Lewis A. Jacobson

Structural and thermodynamic strategies for site-specific DNA binding proteins.

BACKGROUND Site-specific protein-DNA complexes vary greatly in structural properties and in the thermodynamic strategy for achieving an appropriate binding free energy. A better understanding of the structural and energetic engineering principles might lead to rational methods for modification or design of such proteins. RESULTS A novel analysis of ten site-specific protein-DNA complexes reveals a striking correspondence between the degree of imposed DNA distortion and the thermodynamic parameters of each system. For complexes with relatively undistorted DNA, favorable enthalpy change drives unfavorable entropy change, whereas for complexes with highly distorted DNA, unfavorable DeltaH degrees is driven by favorable DeltaS degrees. We show for the first time that protein-DNA associations have isothermal enthalpy-entropy compensation, distinct from temperature-dependent compensation, so DeltaH degrees and DeltaS degrees do not vary independently. All complexes have favorable DeltaH degrees from direct protein-DNA recognition interactions and favorable DeltaS degrees from water release. Systems that strongly distort the DNA nevertheless have net unfavorable DeltaH degrees as the result of molecular strain, primarily associated with the base pair destacking. These systems have little coupled protein folding and the strained interface suffers less immobilization, so DeltaS degrees is net favorable. By contrast, systems with little DNA distortion have net favorable DeltaH degrees, which must be counterbalanced by net unfavorable DeltaS degrees, derived from loss of vibrational entropy (a result of isothermal enthalpy-entropy compensation) and from coupling between DNA binding and protein folding. CONCLUSIONS Isothermal enthalpy-entropy compensation implies that a structurally optimal, unstrained fit is achieved only at the cost of entropically unfavorable immobilization, whereas an enthalpically weaker, strained interface entails smaller entropic penalties.

The autofluorescent lipofuscin granules in the intestinal cells of Caenorhabditis elegans are secondary lysosomes

The nematode Caenorhabditis elegans contains autofluorescent lipofuscin granules, located exclusively in the 32-34 intestinal cells. Using epifluorescence microscopy on live adult animals, we have shown that fluorescent-labeled exogenous probes are taken up by endocytosis and accumulate within the granules. Macromolecular solutes such as proteins and dextran appear to be taken up by fluid-phase pinocytosis. There is no phagocytosis of latex particles with diameter greater than or equal to 0.25 micron. The granules concentrate the lysosomotropic weak base acridine orange, indicating that they have an acidic internal milieu. These observations imply that the lipofuscin granules in the intestinal cells are secondary lysosomes which remain active recipients of endocytosed materials.

Delayed development and lifespan extension as features of metabolic lifestyle alteration in C. elegans under dietary restriction.

SUMMARY Studies of the model organism Caenorhabditis elegans have almost exclusively utilized growth on a bacterial diet. Such culturing presents a challenge to automation of experimentation and introduces bacterial metabolism as a secondary concern in drug and environmental toxicology studies. Axenic cultivation of C. elegans can avoid these problems, yet past work suggests that axenic growth is unhealthy for C. elegans. Here we employ a chemically defined liquid medium to culture C. elegans and find development slows, fecundity declines, lifespan increases, lipid and protein stores decrease, and gene expression changes relative to that on a bacterial diet. These changes do not appear to be random pathologies associated with malnutrition, as there are no developmental delays associated with starvation, such as L1 or dauer diapause. Additionally, development and reproductive period are fixed percentages of lifespan regardless of diet, suggesting that these alterations are adaptive. We propose that C. elegans can exist as a healthy animal with at least two distinct adult life histories. One life history maximizes the intrinsic rate of population increase, the other maximizes the efficiency of exploitation of the carrying capacity of the environment. Microarray analysis reveals increased transcript levels of daf-16 and downstream targets and past experiments demonstrate that DAF-16 (FOXO) acting on downstream targets can influence all of the phenotypes we see altered in maintenance medium. Thus, life history alteration in response to diet may be modulated by DAF-16. Our observations introduce a powerful system for automation of experimentation on healthy C. elegans and for systematic analysis of the profound impact of diet on animal physiology.

Quantitative measures of aging in the nematode Caenorhabditis elegans. I. Population and longitudinal studies of two behavioral parameters.

As a first step in the quantitative characterization of senescence in the nematode Caenorhabditis elegans, we have studied movement wave frequency, defecation frequency, and whole-body water efflux as a function of age. Populations of C. elegans, strain N2, were cultured monoxenically on E. coli lawns at 20 degrees C. The median lifespan in such populations was approximately 12 days. Population mean movement wave frequency declined linearly with age (slope = -4.66 waves/minute per day). The decline in population mean defecation frequency (defecations per minute) was multiphasic, consisting of (1) a rapid decline (slope = -0.233 defecations/minute per day) from day 3 to day 6, (2) no apparent trend from day 6 to day 9, and (3) a gradual decline (slope = -0.089 defecations/minute per day) from 9 to day 14. Animals alive on or after day 15 were not observed to defecate. In longitudinal studies, individual animals exhibited linear declines in movement wave frequency and multiphasic declines in defecation frequency. For future population studies, the age-dependent declines in movement and defecation frequency appear sufficiently large and reproducible to a multiparametric description of senescence in C. elegans. One physiological parameter, 3H2O efflux, was found to be age-independent and to consist of two first-order rates. The half-times of the slow and fast efflux rates were approximately 15 and approximately 2.1 minutes, respectively. The two half-times and the fractions of 3H2O exhibiting the two half-times were invariant with age.

Transgene-Coded Chimeric Proteins as Reporters of Intracellular Proteolysis: Starvation-Induced Catabolism of a lacZ Fusion Protein in Muscle Cells of Caenorhabditis elegans

The product of an integrated transgene provides a convenient and cell‐specific reporter of intracellular protein catabolism in 103 muscle cells of the nematode Caenorhabditis elegans. The transgene is an in‐frame fusion of a 5′‐region of the C. elegans unc‐54 (muscle myosin heavy‐chain) gene to the lacZ gene of Escherichia coli [Fire and Waterston (1989): EMBO J 8:3419–3428], encoding a 146‐kDa fusion polypeptide that forms active β‐galactosidase tetramers. The protein is stable in vivo in well‐fed animals, but upon removal of the food source it is inactivated exponentially (t1/2 = 17 h) following an initial lag of 8 h. The same rate constant (but no lag) is observed in animals starved in the presence of cycloheximide, implying that inactivation is catalyzed by pre‐existing proteases. Both the 146‐kDa fusion polypeptide (t1/2 = 13 h) and a major 116‐kDa intermediate (t1/2 = 7 h) undergo exponential physical degradation after a lag of 8 h. Degradation is thus paradoxically faster than inactivation, and a number of characteristic immunoreactive degradation intermediates, some less than one‐third the size of the parent polypeptide, are found in affinity‐purified (active) protein. Some of these intermediates are conjugated to ubiquitin. We infer that the initial proteolytic cleavages occur in the cytosol, possibly by a ubiquitin‐mediated proteolytic pathway and do not necessarily inactivate the fusion protein tetramer. J. Cell. Biochem. 67:143–153, 1997.

Activation of Ras and the Mitogen-Activated Protein Kinase Pathway Promotes Protein Degradation in Muscle Cells of Caenorhabditis elegans

ABSTRACT To discover and study intracellular signals that regulate proteolysis in muscle, we have employed transgenic strains of Caenorhabditis elegans that produce a soluble LacZ reporter protein limited to body-wall and vulval muscles. This reporter protein is stable in well-fed wild-type animals, but its degradation is triggered upon a shift to 25°C in a strain carrying a temperature-sensitive activating mutation in the Ras oncogene homologue let-60. These mutants are not physiologically starved, inasmuch as growth rates are normal at 25°C. Ras-induced degradation is not prevented by the presence of cycloheximide added at or before the temperature shift and thus uses preexisting proteolytic systems and signaling components. Furthermore, degradation is triggered when adult animals are shifted to conditions of 25°C, confirming that Ras acutely promotes protein degradation in muscles whose developmental history is normal. Reduction-of-function mutations in the downstream protein kinase Raf (lin-45), MEK (mek-2), or mitogen-activated protein kinase (MAPK) (mpk-1) prevent Ras-induced protein degradation, whereas activated MPK-1 is sufficient to trigger degradation, indicating that this kinase cascade is the principal route by which Ras signaling triggers protein degradation in muscle. This pathway is activated in hypodermal cells by the LET-23 epidermal growth factor receptor homologue, but an activating mutation in let-23 does not promote proteolysis in muscle. Starvation-induced LacZ reporter degradation is unaffected by reduction-of-function mutations in Ras, Raf, MEK, or MAPK, implying that Ras activation and starvation trigger proteolysis by mechanisms that are at least partially independent. This is the first evidence that Ras-Raf-MEK-MAPK signaling activates protein degradation in differentiated muscle.

Activated EGL-15 FGF receptor promotes protein degradation in muscles of Caenorhabditis elegans.

Signaling by fibroblast growth factors (FGFs) and their receptors has been previously implicated in control of cell proliferation, differentiation and migration. Here we report a novel role for signaling by the EGL‐15 FGFR of Caenorhabditis elegans in controlling protein degradation in differentiated muscle. Activation of EGL‐15, by means of a reduction of function mutation (clr‐1) affecting an inhibitory phosphatase, triggers protein degradation in adult muscle cells using a pre‐existing proteolytic system. This activation is not suppressed by mutation in either of the known genes encoding FGF ligands (egl‐17 or let‐756) but is well suppressed when both are mutated, indicating that either ligand is sufficient and at least one is necessary for FGFR activation. Activity of the Ras pathway through mitogen‐activated protein kinase (MAPK) is required to trigger protein degradation. This is the first report that degradation of intracellular protein can be triggered by a growth factor receptor using an identified signal transduction pathway. The data raise the possibility that FGF‐triggered proteolysis may be relevant to muscle remodeling or dedifferentiation.

Proteases of the nematode Caenorhabditis elegans

Crude homogenates of the soil nematode Caenorhabditis elegans exhibit strong proteolytic activity at acid pH. Several kinds of enzyme account for much of this activity: cathepsin D, a carboxyl protease which is inhibited by pepstatin and optimally active toward hemoglobin at pH 3; at least two isoelectrically distinct thiol proteases (cathepsins Ce1 and Ce2) which are inhibited by leupeptin and optimally active toward Z-Phe-Arg-7-amino-4-methylcoumarin amide at pH 5; and a thiol-independent leupeptin-insensitive protease (cathepsin Ce3) with optimal activity toward casein at pH 5.5. Cathepsin D is quantitatively most significant for digestion of macromolecular substrates in vitro, since proteolysis is inhibited greater than 95% by pepstatin. Cathepsin D and the leupeptin-sensitive proteases act synergistically, but the relative contribution of the leupeptin-sensitive proteases depends upon the protein substrate.

Opposed growth factor signals control protein degradation in muscles of Caenorhabditis elegans

In addition to contractile function, muscle provides a metabolic buffer by degrading protein in times of organismal need. Protein is also degraded during adaptive muscle remodeling upon exercise, but extreme degradation in diverse clinical conditions can compromise function(s) and threaten life. Here, we show how two independent signals interact to control protein degradation. In striated muscles of Caenorhabditis elegans, reduction of insulin‐like signaling via DAF‐2 insulin/IGF receptor or its intramuscular effector PtdIns‐3‐kinase (PI3K) causes unexpected activation of MAP kinase (MAPK), consequent activation of pre‐existing systems for protein degradation, and progressive impairment of mobility. Degradation is prevented by mutations that increase signal downstream of PI3K or by disruption of autocrine signal from fibroblast growth factor (FGF) via the FGF receptor and its effectors in the Ras–MAPK pathway. Thus, the activity of constitutive protein degradation systems in normal muscle is minimized by a balance between directly interacting signaling pathways, implying that physiological, pathological, or therapeutic alteration of this balance may contribute to muscle remodeling or wasting.

Calpains Mediate Integrin Attachment Complex Maintenance of Adult Muscle in Caenorhabditis elegans

Two components of integrin containing attachment complexes, UNC-97/PINCH and UNC-112/MIG-2/Kindlin-2, were recently identified as negative regulators of muscle protein degradation and as having decreased mRNA levels in response to spaceflight. Integrin complexes transmit force between the inside and outside of muscle cells and signal changes in muscle size in response to force and, perhaps, disuse. We therefore investigated the effects of acute decreases in expression of the genes encoding these multi-protein complexes. We find that in fully developed adult Caenorhabditis elegans muscle, RNAi against genes encoding core, and peripheral, members of these complexes induces protein degradation, myofibrillar and mitochondrial dystrophies, and a movement defect. Genetic disruption of Z-line– or M-line–specific complex members is sufficient to induce these defects. We confirmed that defects occur in temperature-sensitive mutants for two of the genes: unc-52, which encodes the extra-cellular ligand Perlecan, and unc-112, which encodes the intracellular component Kindlin-2. These results demonstrate that integrin containing attachment complexes, as a whole, are required for proper maintenance of adult muscle. These defects, and collapse of arrayed attachment complexes into ball like structures, are blocked when DIM-1 levels are reduced. Degradation is also blocked by RNAi or drugs targeting calpains, implying that disruption of integrin containing complexes results in calpain activation. In wild-type animals, either during development or in adults, RNAi against calpain genes results in integrin muscle attachment disruptions and consequent sub-cellular defects. These results demonstrate that calpains are required for proper assembly and maintenance of integrin attachment complexes. Taken together our data provide in vivo evidence that a calpain-based molecular repair mechanism exists for dealing with attachment complex disruption in adult muscle. Since C. elegans lacks satellite cells, this mechanism is intrinsic to the muscles and raises the question if such a mechanism also exists in higher metazoans.