.

cis-trans recognition and subunit-specific degradation of short-lived proteins.

The N-end rule, a code that relates the metabolic stability of a protein to the identity of its amino-terminal residue, is universal in that different versions of the N-end rule operate in mammals, yeast and bacteria (unpublished data). The N-end rule-based degradation signal comprises a destabilizing amino-terminal residue and a specific internal lysine residue. We now show that, in a multisubunit protein, these two determinants can be located on different subunits and still target the protein for destruction. Moreover, in this case (trans recognition) only the subunit that bears the lysine determinant is actually degraded. Thus an oligomeric protein can contain both short-lived and long-lived subunits. These insights have functional and practical implications.

Kinetics of homologous pairing promoted by RecA protein: effects of ends and internal sites in DNA.

When recA protein was preincubated with single-stranded DNA in the presence of an ATP-regenerating system prior to the addition of homologous duplex DNA, a slow presynaptic step was eliminated, and the subsequent homologous pairing was revealed as a reaction whose rate exceeds by 1 or 2 orders of magnitude the calculated rate of spontaneous renaturation in 0.15 M NaCl at Tm -25 degrees C. The pairing reaction displayed saturation kinetics with respect to both single-stranded and double-stranded DNA, indicating the existence of a rate-limiting enzyme-substrate complex. The signal observed in the assay of the pairing reaction was due to pairing at free homologous ends of the duplex DNA, as well as pairing in the middle of the duplex molecule, away from a free end. The apparent rate of pairing of circular single strands with linear duplex DNA was equal to the sum of the rates of pairing at sites located at either end of the duplex DNA or at interior sites, but the apparent rates attributable to ends were greater, and nicks also stimulated the apparent rate.

Characterization of novel yeast RAD6 (UBC2) ubiquitin-conjugating enzyme mutants constructed by charge-to-alanine scanning mutagenesis.

Ubiquitination of intracellular proteins by the yeast RAD6 (UBC2) ubiquitin-conjugating (E2) enzyme is required for cellular processes as diverse as DNA repair, selective proteolysis, and normal growth. For most RAD6-dependent functions, the relevant in vivo targets, as well as the mechanisms and cofactors that govern RAD6 substrate selectivity, are unknown. We have explored the utility of "charge-to-alanine" scanning mutagenesis to generate novel RAD6 mutants that are enzymatically competent with respect to unfacilitated (E3-independent) ubiquitination but that are nevertheless severely handicapped with respect to several in vivo functions. Five of the nine mutants we generated show defects in their in vivo functions, but almost all of the most severely affected mutants displayed unfacilitated ubiquitin-conjugating activity in vitro. We suggest that E2 mutants obtained by this approach are likely to be defective with respect to interaction with other, trans-acting factors required for their intracellular activity or substrate selectivity and therefore will be useful for further genetic and biochemical studies of ubiquitin-conjugating enzyme function.

Identification of a portable determinant of cell cycle function within the carboxyl-terminal domain of the yeast CDC34 (UBC3) ubiquitin conjugating (E2) enzyme.

The ubiquitin conjugating (E2) enzyme encoded by CDC34 (UBC3) in Saccharomyces cerevisiae is required for the G1 to S transition of the cell cycle. CDC34 consists of a 170 residue amino-terminal domain that is homologous to that found in other E2s, followed by a 125 residue carboxyl-terminal domain that is specific to CDC34. We found that a truncation mutant of CDC34 which lacked the CDC34 carboxyl-terminal domain could not support the essential function of CDC34 in the cell cycle in vivo. To explore further the role of the carboxyl-terminal domain in determining the cell cycle function of CDC34, we constructed and characterized genes encoding chimeric E2s incorporating sequences from CDC34 and the related but functionally distinct E2 RAD6 (UBC2). We found that a construct encoding a chimeric RAD6-CDC34 ubiquitin conjugating enzyme, in which the 21 residue acidic carboxyl-terminal domain of RAD6 has been replaced with the 125 residue carboxyl-terminal domain of CDC34, performed the essential functions of CDC34 in vivo. This chimeric E2 also complemented the growth deficiency, UV sensitivity and sporulation deficiency of rad6 mutant strains. Deletion analysis of the CDC34 carboxyl-terminal domain in both CDC34 and the RAD6-CDC34 chimeric E2 identified a region comprising residues 171-244 of CDC34 that was sufficient to confer CDC34 function on the amino-terminal domains of CDC34 and RAD6. We suggest that this region interacts with substrates of CDC34 or with trans-acting factors (such as CDC34-specific ubiquitin protein ligases) that govern the substrate selectivity of CDC34. Congruent results demonstrating a positive role for the carboxyl-terminal domain of CDC34 in the essential function of CDC34 have also been obtained by Silver et al. (1992) and are reported in the accompanying paper.

The pairing activity of stable nucleoprotein filaments made from recA protein, single-stranded DNA, and adenosine 5'-(gamma-thio)triphosphate.

Under conditions that diminish secondary structure in single-stranded DNA, stable presynaptic filaments can be formed by recA protein in the presence of the nonhydrolyzable analog ATP gamma S, without the need for Escherichia coli single strand binding protein. Such stable presynaptic filaments resemble those formed in the presence of ATP and pair efficiently with homologous duplex DNA. Since this kind of stable filament does not displace a strand from the duplex molecule, it provides a model substrate to study synapsis independent of the earlier and later stages of the recA reaction. Even though detectable strand displacement did not occur in the presence of ATP gamma S, both single strand and double strand breaks in duplex DNA stimulated homologous pairing. These and related observations support the view that the presynaptic nucleoprotein filament and naked duplex DNA intertwine to form a nascent joint in which the duplex DNA is partially unwound, i.e. in which the pitch of the involved duplex segment is reduced.

Universality and structure of the N-end rule.

Our previous work has shown that, in the yeast Saccharomyces cerevisiae, any of the eight stabilizing amino-terminal residues confers a long (greater than 20 h) half-life on a test protein beta-galactosidase (beta gal), whereas 12 destabilizing amino-terminal residues confer on beta gal half-lives from less than 3 min to 30 min. We now show that an analogous single-residue code (the N-end rule) operates in an in vitro system derived from mammalian reticulocytes. We also show that the N-end rule has a hierarchical structure. Specifically, amino-terminal Glu and Asp (and also Cys in reticulocytes) are secondary destabilizing residues in that they are destabilizing through their ability to be conjugated to primary destabilizing residues such as Arg. Amino-terminal Gln and Asn are tertiary destabilizing residues in that they are destabilizing through their ability to be converted, via selective deamidation, into secondary destabilizing residues Glu and Asp. Furthermore, in reticulocytes, distinct types of the N-end-recognizing activity are shown to be specific for three classes of primary destabilizing residues: basic (Arg, Lys, His), bulky hydrophobic (Phe, Leu, Trp, Tyr), and small uncharged (Ala, Ser, Thr). Features of the N-end rule in reticulocytes suggest that the exact form of the N-end rule may depend on the cell's physiological state, thereby providing a mechanism for selective destruction of preexisting proteins upon cell differentiation.

Mammalian ubiquitin-conjugating enzyme Ubc9 interacts with Rad51 recombination protein and localizes in synaptonemal complexes.

Hsubc9, a human gene encoding a ubiquitin-conjugating enzyme, has been cloned. The 18-kDa HsUbc9 protein is homologous to the ubiquitin-conjugating enzymes Hus5 of Schizosaccharomyces pombe and Ubc9 of Saccharomyces cerevisiae. The Hsubc9 gene complements a ubc9 mutation of S. cerevisiae. It has been mapped to chromosome 16p13.3 and is expressed in many human tissues, with the highest levels in testis and thymus. According to the Ga14 two-hybrid system analysis, HsUbc9 protein interacts with human recombination protein Rad51. A mouse homolog, Mmubc9, encodes an amino acid sequence that is identical to the human protein. In mouse spermatocytes, MmUbc9 protein, like Rad51 protein, localizes in synaptonemal complexes, which suggests that Ubc9 protein plays a regulatory role in meiosis.

The mouse and human genes encoding the recognition component of the N-end rule pathway.

The N-end rule relates the in vivo half-life of a protein to the identity of its N-terminal residue. The N-end rule pathway is one proteolytic pathway of the ubiquitin system. The recognition component of this pathway, called N-recognin or E3, binds to a destabilizing N-terminal residue of a substrate protein and participates in the formation of a substrate-linked multiubiquitin chain. We report the cloning of the mouse and human Ubr1 cDNAs and genes that encode a mammalian N-recognin called E3alpha. Mouse UBR1p (E3alpha) is a 1,757-residue (200-kDa) protein that contains regions of sequence similarity to the 225-kDa Ubr1p of the yeast Saccharomyces cerevisiae. Mouse and human UBR1p have apparent homologs in other eukaryotes as well, thus defining a distinct family of proteins, the UBR family. The residues essential for substrate recognition by the yeast Ubr1p are conserved in the mouse UBR1p. The regions of similarity among the UBR family members include a putative zinc finger and RING-H2 finger, another zinc-binding domain. Ubr1 is located in the middle of mouse chromosome 2 and in the syntenic 15q15-q21.1 region of human chromosome 15. Mouse Ubr1 spans approximately 120 kilobases of genomic DNA and contains approximately 50 exons. Ubr1 is ubiquitously expressed in adults, with skeletal muscle and heart being the sites of highest expression. In mouse embryos, the Ubr1 expression is highest in the branchial arches and in the tail and limb buds. The cloning of Ubr1 makes possible the construction of Ubr1-lacking mouse strains, a prerequisite for the functional understanding of the mammalian N-end rule pathway.